CA2257353A1 - Liver retention clearing agents - Google Patents

Abstract

Liver retention clearing agents (LRCAs) and the use thereof are discussed. LRCAs are composed of a hepatic clearance directing component which directs the biodistribution of a LRCA-containing construct to hepatic clearance; a binding component which mediates binding of the LRCA to a compound for which rapid hepatic clearance is desired; a liver retention component which diminishes access of binding component-containing metabolites to target sites; and a structural component to provide a scaffold for the other components.

Description

CA 022~73~3 1998-12-01 L~VER RETENTION CLEARING AGENTS

s Technical Field The present invention relates to liver retention clearing agents (LRCAs), reagents for the plepardlion 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 o 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.

1S Back~round of the Invention Conventional cancer therapy is limited by the problem that the generally in~ble targeting ratio (ratio of a~mini~tered dose of active agent loc~li7ing totumor versus atlmini~t~red dose circnl~ting in blood) is low. This limitation isgenerally encountered in systemic afim;ni~tration of chemotherapeutic agents as well as in ~tlmini.~tration of monoclonal antibody-active agent conjugates.
~ystemic a~minictration involves exposure of healthy tissue to the active agent.Also, as a result of the relatively long half life of a monoclonal antibody, non-target tissue is exposed to circulating antibody-active agent conjugate.
Improvement in targeting ratio is therefore sought.
2s A method employed to improve targeting ratio is referred to generally as pretargeting. In pretargeting, 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 ~lmini~tered to a recipient, and permitted to localize to the target site with binding at that site mediated by the targeting agent. When target site localization and sufficient elimin~tion of circulating targeting moiety is achieved by the recipient's .. ,.. ~. , ... . , . , .. ... . . .. .. . . . ... .... ,.~

CA 022~73~3 1998-12-01 metabolism, the active agent-ligand is ~minictered The ligand component of the construct binds to the pretargeted receptor, thereby delivering the active agent to the target.
Pretargeting is made more efficient by ~(lminiStration of a clearing agent to facilitate elimin~Sion of circul~ting targeting moiety. Various clearing agents have been disclosed. Galactose-human serum albumin (HSA)-biotin clearing agents have been used in pretargeting protocols employing a monoclonal antibody-streptavidintargeting moiety and a biotin-active agent construct. Such clearing agents are discussed in PCT/US93/05406. Derivatization by galactose facilitates elirnination of o 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.

Summarv ofthe Invention The present invention is directed to liver retention clearing agents (LRCAs) which are designed to reduce the level of serum-associated targeting moiety-anti-ligand comple~c. Preferred LRCAs of the 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-ligand or ligand receptor. LRCAs of the present invention are preferably capable of achieving circul~tinv targeting moiety clearance without compromising the binding potential of the pretargeted targeting moiety, either directly by binding of the 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:
- rapid, efficient complexation with serum-associated targeting moiety-ligand (or anti-ligand) conjugate in vivo;
- rapid clearance from the blood of serum-associated targeting moiety conjugate capable of binding a subsequently administered complementary anti-ligand or livand cont~ining molecule, T

CA 022~73~3 1998-12-01 WO 97/46099 rCT/US97/09400 - high capacity for clearing (or inactivating) large amounts of serum-associated targeting moiety conjugate; and - low immllnogenicity.
An additional preferred characteristic of LRCAs of the present invention is 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 cont~ininp ligand or anti-ligand; and (4) a structural component.
o The structural component serves as a scaffold for binding of the hepatic clearance directing component, the liver retention component and/or the binding component.Preferably the binding component is attached to the structural component through the liver retention component. This construction facilitates the formation of LRCA
metabolites cont~inin~ both the binding component and the liver retention component.
Hepatic clearance directing components of I,RCAs of the present invention promote clearance of moieties to which they are attached to the liver. Preferredhepatic clearance directing components include sugar residues recognized by hepatocyte receptors. Preferred LRCAs of the present invention incorporate from about 15 to about 60 sugar residues, with from about 25 to about 50 residues preferred. Also, pre~ led sugars include galactose and N-acetylgalactosamine.
Preferably, the structural component is derivatized with an appropriate number of sugar residues.
Liver retention components of the present invention are d~ciPned to prevent release of binding component (such as ligand or anti-ligand)-cont~ining LRCA
metabolites to the serum compartment in a manner allowing those metabolites to accrete to pl~Lalg~led receptors. Preferably, liver retention components ofthe present invention are characterized by at least one of the following attributes:1) Resistance to agents that cleave peptide bonds or otherwise promote catabolism of LRCA-cont~inin~ moieties;

2) Retention in the cytoplasmic or a subcellular compartment following internalization into hepatic cells; or CA 022~73~3 1998-12-01 3) Excretion upon metabolism without re-entry, or with delayed or retarded re-entry, into the serum compartment (e.~J., biliary excretion without reabsorplion in the intestine).
Protease-resistant liver retention components of the present invention are useful, because formation of binding component-cont;lining 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. In a preferred embodiment of the present invention, the ligand or anti-ligand binding component of the LRCA is generally retained ino 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. An example of a protease-resistant liver retention agent is a poly-arnino 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. Another example of a metabolism-s resistant liver retention component incorporates at least one tertiary amide (-CO-N(R)-) bond, wherein R is preferably lower alkyl. Such bonds are more highly resistant to hydrolytic enzymatic activity (e. .., biotinidase activity) than secondary amide (peptide, -CO-NH-)bonds.
Liver retention moieties that are characterized by a limited capacity to traverse hydrophobic cellular or subcellular membranes also afford enhanced retention of binding component-cont~ining 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 a~lmini~tration and accretion to targeted receptor of active agent-cont~ining conjugate. Diffusion-restricted liver retention 2s moieties of the present invention are sufficiently polar or sufficiently charged to render passage through the non-polar lipid bilayer membrane structures difficult.
Examples of such 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), phosphates and phosphonates (negative charge), polylysines (positive charge), polyglutamic acids (negative charge) and the like.

. T

CA 022~73~3 1998-12-01 Liver retention component-binding component metabolites that are characterized by relatively rapid e~ccretion, without passage into the serum compartment, are also useful in the practice of the present invention. Metabolites e~ccreted by a hepatobiliary route without reabsorption by the intestines are preferred s for use in this aspect of the present invention. E~camples of rapid excretion-liver retention components are pepstatin, 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetra acetic acid (DOTA) and the like. Pepstatin is rapidly e~ccreted into the bile. When internalized into hepatocytes, DOTA is generally either trapped therein or e~creted via a hepatobiliary route.
o Another group of moieties useful as liver retention components in the practice of the 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, Iysyl-epsilon-amido S-iodo-3-pyridinecarboxylate, other non-m~mm~ n sugars, other radiolabelresidu~ ingmoieties andthelike.
Liver retention components useful in the present invention may combine more than one of the desirable properties set forth above. Preferred LRCAs of the present invention are characterized by the following liver retention components:
- poly-(D) orientation Iysine residues (metabolic stability and positive charge);
- poly- (D) orientation glutamic acid residues (metabolic stability and negativecharge);
- poly- galactose-derivatized (D) orientation Iysine residues (metabolic stability and polarity); and - poly-phosphonate, for example, based upon alpha-phosphonomethyl amino acid compounds, such as the following:
o HOOC-CH(NH2)CH2-P(OH)2 (negative charge).
Each ofthese p.t;r~.-ed liver retention components incorporate from about 2 to about 12 monomers, with from about 3 to about 6 monomers, more preferred. Also preferred as liver retention components in the practice of the present invention are polymers of natural amino acids of the (L) configuration empioyed in combination CA 022~73~3 1998-12-01 with a tertiary amide of a biotin binding moiety.
Binding components of the T RCAs of the present invention are moieties which recognize epitopes or components of molecules to be cleared by the LRCAs.
Preferred binding components of LRCAs of the present invention are members of s 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 I,RCAs of the 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 I to o 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.
Consequently, human proteins, such as human serum albumin, IgG, Igl~/l and the like constitute structural components useful in the practice of the present invention.
Preferred polymeric structural components of the present invention include dextran, hydroxypropylmethylacrylamide (HPMA), hydroxypropylacrylamide, hydroxypropylethylacrylarnide, poly-D-lysine, poly-D-glutamate, poly-D-aspartate, dendrimers (spherical constructs having functional units on the exterior thereof) and the like.

Brief Description of the Drawings 2s Figure 1 illustrates the tumor uptake profile of antibody-streptavidin conjugate (Ab/SA) in comparison to a control profile of native whole antibody (Ab) and streptavidin (SA).
Figures 2a and 2b schematically illustrate the pl epal ation of a liver retention component-binding component construct, N-methyl-N-{5'-[methylester tris-(-(D, L)-3() phosphonoalanyl)-(D)-cystyl]-5-carbamylpentyl } biotinamide.
Figures 3a and 3b schematically illustrate the preparation of a liver retention CA 022~73~3 1998-12-01 component-binding component construct, N-methyl-N-[5-(triglutamylcysteine)-5-carbamylpentyl] -biotinamide.

Detailed Description of the Invention s Prior to setting forth the invention, it may be helpful to set forth definitions of certain terms to be used within the disclosure.
Tar~eting 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.
o 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.
Li .and/anti-ligand pair: A complementary/anti-complementary set of molecules that demonstrate specific binding, generally of relatively high affimty. Exemplary s ligand/anti-ligand pairs include zinc finger protein/dsDNA fragment, enzyme/inhibitor, hapten/antibody, lectin/carbohydrate, ligand/receptor, S-protein/S-peptide, headactivator protein (which binds to itself), cystatin-C/cathepsin B, and biotir~avidin.
Biotin/aYidin is used throughout the specification as a prototypical example of a ligand/anti-ligand pair.
Anti-li and: As defined herein, an "anti-ligand" demonstrates high affinity, andpreferably, multivalent binding of the complementary ligand. Preferably, the anti-ligand is large enou~h to avoid rapid renal clearance, and is multivalent to bind a larger number of ligands. Univalent anti-ligands are also contemplated by the present invention. Anti-ligands of the present invention may exhibit or be derivatized to 2s exhibit structural features that direct the uptake thereof, e.2., galactose residues that direct liver uptake. Avidin and streptavidin are used herein as prototypical anti-ligands.
Avidin: As defined herein, "avidin" includes avidin, streptavidin and derivatives ~ and analogs thereof that are capable of high affinity, multivalent or univalent bindin;, of biotin.
Li~and: As defined herein, a "ligand" is a relatively small soluble molecule that CA 022~73~3 1998-12-01 binds with high affinity by anti-ligand and plefelably 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 Affinitv Li~Jand or Lower Affinity Anti-Ligand: A ligand or anti-ligand s 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. Preferably, lower affinity ligands and anti-ligands e~libit between fromabout 10 to 10 1~1 binding affinity for the native form of the complementary anti-ligand or ligand. For avidinlstreptavidin and other extremely high affinity binding o molecules, however, lower affinity may range between 10 to 10 ~1. Lower affinity ligands and anti-ligands may be employed in clearing agents of the present mventlon.
Active A ent: A diagnostic or therapeutic agent ("the payload"), including radionuclides, drugs, anti-tumor agents, to~cins, superantigens and the like.
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.
Pretar~eting: As defined herein, 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 accun~llation 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 conjugatè at the target site (two-step pretargeting). Three-step and other related methods described herein are also encompassed.
Clearin~ A~ent: An agent capable of binding, complexin, or otherwise associating with an adminictered moiety (e.g., targeting moiety-ligand, targeting moiety-anti-ligand or anti-ligand alone) present in the recipient's circulation, thereby f~cilit~tinv circ~ ting 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, reduced affinity, configuration or a combination thereof, that limit clearing agent access to the CA 022~73~3 1998-12-01 population of target cells recognized by a targeting moiety used in the same treatment protocol as the clearing agent.
Conju ate: A conjugate encompasses chemical conjugates (covalently or non-covalently bound), fusion proteins and the like.
s Liver Retention Clearin~ A~ent (LRCA): A moiety capable of directing the clearance of a moiety to which it is bound upon administration or of a component to which it becomes associated with in vivo. LRCAs of the present invention direct clearance via a hepatic pathway. Preferred LRCAs of the present invention are characterized by a structural component, a hepatic clearance directing component, a o liver retention component and a binding component.
Hepatic Clearance Directin . 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 residuespreferred. The structural component is preferably derivatized with an appropriate .Is number of sugar residues.
Liver Retention Component: A moiety designed to prevent release of ligand- or anti-ligand-cont~ining metabolites of LRCA constructs to the serum compartment in a manner allowing those metabolites to accrete to pretargeted receptors. Preferable liver retention components of the present invention are characterized by at least one 2() of the following attributes:
1) Resistance to agents that cleave peptide bonds or otherwise promote catabolism of LRCA-con~ining 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.
Bindin~ Component: A ligand, anti-ligand or other moiety capable of in vivo association with a previously a-~mini~tered molecule (bearing the complementary ligand or anti-ligand, for example) or with another toxic or potentially toxic molecule 3~ present in the recipient's circulation or extravascular fluid space via recognition by the - binding component of an epitope associated with the previouslv administered moiety CA 022~73~3 1998-12-01 or with the toxic or potentially toxic molecule.
Structural Component: A moiety which serves as a scaffold for bindin~J of the hepatic clearance directing component, the liver retention component and, optionally, the binding component. Preferred structural components include proteinaceous andnon-proteinaceous materials having sufficient reactive groups for such derivatization, such as human proteins and polymers.

