WO2002087497A2 - Therapeutic agent/ligand conjugate compositions, their methods of synthesis and use - Google Patents
Therapeutic agent/ligand conjugate compositions, their methods of synthesis and use Download PDFInfo
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- WO2002087497A2 WO2002087497A2 PCT/US2002/012502 US0212502W WO02087497A2 WO 2002087497 A2 WO2002087497 A2 WO 2002087497A2 US 0212502 W US0212502 W US 0212502W WO 02087497 A2 WO02087497 A2 WO 02087497A2
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
- A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
- A61K51/1093—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
- A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
- A61K51/1027—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against receptors, cell-surface antigens or cell-surface determinants
- A61K51/103—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against receptors, cell-surface antigens or cell-surface determinants against receptors for growth factors or receptors for growth regulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
Definitions
- compositions useful in the treatment of cancer and other diseases and, more specifically, to compositions comprising therapeutic agents
- chemotherapeutic drugs e.g., chemotherapeutic drugs
- other compounds conjugated to ligands useful for the selective delivery of the agent or compound to tumors and other target tissues.
- the invention also relates to methods for synthesizing and using such compositions.
- Cancer chemotherapy is ultimately limited by the toxicity of drugs to normal tissues.
- Selective delivery of drugs to target cells theoretically allows the use of a reduced dose to achieve the same therapeutic response, with a consequent decrease in systemic toxicity.
- a number of methods have been used to selectively target tumors with therapeutic agents to treat cancers in humans and other animals.
- Targeting moieties such as monoclonal antibodies ( Ab) or their fragments have been conjugated to linear polymers via their side chain functional groups.
- this approach usually results in reduced receptor binding affinity either due to changes in the chemical properties of the antibodies or due to folded configuration of polymers that imbed the targeting moiety in the random coiled structure.
- crosslinks and aggregates of polymers may form as a result of side-chain coupling procedures.
- __ ⁇ __munoconjugates have been synthesized by employing intermediate carriers such as dextran, serum albumin, and synthetic polymers to increase the amount of drugs attached to the antibody without significantly impairing its antigen binding activity.
- intermediate carriers such as dextran, serum albumin, and synthetic polymers to increase the amount of drugs attached to the antibody without significantly impairing its antigen binding activity.
- the antibodies were attached to the side chains of the polymer, which is believed to adversely affect the binding affinity of the antibody and the in vivo behavior of the immunoconjugates.
- compositions according the invention include conjugates of a ligand, a polymer spacer, a polymer carrier, and a therapeutic agent or another compound or agent.
- a preferred composition of the invention comprises a conjugate of an antibody, a polyethylene glycol (PEG) spacer, a polymer carrier, and a therapeutic agent.
- the ligand is a monoclonal antibody
- the polymer spacer is a PEG spacer
- the polymer carrier is poly(l-glutamic acid) (PG)
- the therapeutic agent is a chemotherapeutic agent such as Adriamycin or paclitaxel.
- one embodiment of the invention is directed to a conjugate molecule comprising: a ligand; a polymer spacer; a polymer carrier; and a therapeutic agent.
- the ligand is bonded to the polymer spacer, the polymer spacer is bonded to the polymer carrier, and the polymer carrier is bonded to the therapeutic agent.
- the polymer carrier may be bonded to the therapeutic agent with or without the assistance of a linker molecule.
- Another embodiment of the invention is directed to a composition comprising any of the conjugate molecules described herein and a pharmaceutically acceptable carrier.
- Still another embodiment is directed to a method for selectively delivering a therapeutic agent to a target tissue in a patient comprising: administering a conjugate molecule to the patient having said target tissue, wherein the conjugate molecule comprises: a ligand with affinity for the target tissue; a polymer spacer; a polymer carrier; and a therapeutic agent.
- the conjugate molecule comprises: a ligand with affinity for the target tissue; a polymer spacer; a polymer carrier; and a therapeutic agent.
- the ligand is bonded to the polymer spacer, the polymer spacer is bonded to the polymer carrier, and the polymer carrier is bonded to the therapeutic agent.
- a further embodiment is directed to a method of treating a patient having a diseased tissue, the method comprising administering a therapeutically effective amount of a conjugate molecule to the patient, wherein the conjugate molecule comprises: a ligand with affinity for the diseased tissue; a polymer spacer; a polymer carrier; and a therapeutic agent.
- the conjugate molecule comprises: a ligand with affinity for the diseased tissue; a polymer spacer; a polymer carrier; and a therapeutic agent.
- the ligand is bonded to the polymer spacer, the polymer spacer is bonded to the polymer carrier, and the polymer carrier is bonded to the therapeutic agent.
- the invention also includes different methods for synthesizing conjugate molecules of the invention.
- One such method comprises the steps of: providing a polymer spacer-polymer carrier construct having a sulfliydryl-reactive vinyl sulfone group at one end of the polymer spacer; conjugating the therapeutic agent to the polymer carrier to form a vinyl sulfone-polymer spacer-polymer carrier-therapeutic agent construct; pretreating the ligand to introduce a sulfhydryl group on the ligand; and combining the ligand with the vinyl sulfone-polymer spacer-polymer carrier- therapeutic agent construct, wherein the vinyl sulfone group reacts with the sulfhydryl group to form a conjugate molecule comprising the ligand, the polymer spacer, the polymer carrier, and the therapeutic agent, and wherein the ligand is bonded to the polymer spacer, the polymer spacer is bonded to the polymer carrier, and the polymer
- Figure 1 Schematics of conjugate molecules depicting site-specific attachment of homing ligand to one terminus of PEG molecules for targeted delivery of diagnostic and therapeutic agents.
