WO2007097934A2 - Methods and compositions for using erythrocytes as carriers for delivery of drugs - Google Patents

Methods and compositions for using erythrocytes as carriers for delivery of drugs Download PDF

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Publication number
WO2007097934A2
WO2007097934A2 PCT/US2007/003636 US2007003636W WO2007097934A2 WO 2007097934 A2 WO2007097934 A2 WO 2007097934A2 US 2007003636 W US2007003636 W US 2007003636W WO 2007097934 A2 WO2007097934 A2 WO 2007097934A2
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WIPO (PCT)
Prior art keywords
monoclonal antibody
rbc
rbc binding
fragment
binding conjugate
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PCT/US2007/003636
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French (fr)
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WO2007097934A3 (en
Inventor
Elizabeth G. Posillico
George L. Spitalny
Steven E. Pincus
Lihsyng S. Lee
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Elusys Therapeutics, Inc.
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Publication of WO2007097934A2 publication Critical patent/WO2007097934A2/en
Publication of WO2007097934A3 publication Critical patent/WO2007097934A3/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/49—Urokinase; Tissue plasminogen activator
    • 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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6815—Enzymes
    • 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
    • A61K47/6835—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/035—Fusion polypeptide containing a localisation/targetting motif containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • the invention relates to methods and compositions for using the erythrocyte, or red blood cell (RBC) as a carrier for delivery of one or more biologically active molecules, e.g., enzymes or mammalian serum proteins.
  • RBC red blood cell
  • the invention relates to methods of using the RBC binding conjugate for preventing and treating diseases.
  • the invention also relates to methods of preparing the RBC binding conjugate.
  • erythrocytes or red blood cells (RBCs)
  • RBCs red blood cells
  • the formation of an immune complex in the circulatory system activates the complement factor C3b in primates and leads to the binding of C3b to the immune complex.
  • the C3b/immune complex then binds to the type 1 complement receptor (CRl), a C3b receptor, expressed on the surface of erythrocytes via the C3b molecule attached to the immune complex.
  • the immune complex is then chaperoned by the erythrocyte to the reticuloendothelial system (RES) in the liver and spleen for neutralization.
  • RES reticuloendothelial system
  • the RES cells most notably the fixed-tissue macrophages in the liver called Kupffer cells, recognize the C3b/immune complex and break this complex from the RBC by severing the C3b receptor-RBC junction, producing a liberated erythrocyte and a C3b/immune complex which is then engulfed by the Kupffer cells and is completely destroyed within subcellular organelles of the Kupffer cells.
  • Taylor et al. discloses a method of utilizing the RBCs for removing pathogens from the circulatory system (U.S. Patent Nos. 5,487,890; 5,470,570; and 5,879,679). Taylor et al. have shown that chemical crosslinking of a first monoclonal antibody (mAb) specific to a primate C3b receptor to a second monoclonal antibody specific to a pathogenic antigenic molecule creates a bispecific heteropolymeric antibody (HP) which offers a mechanism for binding a pathogenic antigenic molecule to a primate's C3b receptor without complement activation. A HP that can be used to remove a pathogenic antigen specific autoantibody from the circulation is also reported.
  • mAb monoclonal antibody
  • HP bispecific heteropolymeric antibody
  • Such a HP also referred to as an "Antigen-based Heteropolymer” (AHP) contains a CRl specific monoclonal antibody covalently-conjugated to an antigen (see, e.g., U.S. Patent No. 5,879,679; Lindorfer, et al., 2001, Immunol i?ev.l83: 10-24; Lindorfer, et al., 2001, J Immunol Methods 24%: 125-138; Ferguson, et . al., 1995, Arthritis Rheum 38: 190-200).
  • AHP Antigen-based Heteropolymer
  • bispecific molecules that have a first antigen recognition domain which binds a C3b-like receptor, e.g., a complement receptor 1 (CRl), and a second antigen recognition domain which binds an antigen can also be produced by methods that do not involve chemical cross-linking (see, e.g., PCT publication WO 02/46208; and PCT publication WO 01/80883).
  • PCT publication WO 01/80833 describes bispecific antibodies produced by methods involving fusion of hybridoma cell lines, recombinant techniques, and in vitro reconstitution of heavy and light chains obtained from appropriate monoclonal antibodies.
  • PCT publication WO 02/46208 describes bispecific molecules produced by protein trans-splicing.
  • PCT publication WO 2004/024889 describes bispecific molecules comprising a polyethylene glycol aldehyde/hydrazide linkage.
  • a variety of diseases may be treated by therapeutical agents that are biologically or chemically active in the blood. It is often desirable that such agents remain in the blood for a sufficiently long period of time without degradation and/or loss due to uptake by other tissues. For certain agents, it may also be desirable to limit their tissue uptake to eliminate or reduce harmful side effects. Attaching such agents to red blood cells offers an attractive means to achieve these goals.
  • many disease conditions are a result of the clogging of blood vessels by intravascular clots, including myocardial infarction, disseminated intravascular coagulation, stroke and pulmonary embolism, as well as less acute conditions, such as deep venous thrombosis and peripheral vascular diseases, e.g., atherosclerosis, Raynaud's disease, and ischaemias.
  • Pulmonary thromboembolism a condition of high mortality, is often a result of deep venous thrombosis, as pulmonary emboli often result from thrombi in the deep venous system.
  • Anticoagulant or antithrombotic agents such as heparin, dicumarol, antithrombin concentrates and hirudin
  • Fibrinolytics such as streptokinase, staphylokinase, tissue-type plasminogen activator or tPA, and urokinase
  • These agents act to dissolve intravascular clots by activating plasmin, a protease that digests fibrin.
  • Plasminogen the inactive precursor of plasmin, is converted to plasmin by cleavage of a single peptide bond. Plasmin itself is a nonspecific protease that digests fibrin clots as well as other plasma proteins, including several coagulation factors.
  • fibrinolytic agents to dissolve clots formed in other vascular areas such as deep venous areas has been less successful due partly to the rapid elimination and inactivation of the fibrinolytics agents (Plow, et al., 1995, FASEB J. 9:939-945; Narita, et al. 1995, J. Clin. Invest. 96:1164-1168).