The LRCAs of the present invention are preferably employed in pretargeting protocols. "Two-step" pretargeting procedures feature targeting moiety-ligand orlo targeting moiety-anti-ligand (targeting moiety-receptor) administration, followed by a~mini~tration of active agent conjugated to the opposite member of the ligand-anti-ligand pair. As step "1.5" in the two-step pretargeting methods ofthe present invention, a LRCA is a~ministered to facilitate the clearance of circul~tin~ targeting moiety-receptor conjugate.
s In the two-step pretargeting approach, 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 pl ~L~ ing 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-circul~tin~ avidin-targeting moiety complexes and substantially precludes association with the avidin-targeting moiety conjugates bound at target cell sites. (See, Goodwin, D.A., Antibod. Immunoconj. Radiopharm., 4: 427-3~, 1991).
LRCAs of the present invèntion contain a hepatic clearance directing 2s component, a liver retention component, a binding component and a structural component. Thus, 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 il~ vivo binding or association with molecules to be cleared and interaction with hepatic receptors to effect clearance of LRCA-cont~ining constructs by that route. Preferred LRCAs of the present invention are suitable for use as a CA 022~73~3 1998-12-01 clearing agent in p.eta~geling protocols, in~lu-lin~ two step protocols.
Clearing agents usefill in the practice of the present invention preferably exhibit one or more of the following characteristics:
- rapid, efficient complexation with serum-associated targeting moiety-ligand (or anti-ligand) conjugate in vivo;
- rapid clearance from the blood of serum-associated tar8eting moiety conjugate capable of binding a subsequently administered complementary anti-ligand or ligand con~inin,, molecule;
- high capacity for clearing (or inactivating) large amounts of serum-associatedo targeting moiety conjugate; and - low immunogenicity.
Clearing agents previously developed by the assignee of this patent application incorporated, for example, a structural component of human serum albumin (HSA), a plurality of hexoses and a plurality of 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 ~5 and wherein the hexose is recognized by liver receptors, e. ., 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-cont~ining complexes, and induce endocytosis into the hepatocyte, leading to fusion with a Iysosome and recycling of the receptor back to the cell surface. This clearance mech~nism 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 decigned to meet the four criteria set forth above as well. Two additional performance criteria were instituted for LRCAs:
- operability over a wide LRCA dose range to avoid the desirability of extensivedose optimization; and - low ability to compromise pretargeted receptor.
Preferred LRCAs of the present invention are therefore characterized by a liver CA 022~73~3 1998-12-01 retention component which (limini~h~5 the ability of LRCA metabolites, particularly binding component-containing metabolites, to accre~e to pretar~eted receptor. This feature also serves to increase the dose range over which the LRCA may be employed, because compromise of pretargeted receptor by such binding component-s cont~inin~ metabolites is decreased or avoidecl. Preferred LRCAs of the present invention also incorporate a structural component of proteinaceous or non-protein~ceous composition. Such preferred LRCAs exhibit physical properties facilitating use for in vivo complexation and blood clearance of anti-ligand/ligand-targeting moiety conjugates.
o Other embodiments of the present invention involve the preparation and use of LRCAs in clearance of other previously ~(lmini~tered molecules or to~ic or potentially toxic molecules generated i~l vivo, which compounds to be cleared are present in a patient's circulation or extravascular fluid space. Previously allmini~tered molecules may include active agent-con~ining conjugates (e. .., radionuclide-chelate-antibody which can be cleared by a LRCA cont~ining an anti-chelate or anti-antibody binding moiety; or radionuclide-chelate-antibody-biotin binding protein which can be cleared by a biotin-cont~ining LRCA); targeting moiety-receptor conjugates; or the like.Preferred LRCAs of the present invention are ~ministered and permeate the circulation. Consequently, previously ~mini.~tered compounds or toxic or potentially toxic moieties that are present in the circulation are accessible to the LRCAs of the present invention. Circulating compounds are removed from the serum via association with the LRCA and processing by liver receptors. Previously ~cimini~tered compounds or toxic or potentially toxic moieties, present in extravascular fluid space but not associated with a target cell or epitope, are removed 2s by the LRCAs of the present invention via liver receptors as such compounds dif~use back into the circulation and become associated with LRCAs. Toxic or potentiallytoxic molecules that may be removed from a recipient's circulation or e~ctravascular fluid space include: chemotherapeutics e.g., alkylators, heavy metals and the like.
Binding components useful in the practice of the present invention are capable of associating with the molecule to be cleared. Suitable bindino components therefore include those moieties that are capable of associating with to~;ic or potentially toxic CA 022~73~3 1998-12-01 molecules present in the recipient's circulation, which include antibodies or fr~gm~ntc thereof directed to epitopes that are characteristic of such to:cin or poter,tial to.Yin.
Other useful binding components include oligonucleotides, ligands or anti-ligands.
Ligands and anti-ligands are preferred binding components of the present in~ ention.
s A particularly preferred bindina component for use in the practice of the present invention is biotin or a derivative or analog thereof.
Characteristics of useful binding components are discussed below. The binding between the binding component of the LRCAs of the present invention and the molecule to be cleared from the circulation need only be transient, i e., e,~cists for a u sufficient amount of time to clear the molecule to the liver and for hepatocyte internalization. Also, it should be noted that the binding constant of the binding component is determined with regard to the LRCA as a whole. That is, a biotin-containing LRCA is e~cpected to bind to avidin or streptavidin with a binding constant less than that of biotin itself. Experim~nt~tion has revealed that the LRCAs of the present invention are capable of clearance.
In general, 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 e:ccess of about I oB are preferred.
For use in an LRCA of the present invention, 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 substitute2s acceptable moieties for the binding components ~iccussed specifically herein.
Preferred 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 fi~nctional groups to allow for ~tt~chrnent of other molecules of interest, e.u., peptides, proteins, nucleotides, and other small molecules.
LRCAs of the present invention are designed to interact with hepatic receptors to facilitate clearance of LRCA-containin, constructs via that route The hepatic . . ... . .. .. . . .

CA 022~73~3 1998-12-01 clearance directing component of the LRCA is included for this purpose.
Hepatocyte receptors which provide for effective clearance include in particularAshwell receptors, mannose receptors associated with endothelial cells and/or ICupffer cells of the liver, the mannose 6-phosphate receptor, and the like. Hexoses ~vhich may be employed in the LRCA structure include by way of example galactose, mannose, maMose 6-phosphate, N-acetylgalactosamine, pent~m:-nnosyl-phosphate, and the like. Hexoses recognized by Ashwell receptors include glucose, galactose, galactosamine, N-acetylgalactosamine, p~nt~m~nnosyl phosphate, mannose 6-phosphate and thioglycosides of galactose, galactosides, galactosamine, and N-lo acetylgalactosamine. A sufficient number of he~cose residues are incorporated into the LRCA to provide for effective clearance, e.g., via the Ashwell receptors located on the surface of hepatocytes.
Preferable hepatic clearance directing components of this embodiment of the LRCAs of the present invention constitute between about 15 and about 60 he,Yose residues, e.g., galactose residues or N-acetylgalactosamine residues. ~lore preferably hepatic clearance directing components of the present invention constitute between about 25 and 50 hexose residues. Preferably, the LRCA structural component is derivatized by an appropriate number of sugar residues. However, 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-cont~ining metabolites of LRCA constructs to the serum compartment in a manner allowing those metabolites to accrete to pretargeted receptors. Preferably, liver retention components of the present invention are characterized by at least one of the following attributes:
1) ~esi.ct~nce to agents that cleave peptide bonds or otherwise promote the catabolism of LRCA-con~ g moieties, 2) Retention in the cytoplasmic or a subcellular compartment following internalization into hepatic cells; or 3~ 3) E~ccretion upon metabolism without re-entry, or with delayed or retarded re-entry, into the serum compartment.
T

CA 022~73~3 1998-12-01 WO 97/46099 PCTtUS97/09400 Protease-resistant liver retention components of the present invention are useful, because formation of binding component-containing metabolites of LRCP~ constructs capable of accecsing target-associated ligand or anti-ligand is not favored. That is, any such metabolites are unlikely to accrete t'rom hepatocytes in a manner permitting access to pretargeted receptors. In a preferred embodiment of the present invention, the ligand or anti-ligand binding component of the LRCA is generally retained inhepatocytes for a time sufficient to allow active agent-ligand or active agent-anti-ligand construct accretion to target.
An e~cample of a protease-resistant liver retention agent is a poly-amino acid of 0 unnatural (D) orientation. The (D) amino acid sequence provides resistance to catabolic processing, because Iysosomal exopeptidases and endopeptidases recognize peptides of the natural (L) orientation. Thus, 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 i~ 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) aminoacids are charged (D) amino acid polymers, such as poly (D) Iysine, 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 (AIB) and N-alkyl-substituted amino acids which form tertiary arnide bonds rather than secondary amide (peptide) bonds. The alkyl moiety is lower alkyl, preferably methyl, ethyl, propyl or butyl, with methyl most preferred.
Alternatively, the tertiary arnide can be formed using an N-phosphono substitution.
Liver retention moieties that are characterized by a limited capacity to traverse ~5 hydrophobic cellular or subcellular membranes also afford ~nh~nced retention of binding component-cont~inin~ 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-cont~ining conjugate.
3() Diffusion-restricted liver retention moieties of the present invention are sufficiently polar or sufficiently charged to render passage throuol1 non-polar lipid CA 022~73~3 1998-12-01 bilaver membranes difficult under hepatocyte cellular conditions, pH 6-7. E~amples of such liver retention components are moieties characterized by posi~ive charoe, negative charge or neutral charge combined with hydrophilicitv. Preferred liver retention components of this type are saccharides (neutral char;,e/hydrophilic),phosphates or phosphonates (negative charge), polylysines (positive charge), polyalutamic acids (negative charge) and the like.
With regard to subcellular membranes, diffusion-restricted liver retention components are sufficiently polar or sufficiently charged to render passage through the non-polar lipid bilayer membranes difficult under subcellular (e. a, Iysosomal) 0 conditions. For example, Iysosomes are characterized by a pH of about 5. Thus, moieties expected to be highly charged at acidic pH (basic moieties) are desirable for use as liver Iysosomal retention components. Also, basic proteins are generallv catabolized more slowly than acidic proteins. As a result, liver retention components made up of basic amino acids, such as Iysine, histidinet arginine, ornithine and the like, are useful Iysosomal 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: polyglutamic acids~ poly-phosphates, polyphosphonates, such as poly-alpha-phosphonomethyl amino acids, polyaspartates and the like.
Neutral charge/hydrophilic, diffusion-resistant liver retention a~ents useful in the practice of the present invention include the following: ~ cçk~rides such as cellobiose and lactose, deoxysorbitol, dilactitol, amino-naphthaltyrimide-deoxysorbitol (ANTDS)t unnatural polysaccharides, D-poly amino acid saccharide derivatives andthe like. See, for example, Ali et al., "Synthesis and Radioiodination of Tyramine Cellobiose for Labeling l~lonoclonal Antibodies," Nucl. ~1ed. Biol.~ 1'(5!: 5~7-61, 1988, and Demignot et al., "Differences between the catabolism and tumour distribution of intact monoclonal antibody (791T/36) and its Fab/c fra~ ent in mice CA 022~73~3 1998-12-01 with tumour xenografts revealed by the use of a resi~ li7ino radiolabel (dilactitol-' 5-I-tyramine~ and autoradio ,raphy," Cancer Immunol. Immunother. ~,: iS9-66, 1991.
For liver retention components involving poly-amino acids, the number of such s amino acids is selected to prevent passive diffusion of the binding component-liver retention component metabolite of the LRCA from hepatocyte Iysosomes. Literatureindicates that certain dipeptides diffuse freely, while tripeptides generaily do not.
Consequently, liver retention components having three or more amino acids are generally preferred. Also, lengthy poly-amino acids present some synthetic o 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 e~cretion, without passa~e into the serum compartment are also useful in the practice of the present invention. ~etabolites s e:ccreted by a hepatobiliary route without reabsorption by the intestines are preferred for use in this embodiment of the present invention. E~camples of e~ccretion-liver retention components are pepstatin, 1,4,7,10-tetraazacyclododecane-N,N',~",N"'-tetra acetic acid (DOTA) and the like. Pepstatin is rapidly e~ccreted into the bile. DOTA is excreted via a hepatobiliary route following internalization by hepatocytes. Other rapid excretion liver retention agents useful in the practice of the present invention include the following- diethylene triamine penta-acetic acid (DTPA), ethylene diamine tetra-acetic acid (EDTA), ethylene glycol bis-(beta-aminoethylether)-N,N,~',N'-tetraacetic acid (EGTA) and the like.
Another group of moieties useful as liver retention components in the practice 2s of the 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 lysosomaVintracçll~ r retention and decreased susceptibility to metabolic degradation. Examples of such liver retention components are cellobiose, dilactitol, lysyl-epsilon-amido S-iodo-3-pyridinecarbo~{ylate. other non-mammalian CA 022~73~3 1998-12-01 sugars, other radiolabel r~sidu~ii7ing 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 of the present invention are characterized by the following liver retention components:
s - poly-(D) orientation Iysine residues (metabolic stability and positive charge);
- poly- (D) orientation glutamic acid residues (metabolic stability and negativecharge);
- poly- galactose-derivatized (D) orientation Iysine residues (metabolic stability and polarity); and o - poly-phosphonate, for e~cample, based upon alpha phosphonomethyl amino acids, such as o HOOC-CH(NH,)CH,-P(OH), (negative charge).
Arnino acids of natural (L) configuration may be employed in the preferred liverretention components; provided that at least one bond in the liver retention component-binding moiety construct is a tertiary amide bond. Such tertiary amidebonds 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 of the liver retention component to prevent access of the binding moiety to pretargeted receptor.
When a biotin binding moiety, for e~ample, is employed in a LRCA of the present invention, polyamino acids of natural (L) orientation mav be employed, provided that the amide bonds between the biotin and the polyamino acid and between the individual amino acids of the polyamino acid incorporate a tertiary arnine.
2s 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.
Preferably 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 E~camples of specific preferred embodiments of biotin bindir.g component/liver retenti~n component combinations for use in LRCAs of the presentinvention are shown below s /O R- \ O R 0 /S (I) HS~\NHtC\~N--C(CH:)s-N--C-(CH~)4 ~ ~ (d--Iys)nBT

N H ~--C ~:~ N ~C(C H 2)s- N--C - (C H 2) 4~ ~d--Iys-gal)nBT
~ (CH2hor~ ~ HN~NH

CO2H / 1~l R~\ O R O S (3) HS~ ~C~N--~(CH2)5-N--C-(CH2)4~(d--glu)n BT

C~2 ~ HN~NH

eO ~ N--C(cH2)s-N--C-(CH ~ (ph s~-BT

(CH2) HN~f NH

CA 022~73~3 1998-12-01 CO2Me /~ R O S (5) HS~"~ C~N~CtCH2)s~t~1--C-(CH2)4~ (Ph~s)n BTII

( I H2)t-3 / HN NH
PO:~ H / ~I~

where 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 alk~ l from 1 to about 6 carbon atoms.
s Biotinidase generally cleaves a secondary amide bond adjacent to the biotin. In conducting research relating to active agent constructs involving DOT~-biotin, it was discovered that substitution of the aMide nitrogen with, for e:cample, an alkyl moiety resulted in stability with respect to biotinidase cleavage. Consequently, preferred liver retention component-biotin constructs employ a tertiary amide nitrogen bearing an zo alkyl substituent, preferlbly 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-proteinaceous composition. Preferred structural components are characterized by or are derivatized to contain sufficient reactive groups for binding with hepatic cle~rance 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 fu;lctional ~roups. Tllese ranges are derived as tollows:
Preferred hepatic clearance directing component derivatization is from about 15 to about 60 hexoses (more preferred, from about ''5 to about 50); and preferred binding component/liver retention component derivatization is from about 1 to about 10 ~o CA 022~73~3 1998-12-01 (most preferred, from about 1 to about 2).
Derivatization with both he:cose and binding component are conducted in a manner sufficient to produce individual clearing agent molecules with a ranoe ofderivatization levels that averages a recited whole number. For e~cample, biotinylation s 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 mi~cture 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 0 derivatized. The whole numbers used in this description refer to the average derivatization of the LRCAs under ~i~Cll~cion.
One embodiment of LRCA of the present invention incorporates a proteinaceous structural component of intermediate molecular weight (ranoin, from about 40,000 to about 200,000 Dal), such as asialoorosomucoid, human serum lS albumin or other soluble natural protein, preferably those having low immunogenicity when ~ministered to hllm~nc Alternatively, 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 Igi~ or IgG
(app-o~,mately 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, hydroxypropylmeth~rrylamide polymers, polyvinylpyrrolidone-polystyrene copolymers, divinyl ether-maleic acid copolymers, pyran copolymers, or polyethylene 2s glycol (PEG), also have utility as structural components of LRCAs of the present invention. In addition, liposomes (high molecular weight moieties with poor target access) can be used as a structural component of LRCAs of the present invention.LRCAs having a human protein as the structural component thereof are preferred for use in the practice of the present invention. Human proteins, especially human serum proteins, such as, for example, orosomucoid and human serum albumin,human IgG, human-anti-antibodies of IgG, Ig~ and Ig~I class, and the like. are less , . , .. . . . . .... . . ~ .