- Figure 2 Synthetic scheme for the synthesis of niAb-PEG-PG-Drug conjugates.
- FIG. 1 GPC elution profile of Herceptin (A), VS-PEG-PG-TXL (B), and purified Herceptin-PEG-PG-TXL conjugate (C) using a Superdex 200 column (1.0 x 30 cm).
- FIG. 1 Gel permeation chromatography of C225 (A), PEG-PG-Adr (B), and purified C225-PEG-PG-Adr conjugate (C) using a Superdex 200 column (1.0 x 30 cm).
- Figure 9 Volume-weighted Gaussian Analysis showing particle size and size distribution of C225-PEG-PG-Adr.
- FIG. 1 Hypothetical structure of polymeric nanoparticles (A) and targetable polymeric nanoparticles (B) from amphiphilic block copolymer PEG-PG- Adr.
- FIG. 11 Graphs showing cytotoxicity of Herceptin-PEG-PG-TXL in MDA-MB- 468 (Her 2/neu-) cells (A); and SKOVipl (Her 2/neu+) cells (B).
- FIG. 12 Graphs showing cytotoxicity of Herceptin-PEG-PG-TXL in MDA 435/neo cells (A) and MDA 435/e B2 cells (B).
- the present invention is directed to novel conjugates useful for the selective delivery of therapeutic agents (e.g., chemotherapeutic drugs, hormonal agents and diagnostic agents) and other compounds and agents to tumors or another target tissue.
- therapeutic agents e.g., chemotherapeutic drugs, hormonal agents and diagnostic agents
- the invention is also directed to novel methods of synthesizing and using such conjugates.
- Preferred embodiments of the invention comprise a ligand, such as a monoclonal antibody (e.g., C225 or Herceptin), indirectly coupled to a therapeutic agent, such as a chemotherapeutic drug.
- the coupling is achieved by conjugating the ligand site-specifically to the termini of a polymer-therapeutic agent conjugate using a polymer spacer or linker (e.g. , a PEG spacer).
- PEG polyethylene glycol
- mAb monoclonal antibody
- FIG. 1A a polyethylene glycol (PEG) conjugated monoclonal antibody (mAb) with a radionuclide attached to one terminus of the PEG chain and the antibody to the another terminus of PEG chain was designed and synthesized (See also, X-X. Wen et al, Polyethylene glycol) conjugated anti-EGF receptor antibody C225 with radiometal chelator attached to the termini of polymer chains.
- the polymeric immunoconjugates maintained the binding affinity of the corresponding mAbs. Specifically, C225 and Herceptin conjugates bound to target cell surfaces. In addition, the C225 conjugate appeared to be internalized. As shown in the biologic assays, the attachment of drugs to the polymeric carrier through hydrolytically stable amide linkage and the efficient cellular internalization yielded significantly increased selective cytotoxicity against target cells. Further, targetable polymeric nanoparticles formed when Adriamycin was used as the drug to conjugate to mAb-PEG-PG carrier.
- one embodiment of the invention is directed to a conjugate molecule comprising: a ligand; a polymer spacer; a polymer carrier; and a therapeutic agent.
- the conjugate molecule is useful for the selective delivery of the therapeutic agent to tumors or other tissues with biological receptors.
- the ligand is bonded to the polymer spacer
- the polymer spacer is bonded to the polymer carrier
- the polymer carrier is bonded to the therapeutic agent.
- bonded refers to any physical or chemical attachment, including, but not limited to, covalent bonding, ionic or chelating interactions.
- the ligand is bonded to the polymer spacer via a covalent bond
- the polymer spacer is bonded to the polymer carrier via a covalent bond
- the polymer carrier is bonded to the therapeutic agent directly via a covalent bond, or indirectly using a linker.
- the ligand and polymer spacer may be joined by an amide bond, a thioether (S-C) bond, a disulfide (S-S) bond, or a thiourethane bond, more preferably, an amide, thioether or disulfide bond, and, most preferably, a thioether bond.
- the polymer spacer and polymer carrier may be joined, for example, by an amide bond, a thioether (S-C) bond, a disulfide (S-S) bond, a thiourethane bond, a carbonate bond or a urethane bond, more preferably, an amide or a urethane bond, and, most preferably, an amide bond.
- the polymer carrier and therapeutic agent may be bonded to each other, for example, by an amide, thioether, disulfide, thiourethane, hydrazone or ester bond, and more preferably, by an amide or ester bond.
- the polymer carrier and therapeutic agent can be bonded or joined using a linker.
- linkers include, but are not limited to, aliphatic chains, lipids, amino acids or peptides.
- the polymer carrier is preferably covalently bonded to the linker, and the linker is preferably covalently bonded to the therapeutic agent.
- more than one polymer spacer-polymer carrier- therapeutic agent construct may be bonded to a single ligand or antibody. Multiple therapeutic agents may be bonded to the polymer carrier. Ligands different from those attached to the PEG chain terminus may be bonded to the side chains of the polymer carrier.
- the polymer carrier to which the therapeutic agent or other compound is attached is preferably poly(l-glutamic acid).
- poly(l-glutamic acid) preferably poly(l-glutamic acid).
- other polymers particularly those which are biocompatible, water-soluble, biodegradable, and have multiple side- chain functional groups that allow attachment of multiple drug molecules, may be used without departing from the scope of the invention.