  • tPA and urokinase undergo rapid inactivation by a circulating plasminogen activator inhibitor.
  • Plasmin itself is inactivated by a circulating glycoprotein, ⁇ -2-antiplasmin (Co lien, 1996, Circulation 93:857-865; Reilly, et al, 1991, Arterioscl. Thromb.
  • ⁇ -2-antiplasmin also inactivates staphylokinase (Collen, et al., 1993, Eur. J. Biochem. 216:307-314).
  • other inhibitors of plasminogen activators e.g., Cl-inhibitor, ⁇ 2-macroglobulin, and antitrypsin may also contribute to the decrease in the fibrinolytic response upon treatment with plasminogen activators (Collen, 1996, Circulation 93:857-865).
  • Such inactivation or degradation of plasminogen activators and plasmin reduces the effectiveness of thrombolytic therapy.
  • U.S. Patent No. 6,488,927 and U.S. Patent Publication 2002/0099000 disclose compositions and methods for prevention and treatment of deep vein thrombosis, pulmonary embolism and other diseases or syndromes resulted from uncontrolled formation of intravascular fibrin clots using a composition that comprises a drug, such as a fibrinolytic or anticoagulant drug, e.g., a plasminogen activator, biocompatibly coupled to the red blood cells.
  • a drug such as a fibrinolytic or anticoagulant drug, e.g., a plasminogen activator
  • the red blood cells serve as carriers of the drug, allowing prolonged circulation and restricted tissue uptake.
  • SA/b-RBC streptavidin
  • U.S. Patent No. 5,840,733 teaches compounds comprising chemically reactive intermediates which can react with available reactive functionalities on blood components to form covalent linkages, where the resulting covalently-bound conjugates are found to have thrombin inhibition activity.
  • the thrombin inhibitor compounds of the '733 patent are derivatives of the known thrombin inhibitor argatroban, which can be covalently linked to chemically reactive functionalities on various blood components.
  • the '733 patent also teaches methods for inhibiting thrombin activity in vivo comprising administering to the bloodstream of a mammalian host the disclosed compounds.
  • U.S. Patent No. 5,843,440 teaches bifunctional reagents useful in reducing the biological effect of an undesirable blood-borne agent.
  • the reagents comprise conjugates of a first binding member specific for a blood-borne agent having a detrimental biological activity in a mammalian host, such as a growth factor, coagulation factor, enzyme, toxin, drug of abuse, microbe, autoreactive immune cell, infected or tumorous cell, joined to an second binding member specific for an anchor, where the anchor is a long-lived blood component, including cells, such as a erythrocyte, platelet or endothelial cell and serum proteins, such as albumin, ferritin, or steroid binding proteins.
  • the ⁇ 440 patent teaches therapeutic uses of the conjugates for coupling blood born agent to the blood component so as to reduce the biological activity or effective concentration of the agent, modulate the volume of distribution of the agent, target the agent to sites of enhanced immune response, or facilitate agent clearance from the bloodstream.
  • PEG Polyethylene glycol
  • PEG linkers are non-immunogenic.
  • the hydroxyl end groups of PEG molecules are first converted into reactive functional groups to generate activated PEG.
  • PEG molecules can be covalently attached to various groups on the surface of a protein. For example, PEG can be attached to amino groups using PEG-succinimide derivatives (see, e.g., U.S. Patent No. 4,179,337).
  • PEG can also be attached to sulfhydryl groups using PEG-maleimide derivatives (see, U.S. Patent No. 4,179,337).
  • the amino groups of many proteins are often associated with moieties responsible for the biological ⁇ activity of the proteins. Modification of amino groups may render these proteins biologically inactive.
  • sulfhydryl groups are also often associated moieties having biological or enzymatic activities, and are not readily available for modification.
  • U.S. Patent No. 4,847,325 discloses covalently attaching PEG to CSF-I by reacting PEG-amine, PEG- hydrazine or PEG-hydrazide with CSF-I that had been oxidized with periodate to convert vicinal diols in, the sugars to aldehydes.
  • PCT publication WO92/16555 discloses a method of producing a biologically active macromolecular conjugate comprising a biologically active polypeptide or glycopolypeptide covalently attached to one or more PEG molecules at a reactive carbonyl or carboxylic acid group of a peptide moiety on the polypeptide or glycopolypeptide by a linkage containing a hydrazide or hydrazone functional group.
  • the linkage is formed by reacting an acyl hydrazine derivative of the PEG with a polypeptide or glycopolypeptide having an activated carboxylic acid group or a reactive carbonyl group.
  • Hydrazides readily form relatively stable hydrazone linkages by condensation with aldehydes and ketones (Andresz, et al., 1978, Makromol. Chem. 179:301). This property has also been used for modification of glycoproteins through oxidized oligosaccharide moieties (Wilchek et al., 1987, Meth. Enzymol. 138:429).
  • Activated PEG-hydrazide reacts with an aldehyde group, which is normally absent on the polypeptide chain of a protein, but can be generated in a protein containing carbohydrate moieties by oxidation of the sugar ring. Methods for activation of immunoconjugates are also described in SeIa et al., 1987, Immuno conjugates, Vogel ed., Oxford University Press.
  • U.S. Patent No. 6,743,908 discloses a method of generating polypeptides having introduced glycosylation sites. This patent also discloses site-specific attachment of PEGs to such inserted glycosylation sites.
  • the invention provides a red blood cell (RBC) binding conjugate comprising a monoclonal antibody or a fragment thereof covalently conjugated to a mammalian serum protein or to an enzyme, wherein the monoclonal antibody or fragment thereof (i) binds a C3b-like receptor on a red blood cell, and (ii) lacks a functional effector domain.
  • the monoclonal antibody can be a murine monoclonal antibody, a humanized monoclonal antibody, or a human monoclonal antibody.
  • the monoclonal antibody or fragment binds a CRl receptor on a red blood cell.
  • the monoclonal antibody can be selected from the group consisting of anti- CRl antibodies H4, H9, H47, H48, 7G9, HB8592, 3D9, 57F, and 1B4.
  • the monoclonal antibody or fragment thereof is selected from the group consisting of an Fab, an Fab', an (Fab ⁇ , and an Fv fragment of an immunoglobulin molecule that binds the C3b-like receptor.