CA 022~73~3 1998-12-01 imrnunogenic upon arlmini~tration into the serum of a human recipient. Human orosomucoid is commercially available frcm, for e,Yample, Sigma Chemical Co, St.Louis, Missouri. Treatment of orosomucoid with neuraminidase removes sialic acidresidues, thereby e~cposing galactose residues, forming asialoorosomucoid. HumanHSA (Cutter Biological) and human IgG, IgA and IgM (Sigma Chemical Co.), for e~ample, are also commercially avai}able. Other proteinaceous structural components include albumin, Ig~l, IgG, asialohaptoglobin, asialofetuin, asialoceruloplasmin and the like.
Human serum albumin is a preferred proteinaceous structural component for o LRCAs ofthe present invention. Other m~mm~ n 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. E.Yamples of such m~mm~ n forms of serum albumin are bovine serum albumin, porcine serum albumin, and the like.
In general, LRCAs of the present invention are prepared in the following s manner:
1 ) The hepatic clearance directing component is conjugated to the structural component;
2) The hepatic clearance directing component-structural component construct is derivatized, preferably with from about 1 to about 10, more preferably from about 1 ~o to about 4, and still more preferably from about 1 to about 2 reactive groups;
3) Binding component/liver retention component constructs are synthe~i7~ d and are either characterized or are derivatized to contain a complementary reactive group to those generated in step (2); and 4) Binding component/liver retention component is conjugated to hepatic clearance directing component-structural component to form an LRCA.
The 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 e~ample, maleimide-sulfhydryl, active ester-amine, isothiocyanate-amine and the like. Selection of useful reactive groups is within the ordinary skill in the art.
Preferred LRCAs of the present invention are shown belo~.v:

Gal~O-,O -HSA-S lc (( N-~C(CH.)s-N--C -(CH7)4 ( 1 ) NH \~ ~ HSA ~d--Ys)n BT

( I H2)3 C~1~7 HN NH
+NH

Gal30-~O-HSA-S NH -c\~N~c(cH2)s-N-c-(cHz)4~>~

O (C112)3 or .1~ HN NH (2) ~ \.JaiS~c--I~H ~ H3A (d--ys-gal)n3T

C02H / r~ R- \ O R O
Gal30-40-HSA~ N---IC(cH2)s-N--C-(CH2)q Ç~

2)1 0~ ¦ HSA (d-giu)n BT
HN\ /NH
co-2 .,. lT

2() S HSA~ ,al~O-.~O
/0 R'~\ O / ~ rJ
MeoLc l \c~NH-clcH2)s-N--C-(CH2) ~ (HSA (phos)n BT
(CH2)~ HN~NH

'5 o Gal30-40 HSA 5~ ~lc N~C(CH2)s~N--C-(CH2)4 ( ) ~ ~ HSA (phos)n BTII
( IC~2)~ J HN NH
/~ ~

CA 022~73~3 1998-12-01 where n is 1 to 50, preferably about 3 to about 1~, and more preferably from about 3 to about 6; R is a lower alkyl moiety of from I to about 6 carbon atoms; ar.d R is hvdrogen or lower alkyl from 1 to about 6 carbon atoms.
Preparation of these preferred LRCAs of the present invention may be conducted as follows:
1) Galactosylation of ~SA, 2) ~Ialeimide derivatization of the galactosylated HSA; and 3) Attachment of biotin-liver retention component via a sulfhydryl group to the maleimide-derivatized galactosylated HSA. An e~ample of this process is set forth in 0 detail in the Example IV.
The preferred LRCAs of the present invention were evaluated using t~vo criteria:
A) Effectiveness at clearing targeting moiety-streptavidin conjugate; and B) Biotin release from the LRCA construct.
These criteria were evaluated in comparison to Galactose-HSA-biotin clearing agents previously developed by the assignee of this patent application as set forth in E~camp}e V; however, evaluation of LRCAs does not require such a comparison. Thus, LRCAs can be evaluated in comparison to each other or to established performance criteria.
All four preferred constructs cleared targeting moiety-streptavidin conjugate ~o comparably to the previously developed conjugate, galactose30.35-human serum albumin-LC-biotin, wherein LC is an aminocaproyl spacer and wherein the number of biotins ranges from 1 to about 6. At low doses of agent, LRCAs appear to releasesignific~ntly less biotin, and the trend continues at higher doses.
One embodiment of the present invention provides LRCAs having physical 2s properties f~rilit~ting use for in vivo comple~ation and blood clearance of anti-ligand/ligand (e.g., avidin/biotin)-targeting moiety (e.~., antibody) conjugates. These L~CAs are useful in improving the target:blood ratio of targeting moiety-containing conjugate. One application in which target:blood ratio improvement is sought is in solid tumor im~sring and therapy.
Other applications of these LRCAs include lesional imaging or therapy involving blood clots and the like, employing antibody or other t,.rgeting vehicle-active agent 2~

CA 022~73~3 1998-12-01 delivery modalities. For eYample, an efficacious anti-clotting agent provides rapid target localization and high target:non-target ratio. Active agents a~mini.~tered in pretargeting protocols of the present invention using efficient clearing agents are targeted in the desirable manner and are, therefore, useful in the im~ing/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 m~mm~ n recipient, which methods include:
~lminictPring to the recipient a first conjugate comprisin~ a targeting moiety and a member of a ligand-anti-ligand binding pair;
o thereafter ~-lmini~tering 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 ~iministering 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.
Clearing agent evaluation experimentation involving galactose- and biotin-derivatized clearing agents is detailed in Example III. The specific clearing agents e~camined during the Example III experimentation are human serum albumin derivatized with galactose and biotin and a 70,000 dalton molecular weight de~tran derivatized with both biotin and galactose. The experimentation showed that proteins and polymers are derivatizable to contain both galactose and biotin and that theresultant derivatized molecule is effective'in removing circulating streptavidin-protein conjugate from the serum of the recipient. Biotin loading was varied to determine the 2s effects on both clearing the blood pool of circulating avidin-containing conjugate and the ability to deliver a subsequently ~riministered biotinylated isotope to a target site recognized by the streptavidin-co~ nillg conjugate. The effect of relative doses of the administered components with respect to clearing agent efficacy was also examined. P,epal~lion of LRCAs ofthe present invention is discllssed in Example IV
below. EYperiment~tion relating to the LRCAs of the present invention is set forth in Example V below.

, . . ~

CA 022~73~3 1998-12-01 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 li~and/anti-ligand pair member (i e., lower affinity ligands or anti-ligands). In embodiments of the present invention employing a biotin-avidin or biotin-streptavidin ligand/anti-ligand pair, 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).
o In two-step p~ ~1a~e~i-1g protocols employing the biotin-avidin or biotin-streptavidin ligand-anti-ligand pair, lower affinity biotin, lower affinity avidin or lower affinity streptavidin may be employed. Exemplary lower affinity biotin molecules, for example, 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 IS streptavidin; are non-toxic to m~mm~ n recipients; and the like. Exemplarv lower affinity avidin or streptavidin molecules, for example, 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 m~mm~ n recipients; and the like.
Exemplary lower affinity biotin molecules include 2'-thiobiotin; 2'-iminobiotin;I'-N-methoxycarbonyl-biotin, 3'-N-methoxycarbonylbiotin; 1-oxy-biotin; 1-oxy-2'-thiobiotin; 1-oxy-2'-iminobiotin; I-sulfoxide-biotin, l-sulfoxide-2'-thiobiotin; 1-sulfoxide-2'-irninobiotin; l-sulfone-biotin; 1-sulfone-2'-thio-biotin; 1-sulfone-2'-iminobiotin; imida_olidone derivatives such as desthiobiotin (d and dl optical isomers), dl-desthiobiotin methyl ester, dl-desthiobiotinol, D-4-n-hexyl-imida_olidone, L-4-n-hexylimid~7olidone~ dl-4-n-butyl-imidazolidone, dl-4-n-propylimidazolidone, dl-4-ethyl-imida_olidone, dl-4-methylimiti~lidone, imidazolidone, dl-4,5-dimethylimid~7.olidone, meso-4,5-dimethylimid~701idone, dl-norleucine hydantoin, D-4-n-hexyl-2-thiono-imida7olidine, d-4-n-hexyl-2-imino-imidazolidine and the like;
oxazolidone derivatives such as D-4-n-hexyl-oxazolidone, D-S-n-hexyioxa7olidone and the like; [5-(3,4-diamino-thiophan-2-yl] pentanoic acid; lipoic acid, 4-hvdroxy-azobenzene-'~'-carboxylic acid; and the like. Preferred lower affinity biotin molecules CA 022~73~3 1998-12-01 for use in the practice of the present invention are 2'-thiobiotin, desthiobiotin, l-oxy-biotin, I-o~cy-2'-thiobiotin, l-sillfoxide-biotin, 1-sulfoxide-2'-tlliobiotin, l-sulfone-biotin, l-sulfone-2'-thiobiotin, lipoic acid and the like. These e~cemplary lower affinity biotin molecules may be produced substantially in accordance with known procedures therefor. Incorporation of the e,Yemplary lower affinity biotin molecules into LRCAs proceeds substantially in accordance with procedures described herein in regard to biotin incorporation.
Much has been reported about the binding affinity of different biotin analogs toavidin. Based upon what is known in the art, the ordinary skilled artisan could readily o select or use known techniques to ascertain the respective binding affinity of a particular biotin analog to streptavidin, avidin or a derivative thereof.
The present invention further provides methods of increasing active agent localization at a target cell site of a m~mm~ n recipient, which methods include:
~mini~terjng to the recipient a first conjugate comprisin_ a targeting moiety t~ 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 ~tlmini~terjng 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, conctit~ltes a native or high affinity form thereof.
The "lalgeLil-g moiety" ofthe present invention binds to a defined target cell population, such as tumor cells. Preferred targeting moieties useful in this regard include antibody and antibody fr~mPntc, peptides, and hormones. Proteins corresponding to known cell surface receptors (including low density lipoproteins, transferrin and insulin), fibrinolytic enzymes, anti-H~R2, platelet binding proteins such as aMe~cins' and biological response modifiers (including interleukin, interferon, er~thropoietin and colony-stimul~ting factor) are also ?referred targeting moieties.

... . . . , , ,, " , . . . ..

CA 022=,73=,3 1998-12-01 Also, anti-EGF receptor antibodies, which internalize following binding to the receptor and traffic to the nucleus to an e~cter,t, are preferred targetino moieties for use in the present invention to facilitate delivery of Auger emitters and nucleus binding drugs to target cell nuclei. Oligonucleotides, e.~J., antisense oli~onucleotides 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 o moieties may be de~igned Functional equivalents of the aforementioned molecules 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 moietv-target cell binding.
Another targeting moiety functional equivalent is a short polypeptide drsign~ted as a "minimal" polypeptide, constructed using computer-assisted molecular modeling and mutants having altered binding aff~nity, which minimal polypeptides e~hibit the binding affinity of the targeting moiety.
Preferred targeting moieties of the present invention are antibodies (polyclonalor monoclonal), peptides, oligonucleotides or the like. Polyclonal antibodies useful in the practice of the present invention are polyclonal (Vial and Callahan, Univ. Mich.
l~led. Bull., 20: 2~,4-6, 1956), affinity-purified polyclonal or fra ,ments thereof (Chao et al., Res. Comm. in Chem. Path. & Pharm.. 2: 749-61, 1974).
Monoclonal antibodies useful in the practice of the present invention include ~5 whole antibody and fragments thereof. Such monoclonal antibodies and fra~mrn~
are producible in accordance with conventional techniques, such as hybridoma synthesis, recombinant DNA techniques and protein synthesis. Useful monoclonal antibodies and fr~gment~ may be derived from any species (inrlu-linP 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.

CA 022~73~3 1998-12-01 Human monoclonal antibodies or "hllm~ni7t?d" murine antibody are also useful as targeting moieties in accordance with the present invention. For e.~ampLe, murine monoclonal antibody may be "hllm~ni7ed" by genetically recombining the nucleotide sequence encoding the murine Fv region (i.e., cont~ining the antigen bindin_ sites) or s the complementarity determining regions thereof with the nucleotide sequence encoding a human constant domain region and an Fc region, e.~., in a manner similar to that disclosed in European Patent Application No. 0,411,893 A2. Some murine residues may also be retained within the human variable region framewor~; domains to ensure proper target site binding characteristics. Hllm~ni7ed targeting moieties are o recognized to decrease the immunoreactivity of the antibody or polypeptide in the host recipient, perrnitting an increase in the half-life and a reduction in the possibility o~ adverse irnmune reactions.
Types of active agents (diagnostic or therapeutic) useful herein 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). Because toxin B chain may mediate non-target cell binding, it is often advantageous to conjugate only the toxin A chain to a targeting protein. However, while elimin~tion 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 Achain or "A chain-like" molecules, such as ricin A chain, abrin A chain, modeccin A
chain, the enzymatic portion of Pseudomonas e~cotoxin A, Diphtheria toxin A chain, the enzymatic portion of pertussis toxin, the enzymatic portion of Shiga to~cin,gelonin, pokeweed antiviral protein, saporin, tritin, barley toxin and snake venom peptides. Ribosomal inactivating proteins (RIPs), naturally occurring protein synthesis inhibitors that lack translocating and cell-binding ability, are also suitable for use herein. E:Ytremely highly toxic toxins, such as palytoxin and the li~;e, are also contemplated for use in the practice of the present invention.