- polymers include, but are not limited to, poly(d-glutamic acid), poly(dl-glutamic acid), poly(l-aspartic acid), poly(d-aspartic acid), poly(dl-aspartic acid), polylysine, polysaccharides, polyhydroxypropylmethacryamide (HPMA), dextran, poly(hydroxypropylglutamine), poly(hydroxyethylglutamine), hyaluronic acid, carboxymethyl dextran, polyacrylic acid and chitosan, and copolymers between two or more of them.
- HPMA polyhydroxypropylmethacryamide
- dextran poly(hydroxypropylglutamine), poly(hydroxyethylglutamine), hyaluronic acid
- carboxymethyl dextran polyacrylic acid and chitosan
- copolymers between two or more of them and copolymers between two or more of them.
- the polymer carrier can generally have any number average molecular weight, and preferably has a number average molecular weight of at least about 1,000 daltons.
- the poly(l-glutamic acid) preferably has a number average molecular weight of about 1,000 daltons to about 100,000 daltons.
- the other polymers listed above as carriers preferably have a number average molecular weight of about 1,000 daltons to about 150,000 daltons.
- the polymer spacer between the ligand and the polymer is preferably PEG.
- linear polymers particularly those which are biocompatible and uncharged, may be used without departing from the scope of the invention.
- These polymers include, but are not limited to, a polyamino acid, such as polyglycine, polytyrosine, polyphenylalanine, dextran, polysaccharides, polypropylene oxide (PPO), a copolymer of polyethylene glycol (PEG) with PPO, polyglycolic acid, polyvinyl pyrolidone, polylactic acid and polyvinyl alcohol.
- a polyamino acid such as polyglycine, polytyrosine, polyphenylalanine, dextran, polysaccharides, polypropylene oxide (PPO), a copolymer of polyethylene glycol (PEG) with PPO, polyglycolic acid, polyvinyl pyrolidone, polylactic acid and polyvinyl alcohol.
- the polymer spacer can generally have any number average molecular weight, and preferably has a number average molecular weight of at least about 1,000 daltons.
- the polyethylene glycol preferably has a number average molecular weight of about 1,000 daltons to about 100,000 daltons.
- the other polymers listed above as spacers preferably have a number average molecular weight of about 1,000 daltons to about 100,000 daltons.
- the ligand (or targeting moiety) can generally be any ligand, and preferably is an antibody or its fragments, a peptide or a protein.
- the antibody can generally be a monoclonal antibody, or a polyclonal antibody.
- useful antibodies include, but are not limited to, C225, Herceptin, Rituxan, phage library antibodies, anti-CD, DC101, antibodies to the integrins alpha v-beta 3 (such as LM609), antibodies to VEGF receptors, antibodies to VEGF, or any other suitable antibody.
- the antibody can be an antibody fragment such as F(ab') 2 , Fab', or ScFv fragment or an antibody fragment such as chimeric (c) 7E3Fab (c7E3Fab) that binds to integrin receptors.
- the antibody can be a humanized antibody.
- the peptide can generally be any peptide, such as a cell surface targeting peptide, and preferably is a growth factor, such as VEGF (Vascular Endothelial Growth Factor)-A, -B, -C or -D, PDGF (Platelet-Derived Growth Factor), Angiopoietin-1 or -2, HGF (Hepatocyte Growth Factor), EGF (Epidermal Growth Factor), bFGF (Basic Fibroblast Growth Factor), cyclic CTTHWGFTLC, cyclic CNGRC, or cyclic RGD-4C.
- VEGF Vascular Endothelial Growth Factor
- PDGF Platinum-Derived Growth Factor
- Angiopoietin-1 or -2 vascular endothelial Growth Factor
- HGF Hepatocyte Growth Factor
- EGF Epidermatitis
- bFGF Basic Fibroblast Growth Factor
- CTTHWGFTLC cyclic CNGRC,
- the protein can generally be any protein, such as annexin V, interferons (e.g., interferon , interferon ⁇ ), tumor necrosis factors, endostatin, angiostatin, or thrombospondin, and preferably is annexin V, endostatin, angiostatin, interferon- ⁇ or interferon- ⁇ .
- the ligand is a monoclonal antibody, such as a C225, Herceptin or c7E3Fab antibody, or a protein, such as annexin V.
- the ligand has affinity for a target tissue. Preferred ligands bind specifically to receptors or other binding partners on the target tissue.
- therapeutic agent broadly includes, but is not limited to, drugs, chemotherapeutic drugs/agents, diagnostic agents, hormonal drugs/agents, and other compounds and compositions useful in the treatment, diagnosis and monitoring of disease.
- the invention is particularly useful for the delivery of chemotherapeutic agents.
- Chemotherapeutic agents useful in the practice of the invention include, but are not limited to, Adriamycin (Adr or doxorubicin), daunorabicin, paclitaxel (Taxol), docetaxel (taxotere), epothilone, camptothecin, cisplatin, carboplatin, etoposide, tenoposide, geldanamycin, methotrexate, maytansinoid DM1 or 5-FU.
- the chemotherapeutic agent is Adriamycin or paclitaxel, and, more preferably, is Adriamycin.
- Gd-DTPA gadolinium-DTPA
- ICG indocyanine green
- Alexa fluor Alexa fluor
- Another embodiment of the invention is directed to a composition comprising a plurality of nanoparticles.