  • the monoclonal antibody or fragment thereof comprises a monoclonal antibody in which the effector domain is inactivated.
  • the effector domain of the monoclonal antibody comprises one or more mutations such that the effector domain loses its effector function.
  • any suitable mammalian serum protein or enzyme can be conjugated to the monoclonal antibody or a fragment to generate the RBC binding conjugate of the present invention.
  • the mammalian serum protein is a human serum protein.
  • the mammalian serum protein comprises a mammalian serum enzyme or a functional fragment thereof.
  • the mammalian serum protein is selected from the group consisting of a tissue-type plasminogen activator, a receptor of a tissue-type plasminogen activator, a streptokinase, a staphylokinase, a urokinase, and Factor VOX
  • the invention also provides a method for treating associated with the formation of clots in its circulation, comprising the step of administering to the mammal a therapeutically effective amount of a RBC binding conjugate which contains such a mammalian serum protein.
  • the mammalian serum protein is ⁇ -glucocerebrosidase.
  • the invention also provides a method of treating a patient having Gaucher disease, comprising the step of administering to the patient a therapeutically effective amount of the RBC binding conjugate in which the mammalian serum protein is ⁇ - glucocerebrosidase.
  • the mammalian serum protein is ⁇ -galactosidase A.
  • the invention also provides a method of treating a mammal having Fabry disease, comprising the step of administering to the patient a therapeutically effective amount of the RBC binding conjugate in which the mammalian serum protein is ⁇ -galactosidase A.
  • the mammalian serum protein is a cytokine.
  • the cytokine can be selected from the group consisting of IFN- ⁇ , IFN- ⁇ , EFN- ⁇ , JL-2, IL-3, IL-4, IL-5, IL-6, BL-7, IL-8, IL-9, IL-IO, IL-12 and IL-15.
  • the invention also provides a method of treating a mammal having cancer or a bacterial or viral infection, comprising the step of administering to the mammal a therapeutically effective amount of the RBC binding conjugate in which the mammalian serum protein is such a cytokine.
  • the mammalian serum protein is a peptide hormone.
  • the peptide hormone can be selected from the group consisting of antimullerian hormone (AMH), adiponectin, adrenocorticotropic hormone (ACTH), angiotensinogen and angiotensin, antidiuretic hormone (ADH), atrial-natriuretic peptide (ANP), calcitonin, cholecystokinin (CCK), corticotropin-releasing hormone (CRH), erythropoietin (EPO), follicle stimulating hormone (FSH), gastrin, glucagon, gonadotropin-releasing hormone (GnRH), growth hormone-releasing hormone (GHRH), human chorionic gonadotropin (hCG), growth hormone (GH), insulin, insulin-like growth factor (IGF), leptin, luteinizing hormone (LH), melanocyte stimulating hormone (MSH or ⁇ -MSH), neuropeptide Y, oxytoc
  • the enzyme is selected from the group consisting of L- asparagine, L-glutaminase-L-asparaginase, L-methioninase, L-phenylalan ⁇ ne ammonialyase, L-arginase, L-tyrosinase, L-serine dehydratase, L-threonine deaminase, indolyl-3-alkane hydroxylase, neuraminidase, ribonuclease, a protease, pepsin, and a carboxypeptidase.
  • the invention provides a method of treating a mammal having a cancer, comprising the step of administering to the mammal a therapeutically effective amount of the RBC binding conjugate which contains such an enzyme.
  • the enzyme is lysostaphin.
  • the invention also provides a method of treating a mammal having a bacterial infection, comprising the step of administering to the mammal a therapeutically effective amount of the RBC binding conjugate in which the enzyme is lysostaphin.
  • the mammalian serum protein or the enzyme is covalently conjugated at a selected residue to the monoclonal antibody or fragment thereof.
  • the selected residue is selected from the group consisting of a cysteine residue, a residue comprising a reactive carbonyl or carboxylic acid group when oxidized, and a lysine residue.
  • the RBC binding conjugate preserves at least 5%, 15%, 25%, 50%, 90%, or 99% of the biological activity of the mammalian serum protein or the enzyme when unconjugated.
  • the RBC binding conjugate further comprises a polymer linker, wherein the mammalian serum protein or the enzyme is covalently conjugated to the monoclonal antibody or fragment thereof via the polymer linker.
  • the polymer linker can be selected from the group consisting of polypeptide, polyalkylene oxide, polyoxyethylenated polyol, polyacrylamide, polyvinyl pyrrolidone, polyvinyl alcohol, and dextran.
  • the polymer linker is a polyethylene glycol (PEG) linker.
  • the polymer linker is a PEG hydrazide/aldehyde linker.
  • the RBC binding conjugate is a single polypeptide comprising the monoclonal antibody or fragment thereof fused to the mammalian serum protein or the enzyme.
  • the invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the RBC binding conjugate of the invention and a pharmaceutically acceptable carrier.
  • RBC binding conjugate in the pharmaceutical composition is purified.
  • the invention also provides a method of producing a red blood cell (RBC) binding conjugate.
  • RBC red blood cell
  • the method comprises contacting a monoclonal antibody or fragment thereof with a mammalian serum protein or with an enzyme, wherein the monoclonal antibody or fragment binds a C3b-like receptor on a red blood cell, wherein the monoclonal antibody or fragment or the mammalian serum protein or enzyme is derivatized with a bifunctional polymer linker such that the monoclonal antibody or fragment thereof or the mammalian serum protein or enzyme comprises a reactive hydrazide group, and wherein (i) the mammalian serum protein or enzyme, if the monoclonal antibody or fragment is derivatized, or (ii) the monoclonal antibody or fragment thereof, if the mammalian serum protein or enzyme is derivatized, comprises a reactive carbonyl or carboxylic acid group, under conditions conducive for reaction between the reactive hydrazide group and the reactive carbonyl or carboxylic acid group, thereby producing the RBC binding conjugate.
  • the reactive hydrazide group and the reactive carbonyl or carboxylic acid group is attached to an aromatic ring.
  • the polymer is polyethylene glycol (PEG).