CA 022~73~3 1998-12-01 Preferred drugs suitable for use herein include conventional chemotherapeutics, such as vinblastine, doxombicin. bleomycin, methotrexate, 5-fluorouracil, 6-thioguanine, cytarabine, cyclophosphamide and cis-pl~tinllm, as well as other conventional chemotherapeutics as described in Cancer: PrinciDles and Practice of Oncolo~y, 2d ed., V.T. DeVita, Jr., S. Hellman, S.A. Rosenber" J.B. Lippincott Co., Philadelphia, PA, 1985, Chapter 14. A particularly preferred druu within the present invention is a trichothecene.
Trichothecenes are drugs produced by soil fungi of the class F~ gi imperfecli orisolated from Baccharus ntegapo~mlca (Bamburg, J.R. Proc. ~lolec. Subcell.
o Biol. ~:41-110, 1983; Jarvis & Mazzola, Acc. Chem. Res. 15:338 395, 1982). They appear to be the most toxic molecules that contain oniy carbon, hydrooen and oxygen (Tamm, C. Fortschr. Chem. Or~. Naturst. 31 :61 -117, 1974). They are all reported to act at the level of the ribosome as inhibitors of protein synthesis at the initiation, elongation, or termination phases.
There are two broad classes of trichothecenes: those that have oniy 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). Representative ~o examples of Group A simple trichothecenes include: Scirpene, Roridin C, dihydrotrichothecene, Scirpen-4, 8-diol, Verrucarol, Scirpentriol, T-2 tetraol, pentahydroxyscirpene, 4-deacetylneosolaniol, trichoderrnin, deacetylcalonectrin,calonectrin, diacetylverrucarol, 4-monoacetoxyscirpenol, 4,15-diacetoxyscirpenol, 7-hydroxydiacetoxyscirpenol, 8-hydroxydiacetoxy-scirpenol (Neosolaniol), 2s 7,8-dihydroxydiacetoxyscirpenol, 7-hydroxy-8-acetyldiacetoxyscirpenol, 8-acetylneosolaniol, NT-l, NT-2, HT-2, T-2, and acetyl T-2 toxin.
Representative examples of Group B simple trichothecenes include:
Trichothecolone, Trichothecin, deoxynivalenol, 3-acetyldeoxynivalenol, 5-acetyldeoxynivalenol, 3,15-diacetyldeoxynivalenol, Nivalenol, ~-acetylnivaienol (Fusarenon-X), 4,15-idacetylnivalenol, 4,7,15-triacetylnivalenol. and tetra-acetylnivalenol. Representative examples of Group C simpie trichothecenes include:

CA 022~73~3 1998-12-01 Crotocol and Crotocin. Representative macrocyclic trichothecenes include Verrucarin A, Verrucarin B, Verrucarin J (Satratoxin C), Roridin A, Roridin D, Roridin E (Satrato:~in D), Roridin H, Satratoxin F, Satrato,Yin G. Satratoxin H,Vertisporin, ~vlytoxin A, Mytoxin C, i~ytoxin B, Myrotoxin A, l~/Iyroto~cin B, ~Iyrotoxin C, ~fyroto,Yin D, Roritoxin A, Roritoxin B, and Roritoxin D. In addition, the general "trichothecene" sesquiterpenoid ring structure is also present in compounds termed "baccharins" isolated from the higher plant Baccharis n~egapotan?ica~ and these are described in the literature, for instance as disclosed by Jarvis et al. (Chemistry of Alleopathy, ACS Symposium Series No. 268: ed.
lo A.C. Thompson, 1984, pp. 149-159).
Experimental drugs, such as mercaptopurine, N-methylformamide, 2-amino-1,3,4-thi~ 701e, melphalan, hexamethylmel~mine, gallium nitrate, 3% thymidine, dichloromethotrexate, mitoguazone, suramin, bromodeoxyuridine. iododeo~vuridine,s~m~lstine, 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea, N,N'-1~ hexamethylene-bis-acetamide, azacitidine, dibromodulcitol, Erwinia asparaginase, ifosf~mid~, 2-mercaptoethane sulfonate, teniposide, taxol, 3-deazauridine, soluble Baker's antifol, homoharringtonine, cyclocytidine, acivicin, ICRF-187, spiromustine, levarnisole, chlorozotocin, aziridinyl benzoquinone, spirogerm~nillm aclarubicin, pentostatin, PA~A, carboplatin, amsacrine, caracemide, iproplatin, misonidazole,o dihydro-S-azacytidine, 4'-deoxy-doxorubicin, menogaril, triciribine phosphate.
fazarabine, tiazofurin, teroxirone, ethiofos, N-(2-hydroxyethyl)-2-nitro-lH-imidazole-I-~cet~mide, mitoxantrone, acodazole, amonafide, fludarabine phosphate, pibenzimol, ~id~mnin B, merbarone, dihydrolenperone, flavone-8-acetic acid, oxantrazole, ipomeanol, trimetrexate, deoxyspergualin, echinomycin, and dideoxycytidine (see NCI
2s Investigational Dru~s Pharmaceutical Data 1987. NIH Publication No. 88-2141, Revised November 1987) are also p,~fe,-ed.
Radion~ ides useful within the present invention include garnma-emitters, positron-emitters, Auger electron-emitters, X-ray emitters and fluorescence-emitters, with beta- or alpha-emitters preferred for therapeutic use. Radionuclides are well-123 125 130 131 133 135 47 7' 72 knownintheart andlnclude I, I, I, I, I, I, Sc, As, Se, 90 88 97 100 d lolmRh 119Sb 128ga. Hg. At, Bi, S

... , . .. , , , ~ . ... ~.. . .. .. .

CA 022~73~3 1998-12-01 169 212 109 111 67 68 64 67 75 76 77 99m Eu, Pb, Pd, In, Ga, Ga, Cu, Cu, Br, Br Br, Tc, C, N, O, Ho and F. Preferred th~rapeutic radionuclides include Re, Re, Pb, Pb, 1-Bi 109Pd 6~Cu 67cu 90Y 1~5I 131 77 Ru, Rh, Au and Ag, Ho or Lu.
Other anti-tumor agents, e.l~., agents active against proliferating cells, are ~rlmini.ctrable in accordance with the present invention. Exemplary anti-tumor agents include cytokines, such as ~L-2, tumor necrosis factor or the like, lectin infl~mm~tory response promoters (selectins), such as L-selectin, E-selectin, P-selectin or the like, and like molecules.
T.ig~n~l.s suitable for use within the present invention include biotin, haptens, lectins, epitopes, dsDNA fr~gm~nt~, enzyme inhibitors and analo ,s and derivatives thereof. Useful complementary anti-ligands include avidin (for biotin), carbohvdrates (for lectins) and antibody, fragments or analogs thereof, includin;, mimetics (for haptens and epitopes) and zinc finger proteins (for dsDNA fra~ments) and enzymes(for enzyme inhibitors). Preferred ligands and anti-ligands bind to each other with an affinity of at least about kD 10 M. Other useful ligand/anti~ and systems include S-protein/S-peptide, head activator protein (which binds to itself)7 cystatin-C/cathepsin B, and the like.
One pl ~fe- I ed chelate system for use in the practice of the present invention is 0 based upon a 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetra acetic acid (DOTA) construct. Because DOTA strongly binds Y-90 and other radionuclides, it has beenproposed 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 ~tt~.hed to an effector, such as a ligand or an anti-ligand.
The strategy for design of prere~l ed DOTA molecules incorporating biotin for use in the practice of embodiments of the present invention involved three primary considerations:
1) in vivo stability (incl~ in~ biotinidase and general peptidase activity r~ict~nce), with an initial acceptance criterion of 100% stability for l hour;
2) renal e~cretion, and 3) ease of synthesis.

CA 022~73~3 1998-12-01 The same or sirnilar criteria are applicable to alternative binding moieties, such as li_ands or anti-ligands, as can be readily ascertained by one of ordinary s~;ill in the art.
The DOTA-biotin conjugates that are preferably employed in the practice of the present invention reflect the implementation of one or more of the t'ollowing strategies:
1) substitution of the carbon ~tljacent to the cleavage susceptible amide nitrogen;
2) alkylation of the cleavage susceptible amide nitro_en;
3) substitution of the amide carbonyl with an alkyl amino group;
o 4) incorporation of D-amino acids as well as analogs or derivatives thereof; or 5) incorporation of thiourea linkages.
DOTA-biotin conjugates in accordance with the present invention are described in published PCT Patent Application No. PCT/US93/05~06. A method of preparing a preferred DOTA-biotin embodiment is described in E~ample II hereof.
i ~ The preferred linkers are useful to produce DOTA-biotin or other DOTA-small molecule conjugates having one or more of the following advantages:
- bind avidin or streptavidin with the same or substantially sirnilar aff~nity as free biotin, - bind metal M ions efficiently and with high kinetic stability;
~0 - are excreted primarily through the kidneys into urine;
- are stable to endogenous enzymatic or chernical degradation (e.g., bodily fluid amidases, peptidases or the like);
- penetrate tissue rapidly and bind to pretargeted avidin or streptavidin; and - are e~ccreted rapidly with a whole body residence half-life of less than about S
hours.
One component to be ~tlminictPred 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 E:cample I below, with the preparation involving the steps of: preparation of SMCC-derivatized streptavidin, preparation of DTT-reduced proteinaceous targeting moiety; conjugation of the two prepared moieties; and purification of the CA 022~73~3 1998-12-01 monosubstituted or disubstituted (with respect to streptavidin) conjugate from crosslinked (antibody-streptavidin-antibody) and aggreQate species and unreactedstarting materials. The purified fraction is preferably further characterized bv one or more of the following teclmiclues: HPLC size e~clusion, SDS-PAGE, imrnunoreactivity, biotin binding capacity and in vivo studies.
LRCAs of the present invention may be a~mini~tered in single or multiple doses or via continuous infusion. A single dose of a biotin-cont~ining LRCA, for e~ample, 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-o 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. Alternatively, LRCA may be infused intravenously for a time period sufficient to clear targeting moiety-streptavidin in a continuous manner.
The dose of LRCAs of the 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 ~flminictered component to be cleared that is either measured or e.Ypected to remain in the serum compartment at the time the ~o LRCA is ~mini~tered. Alternatively, the dose of the LRCA will depend on the measured or expected level of toxic agent to be cleared. Generally, a single LRCA
dose will range from about 200 mg to about 1000 mg, with from about 300 mg to about 700 mg preferred.
One embodiment of the present invention in which rapid acting LRCAs are useful is in the delivery of Auger emitters, such as I-125, I-123, Er-165, Sb-l 19, Hg-197, Ru-97, Tl-201 and Br-77, or nucleus-binding drugs to target cell nuclei. In these embodiments of the present invention, 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. Such internalizing receptors include EGF
receptors, transferrin receptors, HER2 receptors, IL-2 receptors, other interleul;ins 3~

CA 022~73~3 1998-12-01 and cluster differentiation receptors, somatostatin receptors, other peptide binding receptors and the like.
A~er the passage of a time period sufficient to achieve localization of the conjugate to target cells, but insufficient to induce internalization of such targeted conjugates by those cells through a receptor-me~i~ted event, a rapidly actin~ LRCA is ~(lmtnict~red. In a preferred two-step protocol, an active agent-cont~ining ligand or anti-ligand conjugate, such as a biotin-Auger emitter or a biotin-nucleus acting drug, is ~minictered as soon as the LRCA has been given an opportunity to comple~c with circulating targeting moiety-cont~inin~ conjugate, with the time lag between LRCA
0 and active agent ~mini~tration being less than about 24 hours. In this manner, 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 I-123, which is available and capable of stable binding.

The invention is further described through presentation of the following e~camples. These e:camples are offered by way of illustration, and not by way of .
hmltatlon.