- the nanoparticles comprise a plurality of the conjugate molecules described herein.
- the therapeutic agent in the nanoparticles is Adriamycin.
- the polymer spacer and polymer carrier have hydrophilic/hydrophobic characters or hydrophobic/hydrophilic characters.
- the PEG block in PEG-PG-Adr is hydrophilic
- the PG-Adr block in the copolymer is hydrophobic.
- compositions comprising any of the conjugate molecules described herein and a pharmaceutically acceptable carrier.
- pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents and isotonic agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art.
- the carrier may comprise water, alcohol, saccharides, polysaccharides, drugs, sorbitol, stabilizers, colorants, antioxidants, buffers, or other materials commonly used in pharmaceutical compositions. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
- compositions that do not produce an allergic or similar untoward reaction when administered to an animal or a human.
- a preferred composition is a pharmaceutical preparation suitable for injectable use.
- Pharmaceutical preparations of the invention suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersions.
- the preparations are stable under the conditions of manufacture and storage and are preserved against the contaminating action of microorganisms, such as bacteria and fungi.
- the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
- the prevention of the action of microorganisms may be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.
- Sterile injectable solutions may be prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
- dispersions may be prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
- a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
- the preferred methods of preparation include vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
- the solution is preferably suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose.
- a further embodiment of the invention is directed towards methods for selectively targeting tumors or other target tissues with biological receptors using any of the herein described conjugate molecules and compositions.
- one such embodiment is directed to a method for selectively delivering a therapeutic agent to a target tissue in a patient comprising: administering a conjugate molecule to a patient having the target tissue, wherein the conjugate molecule comprises: a ligand with affinity for the target tissue; a polymer spacer; a polymer carrier; and a therapeutic agent.
- the ligand is bonded to the polymer spacer
- the polymer spacer is bonded to the polymer carrier
- the polymer carrier is bonded to the therapeutic agent.
- the ligand is an antibody
- the polymer spacer is polyethylene glycol
- the polymer carrier is poly(l-glutamic acid).
- the therapeutic agent is selectively delivered to the tissue, where it exerts its therapeutic effect.
- the therapeutic agent is a cytotoxic agent which exerts a cytotoxic effect on the target tissue.
- the administering step may be performed parenterally, e.g., by intravascular, intraperitoneal, intramuscular or intratumoral injection.
- the conjugate molecule may be administered by inhalation or another suitable route.
- administration is by intravascular injection.
- the target tissue may be any desired tissue, including, but not limited to, a tumor or other neoplasm, inflammatory, infectious, reparative or regenerative tissue (including post trauma and post surgery tissues).
- tumor includes benign and malignant tumors or neoplasia.
- the target tissue is a solid tumor, such as breast cancer, ovarian cancer, colon cancer, lung cancer, head and neck cancer, a brain tumor, liver cancer, a pancreatic tumor, bone cancer, or prostate cancer.
- the target tumor may be a malignancy such as leukemia or lymphoma.
- the patient can be any animal. Preferably the patient is a mammal.
- the mammal can be a human, a dog, a cat, a horse, a cow, a pig, a rat, a mouse or other mammal. More preferably, the patient is a human.
- patient broadly includes, but is not limited to, a human or any animal being treated, tested or monitored in any kind of therapeutic, diagnostic, research, development or other application.
- Additional embodiments of the invention are directed towards other therapeutic applications using the herein described conjugate molecules.
- One such embodiment is directed to a method of treating a patient having or suspected of having a diseased tissue, the method comprising administering a therapeutically effective amount of a conjugate molecule to the patient, wherein the conjugate molecule comprises: a ligand with affinity for the diseased tissue; a polymer spacer; a polymer carrier; and a therapeutic agent.
- the ligand is bonded to the polymer spacer
- the polymer spacer is bonded to the polymer carrier
- the polymer carrier is bonded to the therapeutic agent.
- the therapeutic agent is selectively delivered to the tissue, where it exerts its therapeutic effect.
- the therapeutic agent is a chemotherapeutic or cytotoxic agent and the diseased tissue is a tumor
- the therapeutic effect may include inhibition or killing of the tumor cells.
- the administering step may be performed parenterally, e.g., by intravascular, intraperitoneal, intramuscular or intratumoral injection.
- the conjugate molecule may be administered by inhalation or another suitable route. Preferably, administration is by intravascular injection.
- the dosage of the conjugate molecule can be increased or decreased to modulate the therapeutic effect on the targeted diseased tissue.
- the patient can generally be any animal.
- the patient is a mammal.
- the mammal can be a human, a dog, a cat, a horse, a cow, a pig, a rat, a mouse or other mammal. More preferably, the patient is a human.
- the diseased tissue may be any type of tissue, including, but not limited to, a tumor or other neoplasm, inflammatory, infectious, reparative or regenerative tissue.
- the diseased tissue is a tumor, and, more preferably, is a solid tumor such as breast cancer, ovarian cancer, colon cancer, lung cancer, head and neck cancer, a brain rumor, liver cancer, a pancreatic tumor, bone cancer, or prostate cancer.
- the target tumor may be a malignancy such as leukemia or lymphoma.
- the term "treating" a tumor is understood as including any medical management of a subject having a tumor. The term would encompass any inhibition of tumor growth or metastasis, or any attempt to visualize, inhibit, slow or abrogate tumor growth or metastasis.
- the method includes killing a cancer cell by non-apoptotic as well as apoptotic mechanisms of cell death.