  • the bifunctional polymer linker is N-hydroxy-succinimidyl polyethylene glycol-benzaldehyde.
  • the reactive hydrozide group is introduced to the monoclonal antibody or fragment thereof or the mammalian serum protein or enzyme by derivatizing the monoclonal antibody or fragment thereof or the mammalian serum protein with succinimidyl C6 4-hydrazino-nictoinamde acetone hydrazone.
  • the method further comprises producing the derivatized monoclonal antibody or fragment thereof or the derivatized mammalian serum protein or enzyme.
  • the invention also provides a red blood cell (RBC)ZRBC binding conjugate that consists essentially of a red blood cell bound to one or more RBC binding conjugate of the invention.
  • the invention also provides a red blood cell (RBC) binding conjugate comprising a monoclonal antibody or a fragment thereof covalently conjugated to a hormone selected from the group consisting of an amine-derived hormone, a steroid hormone, or sterol hormone, wherein the monoclonal antibody or fragment thereof (i) binds a C3b-like receptor on a red blood cell, and (ii) lacks a functional effector domain.
  • the amine-derived hormone can be any one. from the group consisting of catecholamine, epinephrine, dopamine, norepinephrine, melatonin, serotonin, thyroxine and triiodothyronine.
  • the steroid hormone can be any one from the group consisting of glucocorticoid, mineralocorticoid, androgen, estrogen, and progestagen.
  • the androgen can be any one from the group consisting of testosterone, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), androstenedione, and dihydrotestosterone (DHT).
  • DHEA dehydroepiandrosterone
  • DHEAS dehydroepiandrosterone sulfate
  • DHT dihydrotestosterone
  • the sterol hormone can be a Vitamin D derivative.
  • the invention also provides a red blood cell (RBC) binding conjugate comprising a monoclonal antibody or a fragment thereof covalently conjugated to a molecule selected from the group consisting of a DNA damaging agent, an antimetabolite, and an anti-mitotic agent, wherein the monoclonal antibody or fragment thereof (i) binds a C3b-like receptor on a red blood cell, and (ii) lacks a functional effector domain.
  • RBC red blood cell
  • the DNA damaging agent is selected from the group consisting of camptothecin, topotecan, doxorubicin, etoposide phosphate, teniposide, sobuzoxane, anthracycline antibiotic, mitomycin antibiotic, cisplatin, busulfan, cyclophosphamide, bleomycin, and tamoxifen.
  • the anti-metabolite is selected from the group consisting of cytosine, arabinoside, fioxuridine, 5-fluorouracil (5-FU), mercaptopurine, gemcitabine, hydroxyurea (HU), and methotrexate (MTX).
  • the anti-mitotic agent is selected from the group consisting of vinblastine, vincristine, and paclitaxel (Taxol).
  • FIG. 1 Conjugate of tPA and 7G9 using PEG(5K)-aldehyde-hydrazino linker. The sample was analyzed with SDS-PAGE with a 3-8% Tris-acetate gradient gel.
  • the present invention provides methods and compositions for using erythrocytes (red blood cells, RBCs) as carriers for delivery of therapeutic molecules, e.g., therapeutic enzymes or mammalian serum proteins, or diagnostic agents.
  • RBCs red blood cells
  • the invention involves conjugating the drug or the diagnostic agent to an antigen recognition portion that binds RBC, e.g., anti-CRl antibody or anti-glycophorin A antibody.
  • an antigen recognition portion that binds RBC, e.g., anti-CRl antibody or anti-glycophorin A antibody.
  • the conjugate When administered to a patient, the conjugate binds to RBC, and is delivered to desired locations by the RBC.
  • the RBC binding portion and the enzymatic portion in the RBC binding conjugate are linked by a polymer/aldehyde/hydrazide linkage.
  • the polymer/aldehyde/hydrazide linker preferably comprises a water soluble, nonimmunogenic polymer.
  • the polymer is a polyethylene glycol (PEG).
  • the RBC binding conjugate can be produced using a bifunctional linker molecule that comprises either a hydrazide functional group or an aldehyde functional group at one end.
  • the functional group at the other end can be any functional group that can attach to a protein, such as an N-hydroxysuccinimide (NHS).
  • NHS N-hydroxysuccinimide
  • the hydrazide or aldehyde has an aromatic ring attached next to it.
  • the linker molecule is attached to the RBC binding portion or the enzymatic portion via the second functional group.
  • the hydrazide functional group or aldehyde functional group is then allowed to react with an appropriate counterpart portion to form a hydrazide/aldehyde linkage.
  • the RBC binding conjugates of the present invention can be used as a means for delivery of many types of therapeutic molecules.
  • the RBC binding conjugate is used to deliver an anticoagulant or thrombolytic, e.g., a streptokinase, staphylokinase, tissue-type plasminogen activator or tPA, or urokinase, for treating or preventing disease conditions associated with the formation of clots in the blood of a subject.
  • an anticoagulant or thrombolytic e.g., a streptokinase, staphylokinase, tissue-type plasminogen activator or tPA, or urokinase
  • the RBC binding conjugates of the present invention are used to deliver enzymes in an enzyme replacement therapy, e.g., ⁇ -glucocerebrosidase or ⁇ -galactosidase A, for treating disease conditions associated with enzyme deficiency in a subject, e.g., various metabolic diseases, such as Gaucher disease and Fabry disease.
  • an enzyme replacement therapy e.g., ⁇ -glucocerebrosidase or ⁇ -galactosidase A
  • disease conditions associated with enzyme deficiency in a subject e.g., various metabolic diseases, such as Gaucher disease and Fabry disease.
  • the RBC binding conjugates of the present invention are used to deliver an anti-cancer agent, e.g., L-asparaginase, for treating or preventing cancers in a subject.
  • an anti-cancer agent e.g., L-asparaginase
  • the RBC binding conjugates of the present invention are used to deliver an anti-infectious agent, e.g., lysostaphin, for treating or preventing infectious diseases in a subject, e.g., various bacterial or viral infections.
  • an anti-infectious agent e.g., lysostaphin
  • the RBC binding conjugates of the present invention are used to deliver an antidote, e.g., carboxypeptidase Gl for treating an overdose of certain substance in a subject, e.g., an overdose of methotrexate.