E:~ample I
T~rgeting ~Ioiety-~nti-Lig~nd Conjug~te for Two-Step Pret:lrgeting In Vivo ~5 A. PreparationofSMCC-derivatizedstreptavidin.
31 mg (0.48 mol) streptavidin was dissolved in 9.0 ml PBS to prepare a final solution at 3.5 mg/ml. The pH of the solution was adjll~ted to ~.5 by addition of 0.9 ml of 0.5 M borate buffer, pH 8.5. A DMSO solution of SMCC (3.5 mg/ml) was prepared, and 477 1 ~4.8 mol) of this solution was added dropwise to the vortexing protein solution. After 30 rninutes of stirring, the solution was purified by G-'~5 (PD-iO, Pharmacia, Picastaway, New Jersey) column chromatographv to remove CA 022~73~3 1998-12-01 unreacted or hydrolyzed SMCC. The purified SMCC-derivatized streptavidin was isolated (28 mg" 1.67 mg/ml).
B. Preparation of DTT-reduced NR-LU- 10. To 77 mg NR-LU- 10 (0.~" mol) in 15.0 m[ PBS was added 1.5 ml of 0.5 ~I borate buffer, pH 8.5. .~ DTT solution, at s ~00 mg/mi (165 1) was added to the protein solution. After stirring at room temperature for 30 minllteS, the reduced antibody was purified by G-25 size exclusion chromatography. Purified DTT-reduced NR-LU-10 was obtained (7~ mg" ~.17 mg/mi) C. Conjugation of SMCC-st}eptavidin to DTT- reduced ~ LU- 10. DTT-o reduced NR-LU-10 (63 mg, 29 ml, 0.42 mol) was diluted with 4~.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, avaiiable from ivlac~lod). A~Ler approximately 45 minutes, the reaction wass quenched by adding solid sodium tetrathionate to a final concentration of 5 ml\I.
D. Purification of conjuPate. For small scale reactions, monosubstituted or disubstituted (with regard to streptavidin) conjugate was obtained using H~LC
Zorbax (preparative) size exclusion chromatography. The desired monosubstituted or disubstituted conjugate product eluted at 14.0-14.5 min (3.0 ml/min flow rate~, while unreacted NR-LU-10 eluted at 14.5-15 min and unreacted derivatized streptavidin eluted at 19-20 min.
For larger scale conjugation reactions, monosubstituted or disubstituted adduct is isolatable using DEAE ion exchange chromatography. After concentration of thecrude conjugate mixture, free streptavidin was removed therefrom by eluting the 2s 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 colurnn using an increasing salt 8radient in 20 mM diethanolamine adjusted to pH 8.6 with sodium hydroxide.
E. Characterization of Conju~ate.
1. HPLC size e:cciusion was conducted as described above with respect to small scale purification CA 022~73~3 1998-12-01 2. SDS-PAGE analysis was performed using 5% poiyacrylamide gels under non-denaturing conditions. Conjugates to be evaluated were not boiled in sample buffer containing SDS to avoid dissociation of streptavidin into its 15 ~;D subunits.
Two product bands were observed on the gel, which correspond to the mono- and di-sl-hstit~ted conjugates.
~ 3. Tmmllnoreactivity was assessed, for e~ample, by competitive binding ELISA
as compared to free antibody. Values obtained were within 10% of those for the free antibody.
4. Biotin binding capacity was ~s~ssed, for e~cample, by titratin, a known o quantity of conjugate with p-[I- I 25]iodobenzoylbiocytin. Saturation of the biotin binding sites was observed upon addition of 4 equivalences of the labeled biocytin.
5. In vivo studies are usefi~l to characterize the reaction product, which studies include, for e~cample, serum clearance profiles, ability of the conjugate to target antigen-positive tumors, tumor retention of the conjugate over time and the ability of a biotinylated molecule to bind streptavidin conjugate at the tumor. These data facilitate deterrnination that the synthesis resulted in the formation of a 1:1 streptavidin-NR-LU-10 whole antibody conjugate that e~chibits blood clearance properties similar to native NR-LU-10 whole antibody, and tumor uptake and retention properties at least equal to native NR-LU- 10.
O For example, Figure 1 depicts the tumor uptake profile of the NR-LU- 10-streptavidin conjugate (Ab/SA, referred to in this e~cample as LU- 1 0-~trAv) incomparison to a control profile of native NR-LU- 10 whole antibody and a controlprofile 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 eff~ciently as NR-LU- 10 whole antibody itself.

E~cample II
Synthesis of DOTA-Biotin Conjug~tes A. Synthesis of Nitro-Benzyl-DOTA.
The synthesis of aminobenzyl-DOTA was conducted substantially in accordance with the procedure of ~Icl~urry et al., Bioconju(,ate Chem.~ ~: 10~-1 17, 199~. The .. . . . _ ..... . . .

CA 022~73~3 1998-12-01 critical step in the prior art synthesis is the intermolecular cyclization between ~iic~lccinimidyl N-(tert-buto~ycarbonyl)iminodiacetate and N-(''-aminoethyl)-~-nitrophenyl ~l~nin~mide to prepare 1 -(tert-butoxycarbonyl)-5-(4-nitrobenzyl)-3,6, 11-trioxo-1,4,7,10-tetraazacyclododecane. In other words, the critical step is the s interrnolecular cyclization between the bis-NHS ester and the diarnine to give the cyclized dodecane. McMurry et al. conducted the cyclization step on a 1'~0 mmol scale, dissolving each of the reagents in 100 rnl 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 0 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. Consequently on a 30 mmol scale, each of the reagents was dissolved in 500 ml DMF and added via addition funnel over 27 hours to a reaction pot cont~ining 3 liters dioxane. The addition rate of the method employed involved a 5.1~ mmol/hour addition rate and a 0.047 ~I reaction concentration.

B. Synthesis of an N-methyl-~lYcine linked conju~ate.
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.) wascondensed with biotin-NHS ester in DrvIF and triethylamine to obtain N-methyl glycyl-biotin. N-methyl-glycyl biotin was then activated with EDCI and N~S. The resultant NHS ester was not isolated and was condensed in situ with DOTA-aniline2s and excess pyridine. The reaction solution was heated at 60~C for 10 minutes and then evaporated. The residue was purified by p,epaldli~e HPLC to give [(N-methyl-N-biotinyl)-N-glycyl] -arninobenzyl-DOTA.
1. Preparation of (N-methyl)glycyl biotin. DMF (8.0 ml) and triethvlamine (0.61 rnl, 4.3S mmol) were added to solids N-methyl glycine ( 182 mg, 2.05 mmol)and N-hydroxy-suc~ imidyl biotin (500 mg, 1.46 mrnol). The mixture was heated for 1 hour in an oil bath at 85~C durin=, which time the solids dissolved producing a clear 3~

and colorless solution. The solvents were then evaporated. The yellow oil residue was acidified with glacial acetic acid, evaporated and chromatoarapl1ed on a 27 mm column packed with 50 g silica, eluting with 30% MIeOH/EtO~c 1% HO~c to give the product as a white solid (3~3 mg) in 66% yield.
H~ R (DMSO)~ -1.25 (m, 6H, (CH2)3), 2.15, 2.35 (~ i's, 2H, CH2CO), 2.75 (m, 2H~ SCH2), 2.~0, 3.00 (2 s's, 3H, NCH3), ~ 05-3.15 (m, lH, SCH), 3.95, 4.05 (2 s's, 2H, CH2N), 4.15, 4.~ (2 m's, 2H~
2CHN's), 6.35 (s, NH), 6.45 (s, NH).
2. Preparation of [(N-methyl-N-biotinyl)glycyl] aminobenzyl-DOTA. N-o hydroxysusçinimide (10 mg, 0.08 mmol) and EDCI (15 mg, 6.08 mmol) were added to a solution of (N-methylglycyl biotin (24 mg, 0.08 mmol) in DMF (1.0 ml). The solution was stirred at 23 C for 64 hours. Pyridine (0.~ ml) and arninobenzyl-DOTA
(~Omg, 0.04 mmol) were added. The mixture was heated in an oil bath at 63~C for 10 minlltes, then stirred at 23 C for 4 hours. The solution was evaporated. The s residue was purified by preparative HPLC to give the product as an off white solid (~
mg, 0.01 mrnol) in 27% yield.
H-N~ (D20): 1.30-1.80 (m, 6H), 2.40, 2.55 (2 t's, 2H, CH,CO), 2.70~.2 (complex multiplet), 4.35 (m, CHN), 4.55 (m, CHN), 7.30 (m, 2H, benzene hydrogens), 7.40 (m, 2H, benzene hydrogens).
~o EXAMPLE III
Cle:lring Agent Evalu:ltioll Experiment:ltioll A. Galactose- and Biotin-Derivatization of Human Serum Albumin (HSA).
HSA was evaluated because it e~hibits the advantages of being both ine~pensive and non-immunogenic. HSA was derivatized with varying levels of biotin ( l-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 ~.5, followed by dropwise addition of a DMSO solution of NHS-LC-biotin (Sigma Chemical Co.) 3u to the stirred solution at the desired molar offering (relative molar equivalents of reactants). The final percent DMSO in the reaction mi~cture should not e:cceed 5%.

CA 022~73~3 1998-12-01 After stirring for I hour at room temperature, the reaction was complete. A 90%
incorporation efficiency for biotin on HSA was generally observed. As a result, if, molar equivalences ofthe NHS ester of LC-biotin was introduced, about ~.7 biotins per HSA molecule were obtained. Unreacted biotin reagent was removed from the S biotin-derivatized HSA using G-~5 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 galactoseslmolecule.
Galactose derivatization of the biotinylated HSA was performed accordin, to the o procedure of Lee, et al., Biochemistry. 15: 3956, 1976. More specifically, a 0.1 M
m~th~nolic 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 NaO~le 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. 13iotinylated HSA in PBS, buffered with 10% v/v 0.5 M sodium borate, was added to the oily residue.
After stirring at room temperature for 2 hours, the mixture was stored at 4~C for 12 hours. The galactosylated HSA-biotin was then purified by G-~S size e:~clusion chromatography or by buffer exchange to yield the desired product. The same ch~mi~try is exploitable to galactosylating dextran. The incorporation efficiency of galactose on HSA is approximately 10%.
70 micrograms of Galactose-HSA-Biotin (G-HSA-B), with 12-45 galactose residues and 9 biotins, was administered to mice which had been a-lminictered 200 micrograms of StrAv-MAb or 200 microliters of PBS 24 hours earlier. Results 2s indicated that G-HSA-B is effective in removing StrAv-MAb from circulation. Also, the pharmacokinetics of G-HSA-B is unperturbed and rapid in the presence or absence of circulating ~L~b-StrAv.
B. Non-Protein ClearinP A~ent. A commercially available form of dextran, molecular weight of 70,000 daltons, pre-derivatized with approximately l ~
biotins/molecule and havin;, an e(luivalent number of free primary amines was studied.
The primary amine moieties were derivatized with a Qalactosylating rea~Jent, ~o CA 022~73~3 1998-12-01 subst~nsi~lly in accordance with the procedure therefor described above in the discussion of HSA-based clearing agents, at a levei of about 9 ualac,oses/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 ~Iicrograms of galactose-dextran-biotin (GAL-DE~-BT) was then injected i.v. into one group of mice which had received ~00 micrograms MAb-StrAv conjugate intravenously 2~
hours earlier, while ~0 micrograms of GAL-DEX-BT was injected into other such mice. GAL-DEX-BT was rapid and efficient at clearing StrAv-l~lAb conjugate, removing over 66% of circulating conjugate in less than 4 hours after clearing agent ~ministration An equivalent effect was seen at both clearing agent doses, which correspond to 1.6 (40 micrograms) and 3.~ (50 micrograms) times the stoichiometric amount of circuiatin;, StrAv conjugate present.
C. Dose Ran_ing for G-HSA-B ClearinP Agent. Dose ran_ing studies followed the following basic format:
200 micrograms ~LAb-StrAv coniugate ~minictered;
24 hours later, clearing agent a~minict~red; and 2 hours later, 5.7 micrograms PIP-biocytin a-lministered Dose ranging studies were performed with the G-HSA-B clearing agent, starting with a loading of 9 biotins per molecule and 12-45 galactose residues per molecule. Doses of 20, 40, 70 and 120 micrograms were ~imini~tered 24 hours after a 200 microgram dose of MAb-StrAv con~ugate. The clearing agent a-lmini~trationswere followed 2 hours later by ~minictratio" of 5.7 micrograms of I-13 I-PIP-biocytin. Tumor uptake and blood retention of PIP-biocytin was e:camined ~4 hours after a~minictration thereof (46 hours after clearing agent a~ministration). The results showed that a nadir in blood retention of PIP-biocytin was achieved by all dosesgreater than or equal to 40 micrograms of G-HSA-B. A clear, dose-dependent decrease in tumor binding of PIP-biocytin at each increasing dose of G-HSA-B waspresent, however. Since no dose-dependent effect on the localization of ~IAb-StrAv conjugate at the tumor was observed, this data was interpreted as being indicative of 3~ relatively higher blocking of tumor-associated MAb-StrAv conjugate by the release of biotin from catabolized clearing agent. Similar results to those described earlier for CA 022~73~3 1998-12-01 the asialoorosomucoid clearing agent regarding plots of tumor/blood ratio were found with respect to G-HSA-B. in that an optimal oalance between blood clearar.ce andtumor retention occurred around the ~0 microgram dose. Because of the relativelylarge molar amounts of biotin that could be released by this clearing agent at higher doses, studies were undertaken to evaluate the effect ol iower levels of biotinylation on the effectiveness of the clearing agent. G-HSA-B, derivatized with either 9, 5 or 2 biotins/molecule, was able to clear MAb-StrAv con,ugate from blood at e(lual protein doses of clearing agent. All levels of biotinylation yielded effective, rapid clearance of MAb-StrAv from blood.
0 Comparison ofthese 9-, 5-, and 2-biotin-derivatized clearing agents with a single biotin G-HSA-B clearing agent was carried out in tumored rnice, employing a 60 rnicrogram dose of each clearing agent. This e~periment showed each clearing agent to be substantially equally effective in blood clearance and tumor retention of l~/lAb-StrAv conjugate 2 hours after clearing agent administration. The G-HSA-B
with a single biotin was e~camined for the ability to reduce binding of a subsequently admini.~t~red biotinylated small molecule (PIP-biocytin) in blood, while preserving tumor binding of PIP-biocytin to prelocalized MAb-StrAv conjugate. Measured at 44 hours following PIP-biocytin ~clmini~tration, tumor localization of both the MAb-StrAv conjugate and PIP-biocytin was well preserved over a broad dose range of G-~o HSA-B with one biotin/molecule (90 to 180 micrograms). A progressive decrease in blood retention of PIP-biocytin was achieved by increasing doses of the single biotin G-HSA-B clearing agent, while tumor localization remained essentially constant, indicating that this clearing agent, with a lower level of biotinylation, is preferred.
This prerelence arises because the single biotin G-HSA-B clearing agent is both effective at clearing MAb-StrAv over a broader range of doses (potentially Pl;~;t~it~;no 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.
Another way in which to decrease the effect of clearing agent-released biotin onactive agent-biotin conjugate binding to prelocalized targeting moiety-streptavidin conjugate is to attach the protein or polymer or other primary clearing aoent ~2 CA 022~73~3 1998-12-01 WO 97/46099 PCT/US97tO9400 component to biotin us ng a retention linker. A retention linker has a chemical structure that is resistant to agents that cleave peptide bonds and, optionally, becomes protonated when locali7ed to a catabolizina space, such as a Iysosome. Preferredretention linkers of the present invention are short strings of D-amino acids or small S molecules having both of the characteristics set forth above. An exemplary retention linker of the present invention is cyanuric cllloride, which may be interposed between an epsilon amino group of a Iysine of a prot~in~ceous primary clearing agent component and an amine moiety of a reduced and chemically altered biotin carboxymoiety (which has been ~ clls.~ed above) to form a compound of the structure setlo forth below.
N~\N
Lysine--NH~N~LNI l- (CH2)4--When the compound shown above is catabolized in a catabolizing space, the heterocyclic ring becomes protonated. The ring protonation prevents the catabolite from e,Yiting the Iysosome. In this manner, biotin catabolites cont~inin~ the heterocyclic ring are restricted to the site(s) of catabolism and, therefore, do not compete with active-ag~nt-biotin conjugate for prelocalized targeting moiety-streptavidin target sites Coll~p~risolls ~ ionol r~ lal~elc~ l'll'-l)iocylinobserved in the G-HSA-B dose ranging studies showed that optimal tumor to background targeting was achieved over a broad dose range (90 to 180 micrograms), with the results providing the expectation that even larger clearing agent doses would also be effective. Another key result of the dose ranging experimentation is that G-HSA-B
with an average of only I biotin per molecule is presumably only clearing the MAb-StrAv conjugate via the Ashwell receptor mech~nicm only, because too few biotinsare present to cause crcss-linking and aggregation of MAb-StrAv conjugates and clearing agents with such aggregates being cleared by the reticuloendothelial system.
~ D. Tumor Tar~etin Evaluation UsinP G-HSA-B. The protocol for this experiment was as follows:
Time 0: a~minister 400 micrograms MAb-StrAv conjugate;