- a therapeutically effective amount of the conjugate molecules of the invention is preferably administered to achieve the desired effect.
- the actual dosage amount of a composition comprising the conjugate molecule of the present invention administered to the patient to achieve the desired effect can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and route of administration, as well as other factors known to those of skill in the art.
- the practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
- the invention also includes methods for synthesizing the novel conjugates and compositions of the invention.
- One such method for synthesizing a conjugate molecule comprising a therapeutic agent and ligand comprises the steps of: providing a polymer spacer-polymer carrier construct having a sulfliydryl-reactive vinyl sulfone group at one end of the polymer spacer; conjugating the therapeutic agent to the polymer carrier to form a vinyl sulfone-polymer spacer-polymer carrier-therapeutic agent construct; pretreating the ligand to introduce a sulfhydryl group on the ligand; and combining the ligand with the vinyl sulfone-polymer spacer-polymer carrier- therapeutic agent construct, wherein the vinyl sulfone group reacts with the sulfhydryl group to form a conjugate molecule comprising the ligand, the polymer spacer, the polymer carrier, and the therapeutic agent, and wherein the ligand is bonded to
- Another method for synthesizing a conjugate molecule of the invention comprises the steps of: introducing at least one protected sulfhydryl group (SH) to an end of a polymer spacer; conjugating the polymer spacer to a polymer carrier to form a protected SH-polymer spacer-polymer carrier construct; conjugating a therapeutic agent to the polymer carrier to form a protected SH-polymer spacer-polymer carrier- therapeutic agent construct; pretreating a ligand to introduce a sulfhydryl reactive functional group on said ligand; deprotecting the protected SH group to obtain a free SH group; and combining the pretreated ligand with the SH-polymer spacer-polymer carrier-therapeutic agent construct, wherein the SH group reacts with the sulfhydryl reactive functional group to form a conjugate molecule comprising the ligand, the polymer spacer, the polymer carrier, and the therapeutic agent.
- the ligand is bonded to the polymer spacer
- the ligand is preferably pretreated with a suitable agent, such as vinyl sulfone or maleimide to introduce the sulfhydryl reactive functional group.
- a suitable agent such as vinyl sulfone or maleimide
- the SH group is deprotected to obtain a free SH group before combining with the ligand.
- Still another method for synthesizing a conjugate molecule comprises the steps of: providing a polymer-spacer-polymer carrier-therapeutic agent construct; introducing a protected amine to an end of the polymer spacer to form a protected amine-polymer spacer-polymer carrier-therapeutic agent construct; deprotecting the protected amine-polymer spacer-polymer carrier-therapeutic agent construct to obtain a free amine-polymer spacer-polymer carrier-therapeutic agent construct; and combining the free amine-polymer spacer-polymer carrier-therapeutic agent construct with a ligand having a carboxylic acid group.
- the carboxylic acid in the ligand conjugates with the free amine to form an amide bond, thereby forming a conjugate molecule comprising the ligand, the polymer spacer, the polymer carrier and the therapeutic agent.
- the ligand is bonded to the polymer spacer
- the polymer spacer is bonded to the polymer carrier
- the polymer carrier is bonded to the therapeutic agent.
- Ligands, polymer spacers, polymer carriers and therapeutic agents useful in the practice of the foregoing synthetic methods include, but are not limited to, any of the ligands, polymer spacers, polymer carriers and therapeutic agents disclosed herein.
- the therapeutic agent may comprise a contrast agent or a chemotherapeutic drug.
- the ligand is preferably bonded to the polymer spacer via a covalent bond
- the polymer spacer is bonded to the polymer carrier via a covalent bond
- the polymer carrier is bonded to the therapeutic agent directly via a covalent bond or indirectly using a linker.
- conjugate molecules are advantageous over those previously described in the art.
- Prefereed embodiments of the conjugate molecules are useful for the targeted treatment of tumors and other diseased tissue.
- Preferred embodiments have improved in vivo half lives and exhibit reduced or eliminated accumulation in the liver.
- the use of polymers reduces non-specific interaction with non-target tissues and reduces background activity. Attachment of the therapeutic agent and polymer carrier to the ligand with a polymer spacer instead of to the ligand directly improves retention of the ligand' s receptor binding affinity.
- the conjugate molecule design strategy is flexible, and allows for the preparation of a wide array of molecules for different diagnostic and clinical uses. It allows both passive targeting (when ligand is not attached) and active targeting (when ligand is attached).
- Diisopropylcarbodiimide (DIG), dimethylformamide (DMF), poly(l-glutamic acid) (PG, MW 3 IK), p-nitrophenol, p-nitrophenyl chloroformate (PNP), dimehtylaminopyridine (DMAP), 2-isobutoxy-l-isobutoxycarbonyl-l,2- dihydroquinoline) (IIDQ) were purchased from Sigma-Aldrich (Milwaukee, WI). Paclitaxel and Adriamycin-hydrochloride (Adr ⁇ Cl) were obtained from Hande Tech. (Houston, TX). BODIPY FL hydrazide dye was obtained from Molecular Probes (Eugene, OR).
- Vinylsulfonyl N-hydroxysuccinimidyl PEG (VS-PEG- ⁇ HS, MW 3400) and ⁇ H 2 -PEG-OH were obtained from Shearwater (Huntsville, AL).