  • an antidote e.g., carboxypeptidase Gl
  • an overdose of certain substance in a subject e.g., an overdose of methotrexate.
  • the RBC binding conjugate of the present invention is a molecule comprising a RBC binding portion that binds a red blood cell, e.g., via a receptor on the red blood cells, and a drug portion, e.g., an biologically active enzymatic portion.
  • the red blood cell membrane possesses a large number of surface antigens.
  • the more abundant human erythrocyte-specific antigens include but not limited to KeIl glycoprotein, Rh glycoprotein, Landsteiner Wiener (LW) glycoprotein, glycophorin A, Band 3, Lutheran glycoprotein, and Duffy (Fy) glycoprotein (see, e.g., Southcott et al., 1999, Blood 93 :4425-35).
  • the RBC binding portion may bind to any suitable blood cell surface antigen.
  • the RBC binding portion binds a CRl receptor.
  • the RBC binding portion binds glycophorin A.
  • C3b-like receptor refers to any mammalian circulatory molecule expressed on the surface of a mammalian blood cell, which has an analogous function to a primate C3b receptor, the CRl, in that it binds to a molecule associated with an immune complex, which is then chaperoned by the blood cell to, e.g., a phagocytic cell for clearance.
  • Glycophorin A is a glycoprotein that spans the plasma membrane of human red blood cell. Glycophorin A is a 131 amino acid protein that spans the membrane once and presents its amino-terminal end at the extra-cellular surface of the red blood cell. Each RBC has some 500,000 copies of the molecule embedded in its plasma membrane.
  • Rh glycoprotein is a hereogenously glycosylated polypeptide that is associated with the Rh polypeptides in the membrane of the red blood cells.
  • the LW antigen is a single spanning glycoprotein.
  • the anti-CRl monoclonal antibody is H4, H9, H47, H48, 7G9, HB8592, 3D9, 57F, or 1B4 (see, e.g., Talyor et al, U.S. Patent No. 5,487,890, which is incorporated herein by reference in its entirety) with the effector domain partially or entirely removed.
  • the RBC binding portion portion is an anti-CRl antibody, including but is not limited to, an Fab, an Fab', an (Fab')2, or an Fv fragment of an immunoglobulin molecule, or a single-chain variable region fragment (scFv) with specificity for a C3b-like receptor.
  • the RBC binding portion can also be a chimeric antibody, such as but is not limited to a humanized monoclonal antibody in which the complementarity determining regions are mouse, and the framework regions are human thereby decreasing the likelihood of an immune response in human patients treated with the antibody (United States Patent Nos. 4,816,567, 4,816,397, 5,693,762; 5,585,089; 5,565,332 and 5,821,337, each of which is incorporated herein by reference in its entirety). Although, for simplicity, this disclosure often makes reference to an RBC binding portion that binds a C3b-like receptor, it will be understood by a skilled artisan that the disclosure is equally applicable to antibodies that binds other red blood cell receptors.
  • RBC binding conjugates can also be attached to a red blood cell via antigens other than a C3b-like receptor.
  • Antibodies that bind other RBC-specir ⁇ c antigens, e.g., glycophorin A, are well-known in the art (see, e.g., Southcott et al., 1999, Blood 93:4425-35, which is incorporated herein in its entirety).
  • the drug portion comprises a polypeptide or glycopolypeptide that is biologically active.
  • the drug portion comprises an enzyme.
  • the enzyme can be, but is not limited to, an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase or a ligase, or any analogue thereof.
  • the enzyme can be but is not limited to asparaginase, arginase, arginine deaminase, adenosine deaminase, superoxide dismutase, catalase, chymotrypsin, lipase, uricase, bilirubin oxidase, glucose oxidase, glucosidase, galactosidase, ghicocerebrosidase, or glucuronidase, or any analogue thereof having enzymatic activity.
  • the drug portion can also comprise a protein selected from among, but not limited to, the following Factor VIH and polypeptide hormones such as insulin, ACTH, glucagon, somatostatin, somatotropins, thymosin, parathyroid hormone, pigmentary hormones, somatomedins, erythropoietin, luteinizing hormone, hypothamic releasing factors, antidiuretic hormones, interleukins, interferons, colony stimulating factors, hemoglobin, prolactin, or anti-angiogenic agents, e.g., angiostatin or endostatin, or any biologically active analogue thereof.
  • Factor VIH and polypeptide hormones such as insulin, ACTH, glucagon, somatostatin, somatotropins, thymosin, parathyroid hormone, pigmentary hormones, somatomedins, erythropoietin, luteinizing hormone, hypothamic releasing factors, antid
  • the drug portion can also comprise a glycopolypeptide such as, but are not limited to, immunoglobulins, chorionic gonadotrophin, follicle-stimulating hormone, thyroid-stimulating hormone, ovalbumin, bovine serum albumin (BSA), lectins, tissue plasminogen activator, glycosylated interleukins, glycosilated interferons or glycosilated colony stimulating factors, or any analogue thereof.
  • the drug portion can also comprise an allergen protein or glycoprotein for use as tolerance inducer in reducing allergenicity of such proteins (see, e.g., Sehon et al., 1987, Pharmacol. Toxicol. Proteins 65:205-19).
  • the drug portion can also comprise a small molecule drug, including but not limited to DNA damaging agents, anti-metabolites, anti-mitotic agents.
  • DNA damaging agents include but are not limited to topoisomerase inhibitor, DNA binding agent.
  • a topoisomerase inhibitor that can be used in conjunction with the invention can be a topoisomerase I (Topo I) inhibitor, a topoisomerase II (Topo II) inhibitor, or a dual topoisomerase I and II inhibitor.
  • a topo I inhibitor can be for example from any of the following classes of compounds: camptothecin analogue (e.g., karenitecin, aminocamptothecin, lurtotecan, topotecan, irinotecan, BAY 56-3722, rubitecan, GI14721, exatecan mesylate), rebeccamycin analogue, PNU 166148, rebeccamycin, TAS-103, camptothecin (e.g., camptothecin polyglutamate, camptothecin sodium), intoplicine, ecteinascidin 743, J- 107088, pibenzimol, camptothecin, topotecan (hycaptamine), irinotecan (irinotecan hydrochloride), belotecan, or an analogue or derivative of any of the foregoing.