Time 24 hours~ mirli~ter 240 micrograms of G-HSA-B with one biotin and 1'-45 galactoses and Time 26 hours: admil1ister 6 miclogral11s of DOTA--C~ ~NH--C~(CHZ)5--N(C~)--C~(Ci!2)4~

HN~NH

Lu-177 is complexed with the DOTA chelate using known techniques therefor.
Efficient delivery of the Lu- 1 77-DOTA-biotin small molecule was observed, 20-25 % il1jected dose/gram of tumor. These values are equivalent with tl1e efficiency of tlle delivery of the MAb-StrAv conju~ate. The AUC tumor/AUC blood obtained for this non-optimized clea:in~ agent dose was 300% greater than that achievable by comparable direct MAb-radiolabel a~lmini~tration. Subsequent experimentation hasresulted in AUC tumor/AUC blood over 1000% 8reater than that achievable by lS comparable conventional MAb-radiolabel a(lmini~tration. In addition, the HSA-based clearing agent is expect-d to exhibit a low degree of immunogenicity in hllm~nc EXAMPLE IV
LRCA Prcp:~r:ltion 2() A. Hepatic Clearance l)irectin~ Moiety-Structural Component-Reactive Group Construct.
1. Galactosvlated HSA. An optimized procedure for the preparation of a (galactose),8-HSA-(maleimide)2 construct is set forth below. To a solution of 236 mg of cyanomethyl-2,3,4,6- tetra-O-acetyl- 1 -thio-D-galactopyranoside (commercially available from Sigma Chemical Co., St. Louis, Missouri) in 5.85 ml of methanol was added 0.5~ ml of 0.1 M NaOMe. The mixture was stirred at 15-25 ~C for 12-16 hours to afford methanolic 2-imino-2-methoxyethyl- I-thio-galactopyranoside. An aliquot (2.37 ml) of the solution was concentrated. To 100 mg of HSA (commercially available from Sigma Cllemical Co.) in 4.08 n1l of 20 mM PBS (pH 7.0) was added 0 92 ml of PBS and 0.5 ml of 0.SM borate buf~er (p~l ~.5). The resultant HSA

CA 022~73~3 1998-12-01 WO 97/46099 PCT/US97tOg400 solution was then added to the concentrated 2-imino-2-methoxyethyl-1-thiogalactose pyranoside. The mixture was rotated at 15-25~C t'or 24 hours. The product was purified on 47 ml of G-25 e:cclusion matri:c (commercially available from Pharmacia, Piscastaway, New Jersey) eluting with PBS to afford 10 ml of 9.3 mg protein/ml of product cont~ining 4~ galactose residue per HSA. The e~tent of galactose loading is assayed by S~fl 05.210.063 Antnrone assay (Viles and Silverman, "Determination of starch and cellulose with anthrone," Anal. Chem., 21: 950-3, Ig49).
2. Galactosylated-HSA-Maleimide. To 7.15 ml of the galactose-HSA solution was added 715 microliters of 0.5M borate (pH 8.0). While rotating, 357 microliters o of s~lc~inimidyl-4-(N-maleimidomethyl) cyclohexane-l-carboxylate (SMCC) solution in DMSO (10.64 mg/ml) was added. The mixture was stirred for 60 minutes at 15-25 ~C. The resultant mi:~ture was purified on 175 ml of G-25 size e~cclusion matri,~, 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 Arnicon filtration(30,000 MW cutoff), commercially available from Amicon, Beverly, Mass., to a conce~ Lion 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.

B. Liver Retention Component-Binding Component Constructs.
B 1. As shown in Figure 2, the liver retention component-binding component construct, N-methyl-N-{5'-[methylester tris-(-(D, L)-phosphonoalanyl)-(D)-cystyl~-5-carbamylpentyl}biotinamide (11), may be prepared as described below.

(D. L)-Methyl-2-amino-3-diethvlphosphono-propionate (1). To a solution of 3.771 g (22.3 mrnole) of (D, L)-2-amino-3-phosphono-propionic acid (Aldrich Chemical, Milwaukee, WI) in 110 ml dry MeOH at -5 ~C was added dropwise 22.3 ml of SOCI,.
The reaction rni~ture was stirred at room temperature under nitrogen overnight, at which time solvent was removed under vacuum to give a methyl ester intermediate.This intermediate was taken up in 200 ml anhydrous HC(OEt)3 and heated at 125-165~C for 4 hours with constant removal of EtOH via a Vigreu~c distilling head CA 022~73~3 1998-12-01 (Chem Glass Inc., Vineland, New Jersey). E~ccess HC(OEt)3 was then removed undervacuum to give a crude intermediate. This crude intermediate was purified bv flash column silica gel chromatography (eluting solvent: CH,Cl,\acetone\ ''-propanol =85: 10:5) to give 2.395 g of the N-formyl derivative of the desired product. This N-formyl deriv~tive was dissoived in dry MeOH and saturated ~.vith HCI gas. To this HCl saturated solution was added 0.6 ml SOCI,. The reaction mi,~ture was stirred at room temperature under N, overnight, at which time solvent was removed under vacuum and the resulting residue was co-evaporated once with dry CH3CN to give 2.364 ~ of the hydrogen chloride salt of the desired product. (39% yield) IH-N~IR
0 (CD30D, o): 4.20 (m, CH & CH.O), 3.~7 (s, OCH3), 2.4~ (m, CH2-P), and 1.33 (t, CH3).

(D L)~l~Iethyl-N-BOC-2-amino-3-diethvlphosphono-propionate (2). To a solution of1.682 g (6 1 mmole) of (1) in 32 ml dry CH3CN and 1.643 ml (2 eq) of dry Et3N was added a solution of 1.392 g (1.05 eq) of BOC anhydride (Aldrich Chemical) in 20 ml dry CH3CN. The reaction mixture was stirred at room temperature overnight under nitrogen, at which time thin layer chromatography (eluting solvent: CH.Cl.\MeOH =
9: 1) showed that the reaction was not yet completed Consequently, to the reaction mixture was added 0. I g of BOC anhydride and 0.2 ml Et3N. The reaction rnhcture0 was allowed to continue to stir at room temperature for 4 hours, at which time solvent was removed under vacuum to give a crude residue. After the usual work-up in EtOAc (i e., washed with water, saturated NaCI, dried over Na2SO." filtered and evaporated), the crude product was purified by flash colurnn silica gel chromatography (eluting solvent: EtOAc) to give 1.206 g pure product (2). (5~%
yield) IH-NMR (CDCI3, ~): 5.70 (d, IH, NH), 4.62 & 4.49 (m & m, IH, CH), 4.10 (m, 4H, CH2O), 3.77 (s, 3H, OCH3), 2.33 (m, 2H, CH2-P), 1.45 (s, 9H, t-butyl) and 1.33 (t, 6H, CH3).

(D~ L)-N-BOC-2-Amino-3-diethylphosphono-propionic acid (3). To a solution of 1.2g (3.53 mmole) of (2) in 7 ml MeOH was added 7.26 ml (1.02 eq) of 0.5N NaOH.
The reaction mi~cture was heated at reflu:c temperature for 3 hours. Solvent was ~6 CA 022~73~3 1998-12-01 removed under vacuum, and the resulting residue was dissolved in 12 ml water ande~tracted twice with 10 ml ether. The water layer was adjusted to pH 3 with 6~ HCI
and e~ctracted with EtOAc. The aqueous layer was then further adjusted to pH I with 6N HCI and e:Ytracted four more times with EtOAc. The EtO~c e.Ytractions were combined, dried over Na,SO" filtered and evaporated to give a crystalline solid (3).
(1.094 g; 95% yield) 'H-NMR (CDCI3, o): 5.74 (d, lH, NH), 4.56 & 4.42 (m ~; m, lH, CH), 4.15 (m, 4H, CH20), 2.50 (dd, 2H, CH,-P), 1.44 (s, 9H, t-butyl) and 1.33 (two sets of t, 6H, CH3).

o N-BOC-(D L)-Diethylphosphono-alanyl-(D~ L)-diethvlphosphono-alanyl methvl ester (4). To a solution of 4.75 mg (1.46 mrnole) of(3) and 839 mg (1.3 eq) of benzotriazolylo~ytris(dimethylamino)phosphonium hexaphosphonate (BOP, cornmercially available from Aldrich Chemical Company and Chem-Impe~c International, Wood Dale, Illinois) in 9 ml dry DMF was added simultaneously a solution of 463 mg (1.68 mmole; 1.15 eq) of (1 ) in 6.3 ml dry D~vIF and 1.02 ml (4 eq) diisopropylethylamine. The reaction mixture was stirred under nitrogen at room temperature for 2-3 hours, at which time solvent was removed under high vacuum to give a crude residue. This residue was purified by flash column silica gel chromatography (eluting solvent: EtOAc\~eOH= 93:7) to give 673 mg pure (4).
(84% yield) 'H-NMR (CDCI3, o): 7.75 (dd, lH, NH), 5.74 (bs, IH, NH), 4.~5 &
4.72 (m & m, lH, CH), 4.44 (m, IH, CH), 4.10 (m, 8H, CH70), 3.76 (s, 3H, OCH3), 2.35 (m, 4H, CH,-P), 1.46 (s, 9H, t-butyl) and 1.33 (m, 12H, CH3).

(D. L)-Diethvlphosphono-alanyl-(D. L)-diethylphosphono-alanvl methvl ester (5). To a solution of 876 mg (1.6 rnrnole) of (4) in 7.2 ml CH7CI7 was added 10.8 ml trifluotoacetic acid (TFA). The reaction mixture was stirred at room temperature for 30 minlltes, at which time solvent was removed under vacuum to give a crude residue. This residue was co-evaporated once with dry toluene and twice with dryCH3CN to give 1.182g of (5) as 2.55 eq TFA salt. 'H-N~IR (CDCl3, o): 8.35 (d, NH), 4.85 (m, CH), 4.60 (m, CH), 4.15 (m, CH70), 3.73 (d, OCH3), 2.43 (m. CH7-P), and 1.42 (m, CH3).

~7 CA 022~73~3 1998-12-01 N-BOC~ L)-Diethvlphosphonoalanvl-(D. L)-diethvlphosphono-alanvl-(D L)-diethvlphosphono-alanine methvl ester (6). To a solution of 4.5O mg ( I .~ mmole) of (3) and 806 mg (1.3 eq) of BOP in 9 ml of dry Dl~/IF was added I .34 rnl (5.5 eq) of diisopropylethylamine and a solution of 1.~ 1 mmole of (5) in 6 ml dry D~IF. Thereaction mixture was stirred at room temperature under nitrogen for ~ hours. Solvent was removed under high vacuum, and the resulting residue was dissolved in 2û0 mlEtOAc. The EtOAc layer was washed twice with water, and the water layer was back washed four times with EtOAc. The EtOAc layers were combined and washed with saturated NaCl, dried over Na2SO~, filtered and evaporated to give a crude 0 product. This crude product was purified by flash column silica gel chromatography (eluting solvent: CH2CI7\acetone\l\/leOH= ~0:15:5) to give 762 mg product (6).
(72% yield) IH-N~ (CDCI3, o): ~.2~ (m, lH, NH), 7.~3 (m, lH, NH), 5.90 (m, IH, NH), 4.90-4.30 (m, 3H, CH), 4.10 (m, 12H, CH,O), 3.70 (d, 3H, OCH3), 2.38 (m, 6H, CH,-P), 1.46 & 1.44 (2s, 9H, t-butyl) and 1.30 (m, l~H, CH3).
I
(D L)-Diethvlphosphono-alanvl-(D. L)-diethylphosphono-alanyl-(D~ L)-diethyl-phosphono-alanine methyl ester (7). To a solution of 753 mg (1 mmole) of (6) in 5 ml CH7C12 was added 7 ml TFA. The reaction mixture was stirred at room temperature for 30 mimltes, at which time solvent was removed under vacuum to give a crude residue. This residue was co-evaporated twice with dry CH3CN to give 1.095g gummy (7) as 3 eq of TFA salt. 'H-NMR (CDC13, o): 9.17, 8.92, 8.11, 8.00,7.83 and 7.55 (NH), 4.90-4.35 (m, CH), ~.10 (m, CH,O), 3.73 (d, OCH3), 2.45 (m, CH,-P), and 1.30 (m, CH3).

N-Methyl-N-(5-(D)-S-tritylcystyl-5-carbamylpentyl)biotinamide (~). N-methyl-N-(5-NHS-carbonyl-pentyl)biotin~mide is prepared from biotin (Sigma Chemical Company,St. Louis, Missouri) by the following process: First is BOP-mediated coupling ofbiotin and N-methyi-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-hydroxys~lcrinimide in the presence of DCC in D~fF at room temperature overnight. To a solution of 2.~6 g (5."5 mmole) of N-methyl~ (5-NHS-carbonyl-pentyl)biotinamide in ~.5 ml dry D~F was added a suspension of 1.815 g (5 mmole) of (D)-S-trityl cysteine (Bachem Bioscience, Inc., King of Prussia~
PeMsylvania) and 1.74 ml di-isopropylethylamine (DEA) (2 eq) in 30 ml of dry Dl~/IF. The reaction mixture was stirred at room temperature overnight. Solvent was removed under high vacuum to one-fourth of its original volume. The resulting solution was added dropwise to a stirred 600 ml ether solution to cause precipitation.
The precipitate was collected by filtration and partitioned in 70 ml water (pH adjusted 0 to 2 with 0. lN HCI) and 80 ml EtOAc. The water layer was e~tracted 5 more times with 80 ml EtOAc. The EtOAc layers were combined, brined and dried over with MgSO~, and filtered. The MgSO~ was extensiveiy washed with EtOAc to get all of the product out. The EtOAc fiitrate and washes were combined and evaporated to one-third of original volume. Precipitate formed and was collected by filtration to give 2.601 g of off-white solid as the desired product (8). Additional 55~ m~ ofproduct was obtained after silica gel flash column purification (eluting solvent:
CH2CI2\MeOH\HOAc = 8~: 15: 1) of the concentrated residue from the EtOAc filtrate.
(88% yield) IH-NMR (CDCI3, o): 7.30 (m, 15H, ArH), 6.96, 6.43, 6.01 8~ 5.10 (NH, 3H), 4.60, 4.48 & 4.30 (3m, 3H, CH), 3,33 (m, lH, CHS), 3.15 (m, 2H, 0 CH2N), 3.00-2.65 (m, 4H, CH,S), 2.90 & 2.85 (2s, 3H, CH3N), ~.25 (m, ~H, CH2CO) and 1.80-1.20 (m, 12H, CH2).