- N- succinimidyl S-acetylthioacetate (SATA), ⁇ -maleimidobutyric acid ⁇ - hydroxysuccimmide ester (GMBS), ⁇ -succinimidyl 3-[2-pyridyldithio]propionate (SPDP), dithiothreitol (DTT), and hydroxyamine were obtained from Pierce Chemical Co. (Rockford, IL).
- C225 is a human-mouse chimeric monoclonal antibody that targets epidermal growth factor receptor (EGFR or EGF receptor) and was kindly provided by ImClone Systems Inc. (New York, NY). Herceptin (Trastuzumab) was obtained from Genentech (San Francisco, CA).
- the system consisted of an AKTA fast protein liquid chromatography (FPLC) (Amersham Pharmacia Biotech) and a Resource Q anion exchange column (Amersham Pharmacia).
- the mobile phase was run from Buffer A (20 mM Tris buffer, pH 7.5) to Buffer B (20 mM Tris buffer containing 0.15 or 1.0 N NaCl, pH 7.5) in a linear fashion at a flow rate of 3 ml/minute for 20 ml (6.67 minutes).
- the column was eluted with 100% Buffer B for the rest of the chromato graphic period.
- the degree of substitution of C225 by SATA was estimated by measuring the changes in the concentrations of free arnino groups using 2,4,6- trinitrobenzenesulfonic acid (TNBS) assay, and by monitoring the presence of sulfhydryl groups using Ellman's test (GT Hermanson, ed., Amine detection reagents. Bioconjugate Techniques. San Diego, Academic Press, pp. 112-114 and pp. 88-90, 1996).
- TNBS 2,4,6- trinitrobenzenesulfonic acid
- the concentration of each component of the conjugate was determined and the molar ratio was calculated.
- the concentration of the antibody was measured by UV at 650 nm using the Bio-Rad Laboratory protein assay kits (Hucoles, CA). In these measurements, known concentration of C225 or Herceptin was used as a reference standard and PEG-PG-BODIPY FL was used as the background. Taxol, Adr, and BODIPY FL concentrations were quantified by determining the absorbance at 230 nm, 480 nm, or 503 nm.
- the concentration of paclitaxel in Her-PEG-PG-TXL or C225-PEG-PG-TXL was further determined by a hydrolysis/HPLC method.
- the concentration of TXL in the immunoconjugates was estimated by assuming a molar ratio of 1:1 between mAb and PEG-PG-TXL.
- the assumption is based on UV absorbance of niAb-PEG-PG-BODIPY FL where the concentration of BODIPY FL, and hence the molar ratio between mAb and PEG-PG, could be conveniently determined by UV measurements, f. Quantification of TXL concentration in mAb-PEG-PG-TXL by Hydrolysis and HPLC Analysis
- Taxol concentration was obtained from HPLC analysis with the following conditions: 1 mL/minute flow rate, gradient of water/CH 3 CN (changing acetonitrile from 0% to 40%), column: Nova Pak (3.9 x 150 mm) and UV detector at 228 nm.
- a standard curve was constructed using a range of Taxol solution in methylene chloride with concentrations ranging between 0.5 and 6 mg/ml. Ten ⁇ l aliquot was injected from each standard and each extracted solution.
- a linear PEG-PG conjugate that contains a sulfhydryl reactive vinyl sulfone (VS) group at the end of the PEG block of the copolymer was synthesized.
- the anticancer agent Adriamycin (Adr) or paclitaxel (Taxol, TXL) was conjugated to the side chain carboxyl groups in the PG block of VS-PEG-PG via p-nitrophenol activated esters, IIDQ, or carbodiimide-mediated reaction.
- mAb-PEG-PG-drug The synthetic scheme is shown in Figure 2.
- the mAb used in this study included C225, a mAb directed against epidermal growth factor receptor (EGFR) and Herceptin, a mAb directed against Her-2/neu receptor. Both receptors are overexpressed in a variety of solid tumors.
- EGFR is overexpressed on the cells of over one-third of all solid tumors, including bladder, breast, colon, ovarian, prostate, renal cell, squamous cell, non-small cell lung, and head and neck carcinomas.
- the concentrations of the compounds were monitored by RI detector. (Conditions: Flow Rate - 1 mL/minute; Column - Superdex 200; Buffer - PBS with 0.1% LiBr).
- Paclitaxel content 21.6% (w/w) based on UV measurement at 230 nm. Each polymer chain contained about 11 TXL molecules. TXL yield: 54.6%. No free paclitaxel was detected by silica gel thin layer chromatography (MeCl 2 /methanol, 4/1, v/v) and by GPC (system 1).
- Adr was conjugated to VS-PEG-PG via the DIC-mediated coupling reaction using similar procedures.
- the structure of Adr is shown in Figure 5; the drug was conjugated to VS-PEG-PG polymer through its amino groups on the sugar moiety.
- Adr free amine 40 mg, 74 mol
- 30 ⁇ l DIC 304.3 mg, 192 mol
- 100 ⁇ l pyridine 100 ⁇ l pyridine
- trace amount of DMAP was obtained by extracting an aqueous solution of Adr ⁇ Cl and triethylamine (molar ratio 1:3) with chloroform.
- the reaction mixture was worked up as follows: The aqueous solution of polymer conjugate was acidified with 1.0 HCI. The precipitate was collected by centrifugation, washed with water, re- dissolved in 0.1 N NaHCO 3 , and dialyzed. GPC (system 2) revealed the absence of free Adr in the isolated product. The amount of Adr in the polymer was estimated to be 15% (w/w) as measured by UV at 480 nm. Each polymer chain contained about 11 Adr molecules. Yield: 120 mg polymer conjugate, Yield of Adr, 45%.