  • camptothecin analogue e.g., karenitecin, aminocamptothecin
  • Topo ⁇ inhibitors include but not limited to anthracycline antibiotics (e.g., carubicin, pirarubicin, daunorubicin citrate liposomal, daunomycin, 4-iodo-4- doxydoxorubicin, doxorubicin, n,n-dibenzyl daunomycin, morpholinodoxorubicin, aclacinomycin antibiotics, duborimycin, menogaril, nogalamycin, zorubicin, epirubicin, marcellomycin, detorubicin, annamycin, 7-cyanoquinocarcinol, deoxydoxorubicin, idarubicin, GPX-100, MEN-10755, valrubicin, KRN55OO), epipodophyllotoxin compound (e.g., podophyllin, teniposide, etoposide, GL331, 2-ethylhydrazide), anthraquinone compound
  • DNA binding agents include but are not limited to a DNA groove binding agent, e.g., DNA minor groove binding agent; DNA cross ⁇ nking agent; intercalating agent; and DNA adduct forming agent.
  • a DNA minor groove binding agent can be an anthracycline antibiotic, mitomycin antibiotic (e.g., porf ⁇ romycin, KW-2149, mitomycin B, mitomycin A, mitomycin C), chromomycin A3, carzelesin, actinomycin antibiotic (e.g., cactinomycin, dactinomycin, actinomycin Fl), brostallicin, echinomycin, bizelesin, duocarmycin antibiotic (e.g., KW 2189), adozelesin, olivomycin antibiotic, plicamycin, zinostatin, distamycin, MS-247, ecteinascidin 743, amsacrine, antbxamycin, and pibenzimol, or an analogue or derivative
  • DNA crosslinking agents include but are not limited to antineoplastic alkylating agent, methoxsalen, mitomycin antibiotic, psoralen.
  • An antineoplastic alkylating agent can be a nitrosourea compound (e.g., cystemustine, tauromustine, semustine, PCNU, streptozocin, SarCNU, CGP-6809, carmustine, fotemustine, methylnitrosourea, nimustine, ranimustine, ethylnitrosourea, lomustine, chlorozotocin), mustard agent (e.g., nitrogen mustard compound, such as spiromustine, trofosfamide, chlorambucil, estramustine, 2,2,2-trichlorotriethylamine, prednimustine, novembichin, phenamet, glufosfamide, peptichemio, ifosfamide, defosfamide, nitrogen mustard, phen
  • Intercalating agents include but are not limited to an anthraquinone compound, bleomycin antibiotic, rebeccamycin analogue, acridine, acridine carboxamide, amonafide, rebeccamycin, anthrapyrazole antibiotic, echinomycin, psoralen, LU 79553, BW A773U, crisnatol mesylate, benzo(a)pyrene-7,8-diol-9,10-epoxide, acodazole, elliptinium, pixantrone, or an analogue or derivative of any of the foregoing.
  • DNA adduct forming agents include but are not limited to enediyne antitumor antibiotic (e.g., dynemicin A, esperamicin Al, zinostatin, dynemicin, calicheamicin gamma II), platinum compound, carmustine, tamoxifen (e.g., 4-hydroxy-tamoxifen), psoralen, pyrazine diazohydroxide, benzo(a)pyrene-7,8-diol-9,10-epoxide, or an analogue or derivative of any of the foregoing.
  • enediyne antitumor antibiotic e.g., dynemicin A, esperamicin Al, zinostatin, dynemicin, calicheamicin gamma II
  • platinum compound e.g., dynemicin A, esperamicin Al, zinostatin, dynemicin, ca
  • Anti-metabolites include but are not limited to cytosine, arabinoside, floxuridine, 5-fluorouracil (5-FU), mercaptopurine, gemcitabine, hydroxyurea (HU), and methotrexate (MTX).
  • Anti-mitotic agents include but are not limited to Vinblastine, Vincristine, and Paclitaxel (Taxol).
  • the drug portion is an amine-derived hormone, such as but not limited to catecholamine; adrenaline (or epinephrine); dopamine; noradrenaline (or norepinephrine); tryptophan derivatives, e.g., melatonin (N-acetyl-5- methoxytryptamine), serotonin (5-HT); or a tyrosine derivative, e.g., thyroxine (T4) and triiodothyronine (T3).
  • catecholamine adrenaline
  • adrenaline or epinephrine
  • dopamine e.g., noradrenaline (or norepinephrine)
  • tryptophan derivatives e.g., melatonin (N-acetyl-5- methoxytryptamine), serotonin (5-HT)
  • a tyrosine derivative e.g., thyroxine (T4) and triiodoth
  • the drug portion is a peptide hormone, including but not limited to antimullerian hormone (AMH, also mullerian inhibiting factor or hormone); adiponectin; adrenocorticotropic hormone (ACTH, also corticotropin); angiotensinogen and angiotensin; antidiuretic hormone (ADH, also vasopressin, arginine vasopressin, AVP); atrial-natriuretic peptide (ANP, also atriopeptin); calcitonin; cholecystokinin (CCK); corticotropin-releasing hormone (CRH); erythropoietin (EPO); follicle stimulating hormone (FSH); gastrin; glucagon; gonadotropin-releasing hormone (GnRH); growth hormone-releasing hormone (GHRH); human chorionic gonadotropin (hCG); growth hormone (GH or hGH); insulin; insulin-like growth factor (IGF, also s
  • the drug portion is a steroid hormone, including but not limited to glucocorticoids, e.g., Cortisol; Mineralocorticoids, e.g., aldosterone; sex steroids, e.g., androgens (testosterone, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), androstenedione, dihydrotestosterone (DHT)), estrogen, e.g., estradiol, progestagen, e.g., progesterone, progestin.
  • glucocorticoids e.g., Cortisol
  • Mineralocorticoids e.g., aldosterone
  • sex steroids e.g., androgens (testosterone, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), androstenedi
  • the drug portion is a sterol hormone, including but not limited to Vitamin D derivatives, e.g., calcitriol.