N-Methyl-N-{5-~methylestertris-(D L)-diethylphosphono-alanyl)-(D)-S-tritvl-cvstyl1-5-carbamylpentyl}biotinamide (9). To a solution of 751 mg (1.05 mrnole) of 2s (8) and 575 mg (1.3 eq) of BOP in 6 ml dry DMF was added a solution of 1 mmole of (7) in 5 ml dry DMF and 0.87 ml (5 eq) of DEA. The reaction rni:cture was stirred at room temperature under nitrogen for 3-4 hours. Solvent was removed under high vacuum, and the resulting residue was worked up in CH2Cl2 in the usual maMer described above for EtOAc to give a crude product. This crude product was purified by flash column silica gel chromatography (eluting solvent: CH,Cl,\MeOH = 95:5;
then 90: 10; and then 87.5: 12.5) to 2ive 1.065 g pure product (g). (79% yield) 'H-~9 .

CA 022~73~3 1998-12-01 NMR (CDCI3-D.O, o): 7.29 (m, 15H, ArH), 4.90-4.20 (m, 5H. CH), 4.02 (m, 12H, CH,O), 3.70 (br s, 3H, OCH3), 3.20 (m, 3H. CH7N ~ CHS), 2.9~-2.50 (m, 7H, CH3N ~ CH,S), 2.50-2.12 (m, 10H, CH~P & CH.CO) and l .~5-1.17 (m, 30H, CH~
& CH~). 3'P-N~R (CDCl3, ppm): 2~.74, 27.95, 27.~ & 26.9~.

N-Methvl-N-t5-~methvlestertris-(-(D. L)-phosphono-alanyl)-(D)-S-tritvl-cystyll-5-carbamvlpentyl}biotinamide (10). To a 25 ml round bottom flask charged with 135 mg (0.1 mrnole) of(9) was added 7.5 ml of 10% (by volume) trimethylbromosilane (Aldrich Chernical) in dry CH~CI,. The reaction mixture was stirred at room 0 te~lpe.d~LIre under nitrogen for 4 hours. Solvent was removed, and the resulting residue was treated with 6 ml of 0.05 N triethylammonium bicarbonate (prepared by bulling CO. gas to 0.05N triethylamine) (pH 7.1) and 6 ml CH3C~. The mixture wasstirred at room temperature for 15 minllte~, at which time solvent was removed, and the resulting residue was dissolved in 20 ml of water and Iyophilized. The lyophilization was repeated again to give 171 mg of crude product (10). 'H-NMR
(CD30D, o): 7.30 (m, ArH), 4.70-4.25 (m, CH), 3.70 (ms, OCH3), 3.30 (m, CH2N &
CHS), 3.20 (~1, triethylammonium) 3.05-2.55 (m, CH3N & CH.S), 2.30 (m, CH2P &
CH2CO), 1.60 (m, CH,) and 1.30 (t, triethylammonium). Mass spect. (FAB-) (MIZ):
1183 [hi-H] and 940 [M-trityl-H] . This crude product was used directly in the following reaction without further purification.

N-Methyl-N- ~ 5-~methvlester tris-(-(D. L)-phosphono-alanvl)-(D)-cystYI~
carbamylpentyl}biotinamide (11). A solution of 100 mg of (10) in 2.3~ ml TFA, 0.1 ml anisole and 25 ,ul eth~nerlithiol was stirred at room temperature under argon for 2 hours. The reaction mixture was then precipitated into 40 ml ice-cold tert-butylmethylether (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. The supernatant was removed by gentle aspiration. The precipitate was resuspended in 30 ml of deoxygenated tert-butylmethylether, centrifuged again at 2,000 g for 10 minllteS~ and then supernatant was removed. This procedure was repeated three more times, and the final precipitate was dried under high vacuum for 2 hours to give a so CA 022~73~3 1998-12-01 crude (11). lH-NMR (D2O, o): 4.75-4.40 (m, CH), 3.78 (br s, OCH3), 3.36 (m, CH,N & CHS), 3.20 (q, triethylammonium TFA salt) 3.0~ & 2.90 (2s, CH3~), 3.04-2.75 (ms, CH2SH), 2.33 (m, CH,P & CH.CO), 1.60 (m, CH,) and 1.''~ (t, triethylammonium TFA salt). Mass spect. (FAB-) (M/Z): 941 IM] . This crude product was used directly in the conjugation reaction witl1 galactose-derivatized HSA.

B2. As shown in Figure 3, the liver retention component-binding component construct, N-methyl-N-[5-(triglutamylcysteine)-~-carbamylpentyl]-biotinamide, may 0 be prepared as described below.

(D)-N-Fmoc-O-t-butyl lutamvl-(D)-S-tritylcytseine (12). To a solution of 2.367 g (4 mmole of (D)-N-Fmoc-y-O-t-butylglut~m~te pentafluorophenol ester (Bachem Bioscience, Inc.) in 20 ml dry D~IF was added a solution of 1.454 g (4 mmole) ofS (D)-S-tritylcysteine (Bachem Bioscience, Inc.) and 1.393 ml (2 eq) of DEA in 20 ml of dry DMF. The reaction mixture was stirred at room temperature under nitrogen overnight, at which time solvent was removed under high vacuum to give a crude residue. This residue was dissolved in EtOAc and washed with 0. lN citric acid (twice), brined and dried over with Na2SO~, filtered and evaporated to give a crude ~0 product. This crude product was purified by flash colurnn silica gel chromatography (eluting first with EtOAc\CH2Cl2 = 9:1 and then with EtOAc\~ eOH = ~5:15) to give 2.266 g of desired product (12). (74~/0 yield). lH-N~ (CDC13, o): 7. 75-7.05 (m,ArH), 6.85 & 6.00 (2br s, NH), 4.20 (m, CH & CH2O), 2.65 (m, CH,S), 2.35 (m, CH2CO & COOH), 1.95 (m, CH2) and 1.40 (s, t-butyl).
(D)-O-t-butyl lutamvl-(D)-S-tritylcytseine (13). A solution of 3.1 g (4.02 mrnole) of (12) in 12 ml piperidine and 48 ml DMF was stirred at room temperature for I hour.
Solvent was removed under high vacuum to give a gummy residue. This residue was solidified while treated with hexane. Hexane was removed by filtration, and the filter was washed six times with hexane to remove the piperidine-Fmoc adduct. The hexane washed solid was purified by flash column reverse-~hase C- 1~

CA 022~73~3 1998-12-01 chromatography (eluting solvent: MeOH:water = 65:35 and then 70:30) to ~ive 2.163 g product (13) as the piperidinium salt form. This product was dissolved in 30 ml ,~/leOH and treated with 3.6 ml of IN NaOH (I eq). Solvent was removed under vacuum, and the resulting residue was dissolved in 5 ml ether. Tl1e ether solution was s precipitated into 700 ml stirring hexane. The precipitate was collected by filtration, washed three times with hexane and dried under vacuum to give 1.911 g product (13) as the sodium salt form. (81%) 'H-N~ (CD30D, ~): 7.30 (m, 15H, ArH), 4.35 (m, lH, CH), 3.32 (m, IH, CH), 2.60 (m, 2H, CH2S), 2.36 (t, 2H, CH,CO), 1.90 (m, 2H,CH.) and 1.39 (s, 9H, t-butyl).

(D)-N-Fmoc-O-t-butylglutamyl-(D)-O-t-butylglutamic acid (14). To a suspension of2.42 g (4.09 mmole) of (D)-N-Fmoc-y-O-t-butylglllt~m~te pentafluorophenol ester and 873 mg (1.05 eq of (D)-y-O-t-butylglutamic acid (Bachem Bioscience, Inc.) In50 ml dry DMF was added 1.493 ml (2.1 eq) of DEA. The reaction mixture was 1~ stirred at room temperature under nitrogen overnight. Solvent was removed under high vacuum, and the resulting residue was dissolved in EtOAc. The EtOAc layer was washed with 0. IN citric acid twice, water once, brined, dried over Na~SO "
filtered and evaporated to give a crude product. This crude product was purified by flash column reverse-phase C-18 chromatography (eluting solvent MeOH\water =
50:50; then 55:45; then 60:40; then 65:35; then 70:30; then 75:25; then 80:20; and then 85: 15) to give 2.163 g of product (14). 'H-N~IR (CDCI3-D~O, o): 7. ~6, 7.59 &
7.35(d,d&m,8H,ArH),4.53(m, lH,CH),4.40-4.16(m,4H,OCH,&CH),2.38 (m, 4H, CH.CO), 2.05 (m, 4H, CH.) and 1.45 & i.42 (2s, 18H, t-butyl).

(D)-N-Fmoc-O-t-butyl lutamyl-(D)-O-t-butyl~lut~m~teNHSester~l5). Toa solution of 2.163 (3.54 mmole) of(14) and 0.428 g (1.05 eq) of N-hydroxysuccinimi~e in 35 ml dry dioxane was added 803 mg DCC, 1,3-dicyclohexyl-carbodiimide (1.1 eq). The reaction mixture was stirred at room temperature overnight. DCU, 1,3-dicyclohexylurea, was removed by filtration, and the filtrate was evaporated under reduced pressure to give 2.9 g crude (15) in a quantitative yield.
~H-N~IR (Cr)C13, 0) 7.76, 7.60 & 7.35 (d, d & m, ArH), 5.87 (br d, NH), 4.91 (m, CA 022 773 73 1998 - I 2 - o I
. , CH), 4.40-4.15 (m, OCH2 & CH), 2.83 (s, NHS ester), 2.52-1.80 (m, CH,CO &
CH7) and 1.46 & 1.44 (2s, t-butyl).

(D)-N-Fmoc-O-t-butvlYlutamvl-(D)-O-t-butvl~lutamvl-(D)-O-t-butyl.lutamYl-~D)-S-tritylcysteine (16). To a solution of 1.358 8 (1.66 mmole) of (15) in 7 ml dry DMF
was added a solution of 0.9 g (1.58 mrnole) of(l3) and 275 ml (1 eq) of DEA in 8ml dry DMF. The reaction mixture was stirred at room temperature overnight.
Solvent was removed under high vacuum, and the resulting residue was dissolved in 200 ml CH7CI2. The CH~CI.. Iayer was washed twice with 0. lN citric acid, once with 0 water7 brined, dried over with Na.. SO." filtered and evaporated to give a crude product. This crude product was purified by flash column reverse-phase C-18 chromatography (eluting solvent MeOH\water = 75:25; then 80:20; then 85:15; and then 90:10) to give 5S7 mg of product (16). (33% yield) lH-N~R (CDCI3-D.O, o):7.7677.51&7.28(d,d&m,23H,ArH),4.43-4.08(m76H,OCH.&CH),3.93 s (m7 IH7 CH)7 2.75 (m7 2H7 CH2S)7 2.50-1.80 (m7 12H7 CH7CO & CH7) and 1.46, 1.41 & 1.36 (3s, 27H, t-butyl).

(D)-O-t-butYl~lutamvl-(D)-O-t-butyl~lutamyl-(D)-O-t-butyl~lutamYl-(D)-S-tritylcysteine ( 17). ~ solution of 0.58 g (0.51 mmole) of (16) in 1.6 ml piperidine and 6.4 ml Di~F was stirred at room temperature for 30 minutes. Solvent was removed under high vacuum7 and the resulting residue was dissolved in a minimllm amount of EtOAc and then pl ecipilated into he~cane. The precipitate was filtered and washed several times with hexane and then vacuum dried to give 449 mg of crude product as the piperidinium salt. This material was dissolved in 40 m~ MeOH and treated with 2S 0.76 ml of lN HCI. Solvent was removed under vacuum7 and the resultin~, residue was dissolved in a minimllm amount of MeOH and p-e~ ; ted into 420 ml of stirring water. The amorphous precipitate was filtered and dried under vacuum to give 396mg of product (17) as the HCI salt form. IH-NMR (CD30D, o): 7.28 (m7 lSH7 ArH)7 4.42 (m7 2H7 CH)7 4.26 (t7 lH7 CH), 3.87 (t, lH, CH), 2.60 (d, 2H, CH7S), 2.36 (rn, 6H, CH2CO), 2.02 (m, 6H, CH ) and 1.44, 1.43 & 1.39 (3s, 27H, t-butyl).

N-Methvl-~- ' 5-~tri-(D)-O-t-butvl~lutamyl)-(D)-S-tritvlcvsteinel- 5-carbamvlpentvl ~ -biotinamide (1~). To a solution of 269 mg (0.27 mmole) of(l7) in 3 ml dry D~lF
was added 113 mg (5 eq) of NaHCO3 followed by addition of a solution of 1.~2 mg (1.1 eq) of N-methyl-N-(5-NHS-carbonylpentyl)-biotinamide, prepared as describedabove in synthesis Bl, in 1.5 ml dry DMF. The reaction mixture was stirred at room temperature under nitrogen for 5 hours. The mixture was filtered, and the filtrate was concentrated under reduced pressure to give a crude product. This crude product was purified by flash column reverse-phase C-18 chromatography (eluting solvent MeOH\water= 70:30; then 75:25; then 80:20; then 85.15; and then 90:10) to give 0 260 mg of product (18). (76% yield) 'H-N~R (CD30D, ~): 7.2S (m, 15H, ArH), 4.50-4.20 (m, 6H, CH), 3.35 (m, 2H, CH,N), 3.18 (mt lH, CHS), 3.01 & 2.88 (2s, 3H, CH3N), 2.90 & 2.1~ (m & m, 4H, CH S), 2.31 (m, 10H, CH,CO), 2.00 (m, 6H, CH,) and 1.80-1.25 (m and 3s, 39H, CH & t-butyl).