- the fluorescent dye BODIPY was conjugated to VS-PEG-PG to facilitate confocal fluorescent microscopic study. Briefly, 5 mg BODIPY-hydrazide (16.3 mol) was conjugated to 120 mg of VS-PEG-PG to yield 150 mg of VS-PEG-PG sodium salt. Approximately 5 dye molecules were attached to each polymer chain, c. Synthesis of Herceptin-PEG-PG-TXL. Herceptin-PEG-PG-BODIPY.
- mAb was mixed with VS-PEG-PG-TXL, VS-PEG-PG-Adr, or VS-PEG-PG-BODIPY with a molar ratio of mAb to polymer of 1:8-1:10.
- the solution was passed through a nickel affinity column (FreeZyme conjugate purification kit, Pierce Chemical Co., Rockford, IL) to remove unreacted polymer, followed by purification with an anion exchange chromatography to remove free mAb from polymer bound mAb.
- the yield of mAb was calculated to be 8-10%.
- the molar ratios of Herceptin to PEG-PG polymer and C225 to PEG-PG were 1 based on the measurements of protein and BODIPY FL concentrations. Using the ratio of Herceptin to PEG-PG of 1, the calculated TXL content in the conjugate was 4.3%. TXL content obtained from hydrolysis/HPLC assay was 6.65%, which suggests a molar ratio of Herceptin to PEG-PG of 1.8. Thus, the molar ratios of mAb to PEG- PG in immunoconjugates varied between 1.0 to 1.8.
- Figure 6 shows the GPC elution profile of Herceptin (A), VS- PEG-PG-TXL (B), and purified Herceptin-PEG-PG-TXL conjugate (C) using a Superdex 200 column (2.4 x 20 cm). The concentrations of the compounds were monitored by RI detector.
- Figure 7 shows the purification of C225-PEG-PG-Adr by FPLC using a Resource Q anion- exchange column. Each fraction was 0.5 ml. From the FPLC elution profile, the fractions (fractions 3-5) corresponding to the first peak were free C225, and the fractions corresponding to the second peak (fractions 14-21) were the desired conjugate (C225-PEG-PG-Adr), which was pooled, concentrated and stored at 4 °C. As confirmed by GPC (system 2) analysis of purified C225-PEG-PG-Adr, the immunoconjugate was free of unconjugated C225 ( Figure 8).
- Figure 8 shows results of gel permeation chromatography of C225 (A), PEG-PG-Adr (B), and purified C225-PEG-PG-Adr conjugate (C) using a Superdex 200 column (1.0 x 30 cm). The compounds were monitored by measuring absorbance at 254 nm. Although C225-PEG-PG-Adr and PEG-PG-Adr was not completely resolved by GPC, the lack of tailing and the absence of a peak at 23.81 minutes in the chromatogram of C225- PEG-PG-Adr suggests that the product was free of unconjugated PEG-PG-Adr.
- the elution curve of VS-PEG-PG- Adr had two peaks with retention times of 15.81 and 23.81 minutes, respectively.
- the first peak at 15.81 minutes in the GPC chromatogram of VS-PEG-PG- Adr appeared at the dead volume of the column, which may be attributed to the formation of polymer aggregates.
- the second peak may be attributed to the soluble form of the PEG-PG-Adr.
- PEG-poly(L-aspartic acid) (PEG-PAA) block copolymer with Adr coupled to the PAA has been shown to form micelles with average diameter of 40-60 nm (M. Yokoyama, et al, Preparation of micelle-forming polymer-drug conjugates. Bioconjugate Chem. 3:295-101, 1992). 2).
- the formation of particles with volume-average diameter of 207 nm was detected by light scattering. The size of the particles was decreased from 207 nm to 16 nm upon conjugation with C225 because of the increase in the hydrophilic segment of the amphiphilic block copolymer (Table 1, Figure 9).
- Adr in PEG-PG-Adr by reducing the molecular weight of PG from 3 IK to 7.7K also resulted in reduction in particle size to 121 nm (Table 1). 3).
- PEG-PG-Adr did not form particles when it was dissolved in DMF.
- a block copolymer 10 (e.g., PEG-PG-Adr) composed of hydrophobic components 12 (e.g., PG-Adr) and hydrophilic components 14 (e.g., PEG) can form a nanoparticle structure 20 as a result of its amphiphilic character.
- hydrophobic components 12 e.g., PG-Adr
- hydrophilic components 14 e.g., PEG
- Attaching C225 to VS-PEG-PG-Adr affected the balance between hydrophilic and hydrophobic segments, resulting in decrease in particle size.
- the present invention describes a method to prepare targetable polymeric nanoparticles.
- Polymeric nanoparticles were obtained from a VS-PEG-PG-Adr copolymer.
- the VS functional groups residing on the surface of the nanoparticles provided a handle to further introduce homing moieties to the surface of the nanoparticles, whereas Adr attached to the core facilitated hydrophobic interactions to stabilize the nanoparticle structure.
- SKOV-3 cells, or human breast cancer MDA-MB-468 cells were grown in DMEM- F12 medium containing 10% fetal bovine serum at 37 °C. a. hnmunoprecipitation and Western Blotting Analysis
- Cell pellets were treated with cold lysis buffer containing lx protease inhibitor cocktails (Sigma, St Louis, MO) on soft ice for 30 minutes, followed by centrifugation to remove cell debris. Each test drug was added into 200 ⁇ l of supernatant in 0.5-ml microcentrifuge tubes. Two microliters of protein A beads (Sigma) were then added into each tube. The microcentrifuge tubes were incubated at 4 °C for 1 hour, centrifuged, and the beads washed 3 times with 0.5 ml lysis buffer.