  • the drug portion is a lipid or phospholipid hormone (eicosanoid), including but not limited to prostaglandin, leukotriene, prostacyclin, or thromboxane.
  • the drug portion is a cytokine such as IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IL "2 , IL-3, IL-4, IL-5, JL-6, JL-7, IL-S, IL-9, IL-IO, IL-12 and IL-15.
  • cytokine such as IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IL "2 , IL-3, IL-4, IL-5, JL-6, JL-7, IL-S, IL-9, IL-IO, IL-12 and IL-15.
  • the drug portion can also be an agonist or antagonist to a cell surface receptor, including but not limited to G-protein-coupled receptors, acetylcholine receptors, adenosine receptors, adrenoceptors (adrenergic receptors), Type-B GABA receptors ( ⁇ - Aminobutyric acid or GABA); angiotensin receptors; cannabinoid receptors; cholecystokinin receptors; dopamine receptors; glucagon receptors; metabotropic ghitamate receptors; histamine receptors; opioid receptors; secretin receptors; serotonin receptors, somatostatin receptors; tyrosine kinase receptors, such as erythropoietin receptor; insulin receptor, growth factors and cytokines receptors; guanylyl cyclase receptors; GC-A & GC-B receptors, Le., receptors of atrial-natriuretic peptide (
  • a RBC binding conjugate can be used in hormone replacement therapy.
  • a RBC binding conjugate comprising an estrogen or a progestagen as the drug portion can be used for contraception.
  • a RBC binding conjugate comprising a thyroxine, e.g., levothyroxine, as the drug portion can be used for treating hypothyroidism.
  • a RBC binding conjugate comprising a steroid as the drug portion can be used for treating autoimmune diseases and certain respiratory disorders.
  • a RBC binding conjugate comprising an insulin as the drug portion can be used for treating diabetics.
  • the drug portion can also be a diagnostic agent, e.g., a contrast agent for magnetic resonance imaging or a radioactive label for imaging of blood vessels.
  • diagnostic agents include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bio luminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. See generally U.S. Patent No. 4,741,900 for metal ions which can be conjugated to proteins for use as diagnostics.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase.
  • suitable prosthetic group complexes include streptavidi ⁇ /biotin and avidin/biotin.
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin.
  • luminescent material include luminol.
  • bioluminescent materials include luciferase, luciferin, and aequorin.
  • suitable radioactive material include I, I, In or Tc.
  • the RBC binding conjugate comprises an RBC binding portion covalently conjugated to one or more drug portions, such as but not limited to, enzymes.
  • the RBC binding conjugate comprises an RBC binding portion covalently-conjugated to at least 1, 2, 3, 4, 5 or 6 drug portions.
  • the drug portions are attached to the RBC binding portion, most preferably at different sites, in such a way that their therapeutic activity is not compromised.
  • the RBC binding conjugate of the invention retain at least 5%, 15%, 25%, 50%, 90% or 99% of the therapeutic activity as compared to the unconjugated drug portion.
  • the drug portion is attached at a selected residue on the RBC binding portion. Preferably, such a selected residue is selected so that the drug portion's therapeutic activity is not comprised.
  • the drug portions can be the same or different. In embodiments in which the drug portions are different drug portions, such drug portions can have different therapeutic activities.
  • the RBC binding portion e.g., an anti-CRl antibody or anti-glycophorin A antibody
  • the drug portion(s) are preferably covalently conjugated by a linker. Any cross-Unking chemistry known in art for conjugating proteins can be used in conjunction with the present invention.
  • the RBC binding portion and the drug portion are produced using cross-linking agents sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (s SMCC) and N-succinimidyl-S-acetyl-thioacetate (SATA).
  • the RBC binding portion and the drug portion are covalently conjugated via a water soluble polymer linker.
  • the polymer linker can be but is not limited to a polypeptide, a polyalkylene oxide, a polyoxyethylenated polyol, a polyacrylamide, a polyvinyl pyrrolidone, a polyvinyl alcohol, and a dextran.
  • the RBC binding portion and the drug portion are conjugated via a poly-(ethylene glycol) linker (PEG).
  • the PEG moiety can have any desired length.
  • the PEG moiety can have a molecular weight in the range of 200 to 40,000 Daltons.
  • the PEG moiety has a molecular weight in the range of 500 to 8000 Daltons.
  • RBC binding conjugate can be produced using cross-linking agents, e.g., N-succinimidyl-S-acetyl-thioacetate (SATA) and a poly(ethylene glycol)-maleimide, e.g., monomethoxy poly(ethylene glycol)-maleimide (mPEG-MAL), NHS-poly(ethylene glycol)-maleimide (PEG-MAL), or PEG hydrazine.
  • cross-linking agents e.g., N-succinimidyl-S-acetyl-thioacetate (SATA) and a poly(ethylene glycol)-maleimide, e.g., monomethoxy poly(ethylene glycol)-maleimide (mPEG-MAL), NHS-poly(ethylene glycol)-maleimide (PEG-MAL), or PEG hydrazine.
  • Such a RBC binding conjugate can also be produced using NHS- PEG-benzaldehyde (PBA) and succinimidyl C64-hydrazino-nictoinamde acetone hydrazone.
  • PBA PEG-benzaldehyde
  • succinimidyl C64-hydrazino-nictoinamde acetone hydrazone Methods of producing PEG-linked polypeptides that can be used are described in WO 2004024889.
  • the RBC binding conjugate is a polypeptide comprising an RBC binding portion and a drug portion, such as but not limited to, an enzyme.
  • a polypeptide can be produced using methods known in the art, e.g., using recombinant techniques (see, e.g., WO 01/80883 and WO 02/46208, each of which is incorporated herein by reference in its entirety).
  • the RBC binding conjugate of the invention is a polypeptide molecule which consists essentially of, or alternatively comprises, a RBC binding domain, e.g., a binding domain that binds a CRl receptor, bound to the amino terminus of a polypeptide drug portion, e.g., an enzymatic portion.
  • the binding domain can be an Fab, an Fab', an ( ⁇ ab%, or an Fv fragment of an immunoglobulin molecule, or a single- chain variable region fragment (scFv), i.e., a V L fused via a polypeptide linker to a V H .
  • the drug portion can be any one disclosed above.