N-Methyl-N- { 5-~tri-~lutamvl)cysteine]-5-carbamylpentYI ~ -biotinamide (19). A
solution of 50 mg (39,umole) of (18) in 0.95 ml TFA, 40,ul anisole and 10,ul eth"nediol was stirred at room temperature for 3 hours. The reaction mi:~ture was precipitated into 30 ml ice-cold deoxygenated tert-butyl methylether in a 50 ml glass centrifuge tube. T'nis tube was capped and centrifuged at 2,000 g for 10 minutes.
The supernatant was removed by gentle aspiration. The precipitate was resuspended in 30 rnl of deoxygenated tert-butylmethylether and centrifuged again at 2,000 g for 10 minlltec, and then supernatant was removed. This procedure was repeated threemore times, and the final precipitate was vacuum dried for 30 minutes to give 21 mg of product (19). 'H-NM:R (CD30D, ~): 4.62-4.26 (m, CH), 3.36 (m, CH,N), 3.20 ~5 (m, CHS), 3.03 & 2.90 (2s, CH3N), 2.94 (m, CH2S), 2.69 (d, CH2S), 2.40 (m, CH2CO), 2.30-1.85 (m, CH2) and 1.80-1.25 (m, CH2). Mass spect. (FAB-) (M/Z):
[M-H] at 860.

C. LRCA Construct.
The galactose-HSA-maleimide construct is conjugated to the sulfhydryl bearing liver retention-biotin constructs under the following conditions.

CA 022~73~3 1998-12-01 Conjugation of (l l) with Galactose and Maleimide-Derivatized HS.~. In a vorteYing 5 ml test tube containing 1 l .5 mg of galactose and maleimide-derivatized HSA
described in Section A of this E~cample in 1. ~ ml sterile water and 0. I g ml of 0.2M
phosphate buffer (pH 6.5) was added dropwise a solution of 3 .7 mg ( 15 eq of free thiol) of ( I I ) in 3 ~0 microliters of sterile water. The reaction mixture was gently shaken at room temperature for 90 min~ s. The mixture was transferred into a centricon tube (30K molecular weight cutoff) and centrifuged at 5000 RPl~l for 10-12 minutes to reduce the mixture volume to 1.2 ml. The mixture plus 100 microlitersrinsing was purified by PD-I0 Column (Pharmacia Biotech, Inc., Piscataway, New Jersey) eluting with 6 ml PBS. The protein cont~ining fractions were pooled, and the total volume was reduced to 1.12 m~ by means of centrifugation in a centricon tube.
This 1.12 ml biotin-HSA conjugate solution plus 100 microliters rinsing were transferred to a dialysis cassette (Pierce, Rockford, Illinois) and dialyzed against I . SL
PBS at 4~C for I day, at which time fresh PBS (1.5L) was used, and the dialysis was allowed to be continued for 2 days. The dialyzed biotin-HSA solution was sterilefiltered and ready to be used as a LRCA. The final protein concentration was determined by W absorption at 280 nm was 7.81 mglml. The molar substitution ratio for biotin/HSA was 2.13 as determined by HABA assay (Green et al., Biochem.
J., 94: 236, 1965).
~o Conjuaation of (19) with Galactose and ~laleimide-Derivatized HSA. In a vortexing S ml test tube containing 11.5 mg of galactose and maleimide-derivatized HSA
described in Section A of this Example in 1.8 ml sterile water and 0.18 ml of 0.2M
phosphate buffer (pH 6.5) was added dropwise a solution of 3 .0 mg ( 15 eq of free thiol) of(19) in 0.5~5 ml sterile water and 15 rnicroliters DMSO). The reaction mixture was gently shalcen at room temperature for 90 minlltes The mi~ture 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 m-icroliters 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 I . I ml by means of centrifugation CA 022~73~3 1998-12-01 in a centricon tube. This I .1 ml biotin-HSA conjugate solution plus 100 microliters rinsing were transferred to a dialysis cassette (Pierce, Rockford, Illinois) and dialyzed against 1.5L PBS at 4~C for I day, at which time fresh PBS (1.5L) was used, and the dialysis was allowed to be continued for 2 days. The dialyzed biotin-HSA solution s was sterile filtered and ready to be used as a LRCA. The final prolein concentration was determined by W absorption at 2~0 nm was 8.74 mg/ml. The molar substitution ratio for biotin/HSA was 1.39 as deterrnined by HABA assay (Green et al., Biochem.
J., 94: 236, 1965).

o EXA~PLE V
LRCA Evalu:ltion All LRCAs were evaluated in terms of two criteria, 1) Effectiveness at clearing targeting moiety-streptavidin conjugate; and 2) Biotin release from the LRCA construct.
These criteria were evaluated in comparison to a control compound, a galactose-HSA-biotin clearing agent previously developed by the ~CciDnee of this patent application, galactose3~35-human serum albumin-LC-biotin, wherein LC is an aminocaproyl spacer and wherein the number of biotins ranges from 1 to about 6.
o Criteria 1 was evaluated by ~mini~tPring I-12~-labeled NR-LU-10-streptavidin conjugate, ~minictering the LRCA 24 hours later, and counting residual conjugate in the blood at a later time point. The animal model employed for this evaluation was female Balb/c rnice (20-25 g) injected i.v. with 400,~Lg I-125-NR-LU-10-streptavidin conjugate prepared s~lbst~nti~lly as described above. Each group of expe.i~ "lal2s animals consisted of 3 mice. Blood was sampled serially from the retro-orbital sinus (10,uL x 2) at 1, 4 and 24 hours. At 24 hours, LRCA or the control compound was injected i.v. at total protein doses of either 220 or 1100,ug in each animal. Blood was then sampled 1, 2, 4 and 6 hours post-LRCA a~minictration.
Criteria 2 was evaluated by indirect means. The biotin of the LRCAs was not radiolabeled. Biotin release would be expected to comprornise or fill biotin-binding sites of residual serum compartment NR-LU-10-streptavidin conjugate. Thus, CA 022~73~3 1998-12-01 ~C5~c.Cment of biotin release from the LRCA was indirectly determined by measuring the amount of In- l 1 l -DOTA-biotin, prepared substantially in accordance with E:cample III hereof, that bound to the residual serum conjugate. Such residual serum conjugate, e~posed to LRCAs releasing less biotin-conrztining metabolites, should exhibit minimal loss of capacity for binding radiolabeled biotin. Criteria 2 wasevaluated using the animal model and e~cperimental procedure described for Criteria 1 evaluation, except that 4 hours after LRCA a~minictration, the animals received 1.0 or 15.0,ug of In- 111 -DOTA-biotin, prepared substantially in accordance with procedures described herein. A blood sample 2 hours after administration of In- 111 o was used to assess residual biotin-binding capacity of circulating I-125-N~-LU- 10-streptavidin conjugate.
Constructs 1 and 2, HSA(d-lys)3BT, ~ISA(d-lys-gal)3BT were compared to the control compound made in accordance with the procedure set forth in E~cample I~
above, with regard to criteria 1 at clearing agent doses of 220 micrograms and 1100 s micrograms. 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 ~00 micrograms of targeting conjugate. The 1100 microgram dose is 5x the optimal dose. At 200 micrograms, all three compounds cleared conjugate to 4.8-5.6% ID 6 hours after clearing agent ~-I",;,-;.~l, dlion. At 1100 micrograms, all three compounds cleared conjugate to 2.6-2.9 ID 6 hours following clearing agent administration. Constructs 1 and 2 and the reference compound cleared monoclonal antibody-streptavidin conjugate comparably.
Constructs 1 and 2 were then compared to the reference compound on the basis of criteria 2. The quantity of In-111-DOTA-biotin bound to residual conjugate was measured 2 hours after ~",;t~ , ation thereof (~ hours after clearing agent ~mini.ctration). Both the 200 microgram and 1100 microgram doses were tested. Atthe low dose, significztntly less blockage of conjugate in the serum (higher In-111 uptake) was observed for constructs I and 2 in comparison to the reference compound. The In- 111 (serum concentration of In- 111 -DOT~-biotin)/l- 125 (serum concentration of monoclonal antibody-streptavidin conjugate) ratio was 2.66 for construct 1, 2.73 for construct 2 and 0.71 for the reference compound. This trend CA 022~73~3 1998-12-01 was also observed at the high dose, with In-l 11/I-125 ratios of 0.~0 for construct 1, 0.25 for construct 2 and 0.05 for the reference compound.
Construct 3, HSA(d-glu)3BT 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 l and 2. At the 2''0 microgram clearing agent dose, construct 3 and the reference compound cleared conjugate to4.g6 and 3.41 %ID/g, respectively, 6 hours after clearing agent a~mini.~tration. At the 1100 microgram dose, construct 3 and the reference compound cleared conjugate to1.19 and 2.02 %ID/g, respectively, 6 hours after clearing agent ~lmini~tration ~0 Construct 3 and the ,e~e~ ce compound cleared monoclonal antibody-streptavidin conjugate comparably.
With regard to criteria 2, the comparison at low clearing agent dose showed that significantly less blockaae of conjugate in the serum (higher In- 1 1 1 uptake) occurred when construct 3 was used rather than the reference compound (In-11 l/I-125 ration of 5.6 for construct 3 and 0.96 for the reference compound). This trend was also observed at the high dose, with In-111/I-125 ratios of 0.~3 for construct 3 and 0.18 for the reference compound.
Construct 4, HSA(phos)3BT 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 equivalentwhen construct 4 was compared to the control compound.
In a tumored animal model, compromise of tumor-associated monoclonal antibody-streptavidin conjugate by biotin released from a clearing agent, such as construct 4 or the control compound, can be measured directly, rather than by inference from serum data. As in prior studies, mice were injected with 400 micrograms of conjugate, followed 24 hours later by 200 micrograms (low dose) or1100 micrograms (high dose) of clearing agent (LRCA or leference compound), followed 4 hours later by 15 micrograms of In-11 l-DOTA-biotin. Animals were sacrificed 2 hours later. Tumor uptake of In-l 1 l-DOTA-biotin was superior at both low and high clearing agent doses (8.89 and 5.01 %ID/g LRCA verses 8.04 and 0.68%ID/g control).

CA 022~73~3 1998-12-01 Targeting of monoclonal antibody-streptavidin to tumor was relatively consistent for all groups of animals at appro~cimately 250 pmoUg. Use of hi;,h doses of the reference compound results in significant compromise of ligand binding capacity relative to a low dose offering thereof (1492 pmol/g versus 1~6 pmol/g).
s Conversely, construct 4 e~chibited only a modest decrease in ligand binding capacity at the tumor in going from low to high LRCA dose (164~ pmollg versus 929 pmollg).
Thus, much greater dosing latitude appears to be possible with the use of construct 4.

Kits cont~ining one or more ofthe components described above are also 0 contemplated. For instance, LRCAs may be provided in a sterile container for use in prelalg~Lillg procedures. Alternatively, such a LRCA may be vialed in a non-sterile condition for use as a reseal ch reagent.

From the foregoing, it will be appreciated that, although specific embodiments s of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited e~cept as by the appended claims.

s~

Claims

WHAT IS CLAIMED IS:

1. A liver retention clearing agent comprising:
(a) a structural component;

(b) a hepatic clearance directing component directly or indirectly associated with the structural component, wherein the hepatic clearance directing component is composed of a plurality of sugar residues recognized by a hepatocyte receptor;

(c) one or more binding components directly or indirectly associated with the structural component, wherein the binding components are capable of in vivo association with a molecule to be cleared; and (d) one or more liver retention components directly or indirectly associated with the structural component or the binding components or both, such that metabolic processing by hepatocytes of liver retention clearing agent preferentially results in metabolites that contain both a binding component and a liver retention component.

2. A liver retention clearing agent of Claim 1 wherein at least one liver retention component is resistant to peptidase cleavage.

3. A liver retention clearing agent of Claim 2 wherein the liver retention component incorporates at least 3 amino acids of unnatural (D) configuration or unnatural armino acids.

. A liver retention clearing agent of Claim 3 wherein the liver retention component incorporates from about 3 to about 6 amino acids of unnatural (D) configuration or unnatural amino acids.

11. A liver retention clearing agent of Claim 10 wherein the liver retention component incorporates between about 3 and about 6 glutamic acid or lysine aminoacids or galactosylated lysine amino acid derivatives.

12. A liver retention clearing agent of Claim 6 wherein the liver retention component incorporates at least three charged phosphonates of amino acids of natural (L) or unnatural (D) configuration.

13. A liver retention clearing agent of Claim 12 wherein the liver retention component incorporates between about 3 and about 6 alpha phosphonomethyl amino acids of the formula:

14. A liver retention clearing agent of Claim 6 wherein the liver retention component incorporates a neutral charge, hydroplhilic disaccharide.

15. A liver retention clearing agent of Claim 1 wherein the liver retention component-binding component is one of the following:

or wherein n is about 3 to about 12; R and R' are lower alkyl groups from 1 to about 6 carbon atoms.

5. A liver retention clearing agent of Claim 2 wherein the binding component is bound to either the structural backbone or to the liver retention component via a stable tertiary amide bond.

6. A liver retention clearing agent of Claim I wherein the liver retention component is preferentially retained within hepatocyte cytoplasm or a subcellular compartment therein.

7. A liver retention clearing agent of Claim 6 wherein the liver retention component is a DOTA macrocycle.

8. A liver retention clearing agent of Claim 6 wherein the liver retention component incorporates at least three positively charged amino acids of unnatural (D) configuration, at least three negatively charged amino acids of unnatural (D) configuration or at least three neutral charge, hydrophilic amino acids of unnatural (D) configuration.

9. A liver retention clearing agent of Claim 6 wherein the liver retention component incorporates at least three positively charged amino acids of natural (L) configuration, at least three negatively charged amino acids of natural (L) configuration or at least three neutral charge, hydrophilic amino acids of natural (L) configuration and wherein at least one stabilized tertiary amide bond is incorporated in the liver retention component or the binding component or in a linker positioned therebetween.

10. A liver retention clearing agent of Claim 8 or Claim 9 wherein the polyaminoacids is polylysine, polyglutamic acid, polyhistidine, polyarginine, polyaspartates, polyornithine or saccharide derivatives thereof.

16. A liver retention clearing agent of Claim 1 wherein the liver retention component-binding component is one of the following:

or wherein n is about 3 to about 12; R and R' are lower alkyl groups from I to about 6 carbon atoms; and Gal is galactose.
17. A liver retention clearing agent of Claim 1 wherein the liver retention component-binding component is one of the following:

or wherein n is about 3 to about 12; R and R' are lower alkyl groups from 1 to about 6 18. A liver retention clearing agent of Claim I wherein the liver retention component-binding component is one of the following:

or wherein n is about 3 to about 12; R and R' are lower alkyl groups from 1 to about 6 carbon atoms.