- the beads were heated at 95 °C in 20 ⁇ l of lx SDS-PAGE laemmli sample buffer (Bio-Rad, Hercules, CA) for 5 minutes, centrifuged, and analyzed by 7% SDS polyacrylamide gel electrophoresis (PAGE).
- Western blot was carried out by electronically transferring the samples into a nitrocellulose membrane and incubation of the membrane for 1 hour with an anti-EGF receptor antibody (Sigma) or anti- Her2/neu receptor antibody (Oncogen, Boston, MA).
- the receptor signals in the membrane were developed by the ECL chemoluminescence detection kit (Amersham Pharmacia Biotech Inc., Piscataway, NJ).
- MDA-MB-468 cells that do not express Her2/neu receptors were used as negative control when studying the binding of Her- PEG-PG-BODIPY to Her2/neu receptors.
- Cells were washed three times with PBS, fixed in 95% ethanol, and then treated with 1 ⁇ M TO-PRO-3 Iodide (Molecular Probes, Eugene, OR) for 15 minutes for nuclei staining. Fluorescent images of cells were analyzed using LMS-510 confocal microscopy (Zeiss, Thornwood, NY).
- One hundred microliter of growth medium suspending 1000-2000 cells per well was plated out in 96-well plates and incubated for 2 days to allow the cells to attach.
- Various dilutions of the drug or conjugates were added to each well and the plates were incubated for 72 hours at 37 °C.
- a 6-hour pretreatment protocol was used.
- Cells were exposed to various concentrations of the drug or conjugate for 6 hours at 37 °C, and then washed twice with the fresh culture medium. The cells were incubated for additional 72 hours. At the end of the incubation period, twenty microliters of MTT solution from Promega Cell Proliferation Assay kit (Madison, WI) were added to the wells.
- microplates were then incubated for 1 hour at 37 °C. Absorbance was measured at 490 nm using a microplate reader (Molecular Devices Corp, Sunnyvale, CA). The data reported represent the means of quadruplicate measurement and the standard errors of the mean were less than 15%. The IC50, concentration exhibiting 50% growth inhibition were calculated from the growth-inhibition curve.
- the first pair of cell lines included SKOV3ipl, a human ovarian cancer cell variant that overexpress Her2/neu, and MDA-MB-468, which does not express Her2/neu receptors. Results are shown in Figures 11 A and 1 IB.
- the graph in Figure 11A represents the MDA-MB-468 cells, and the graph in Figure 11B represents the SKOV3ipl cells.
- the y axis in each graph represents the % viability and the x axis represents the dose in nM.
- the data for PEG-PGT is represented by squares.
- the data for Her-PEG-PGT is represented by triangles.
- the second pair included MDA-MB-435 transfected with neo only (MDA435/neo), which does not express the receptor, and the stable Her2/neu transfectant MDA-MB435/eB2 (MDA 435/eB2).
- Results are shown in Figures 12A and 12B.
- the graph in Figure 12A represents the MDA 435/neo cells; the graph in Figure 12B represents MDA 435/eB2 cells.
- the y axis in each graph represents the % viability and the x axis represents the dose in nM.
- the data for PEG-PGT is represented by diamonds.
- the data for Her-PEG-PGT is represented by squares.
- IC 50 values thus obtained were used to calculate targeting index, defined as the ratio of IC50 values obtained with no-mAb drug conjugate PEG-PG-TXL in target cells and in non-target cells, times the ratio of IC 50 values obtained with mAb- conjugated PEG-PG-TXL in non-target cells and in target cells.
- the targeting index for the first pair and second pair of cell lines were 3.95 and 1.75, respectively. Since TXL is releasable from the immunoconjugates, one would expect that after 72 hours of incubation, a fraction of free TXL released from the conjugates could also contribute to the cytotoxic effect, resulting in reduced targeting index or selective cytotoxicity.
- the y axis represents the % viability and the x axis represents the concentration in ⁇ g/ml.
- the homing ligand may also be introduced to the end of a PEG-PG block copolymer that contains a sulfhydryl group.
- the ligand is pretreated with ⁇ -maleimidobutyric acid N-hydroxysuccinimide ester (GMBS) to introduce thio-reactive maleimide groups to the ligand.
- GMBS ⁇ -maleimidobutyric acid N-hydroxysuccinimide ester
- DTT dithiothreitol
- compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation iri light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention. Not all embodiments of the invention will include all the specified advantages. The specification and examples should be considered exemplary only with the true scope and spirit of the invention indicated by the following claims.
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PCT/US2002/012502 WO2002087497A2 (en) | 2001-04-26 | 2002-04-19 | Therapeutic agent/ligand conjugate compositions, their methods of synthesis and use |
EP02728872A EP1610751A4 (en) | 2001-04-26 | 2002-04-19 | Therapeutic agent/ligand conjugate compositions, their methods of synthesis and use |
CA002444383A CA2444383A1 (en) | 2001-04-26 | 2002-04-19 | Therapeutic agent/ligand conjugate compositions, their methods of synthesis and use |
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US34314701P | 2001-12-20 | 2001-12-20 | |
US60/343,147 | 2001-12-20 | ||
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