  • the RBC binding conjugate can optionally comprises a linker polypepide between the binding domain and the polypeptide drug portion.
  • the RBC binding conjugate of the invention is a dimeric molecule consisting of a first polypeptide consisting essentially of, or comprising, a RBC binding domain, e.g., a binding domain that binds a CRl receptor, bound to the amino terminus of a first linker polypeptide, and a second polypeptide, consisting essentially of, or comprising, a second linker polypeptide with a polypeptide drug portion, e.g., an enzymatic portion, bound to the second linker polypeptide's carboxy terminus, wherein the first and second linker polypeptides are complementary to and can associate with each other.
  • the linkers do not comprise a functional Fc domain.
  • the first polypeptide consists essentially of, or comprising, a variable light chain domain (VL) and constant light chain domain (CL) followed by the first linker molecule.
  • the first polypeptide consists essentially of, or comprising, a scFv bound to the amino terminus of the first linker molecule.
  • the RBC binding conjugate comprises a RBC binding portion that binds CRl or glycophorin A covalently conjugated to a drug portion comprising a tissue-type plasminogen activator, streptokinase, staphylokinase, or urokinase via a PEG linker.
  • the PEG linker is attached to the drug portion at a moiety comprising an activated carboxylic acid group or a reactive carbonyl group.
  • the PEG linker is attached to the drug portion at an oxidized carbohydrate.
  • the RBC binding conjugate comprises a RBC binding portion attached to a PEG linker that links with the drug portion via a hydrazide/aldehyde linkage.
  • the RBC binding conjugate of the invention can be produced in various ways. Examples of methods for production include, but are not limited to, cross-linking, recombinant technique, or protein trans-splicing. These methods as well as methods for producing the RBC binding portions, the drug portions, and methods for purification and characterization of the RBC binding conjugates are described in Sections 5.2.1-5.2.4, infra.
  • the antibodies can be immunoglobulin molecules.
  • the immunoglobulin molecules are encoded by genes which include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant regions, as well as a myriad of immunoglobulin variable regions.
  • Light chains are classified as either kappa or lambda. Light chains comprise a variable light (V L ) and a constant light (C L ) domain.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD and IgE, respectively.
  • Heavy chains comprise variable heavy (VH), constant heavy 1 (CHl), hinge, constant heavy 2 (CH2), and constant heavy 3 (CH3) domains.
  • VH variable heavy
  • CHl constant heavy 1
  • CH2 constant heavy 2
  • CH3 constant heavy 3
  • IgG heavy chains are further sub-classified based on their sequence variation, and the subclasses are designated IgGl, IgG2, IgG3 and IgG4.
  • Antibodies can be further broken down into two pairs of a light and heavy domain.
  • the paired V L and V H domains each comprise a series of seven subdomains: framework region 1 (FRl), complementarity determining region 1 (CDRl), framework region 2 (FR2), complementarity determining region 2 (CDR2), framework region 3 (FR3), complementarity determining region 3 (CDR3), framework region 4 (FR4) which constitute the antibody-antigen recognition domain.
  • a chimeric antibody may be made by splicing the genes from a monoclonal antibody of appropriate antigen specificity together with genes from a second human antibody of appropriate biologic activity. More particularly, the chimeric antibody may be made by splicing the genes encoding the variable regions of an antibody together with the constant region genes from a second antibody molecule.
  • This method is used in generating a humanized monoclonal antibody wherein the complementarity determining regions are mouse, and the framework regions are human thereby decreasing the likelihood of an immune response in human patients treated with the antibody (United States Patent Nos. 4,816,567, 4,816,397, 5,693,762; 5,585,089; 5,565,332 and 5,821,337, each of which is incorporated herein by reference in its entirety).
  • An antibody suitable for use in the present invention may be obtained from natural sources or produced by hybridoma, recombinant or chemical synthetic methods, including modification of constant region functions by genetic engineering techniques (United States Patent No. 5,624,821).
  • the antibody of the present invention may be of any isotype, but is preferably human IgGl.
  • An antibody can also be a single-chain antibody (scFv) which generally comprises a fusion polypeptide consisting of a variable domain of a light chain fused via a polypeptide linker to the variable domain of a heavy chain.
  • scFv single-chain antibody
  • RBC binding portion that binds a human CRl receptor can be produced by known methods.
  • the RBC binding portion e.g., an IgG
  • the RBC binding portion can be prepared using standard hybridoma precedure known in the art (see, for example, Kohler and Milstein, 1975, Nature 256:495-497; Hogg et al., 1984, Eur. J. Immunol. 14:236- 243; O'Shea et al., 1985, J. Immunol. 134:2580-2587; Schreiber, U.S. Patent 4,672,044).
  • a suitable mouse is immunized with an appropriate RBC specific antigen, e.g., human CRl, which can be purified from human erythrocytes.
  • the spleen cells obtained from the immunized mouse are fused with an immortal mouse myeloma cell line which results in a population of hybridoma cells, including a hybridoma that produces an RBC binding portion.
  • the hybridoma which produces the antibody that can be used as a RBC binding portion is then selected, or 'cloned', from the population of hybridomas using conventional techniques such as enzyme linked immunosorbent assays (ELISA).
  • ELISA enzyme linked immunosorbent assays
  • Hybridoma cell lines expressing antibodies for use as RBC binding portions can also be obtained from various sources, for example, the murine monoclonal antibody that binds human CRl described in U.S. Patent 4,672,044 is available as hybridoma cell line ATCC HB 8592 from the American Type Culture Collection (ATCC). The obtained hybridoma cells are grown and washed using standard methods known in the art. The antibodies are then recovered from supernatants. IQ embodiments in which an anti-CRl antibody is used, the Fc domain (also referred to herein as the "effector domain”) is removed or otherwise inactivated, e.g., by introducing one or more mutations into the Fc domain such that it loses the effector function.
  • the Fc domain also referred to herein as the "effector domain
  • nucleic acids encoding the heavy and light chains of an RBC antibody are prepared from the hybridoma cell line by standard methods known in the art.
  • cDNAs encoding the heavy and light chains of the IgG are prepared by priming mRNA using appropriate primers, followed by PCR amplification using appropriate forward and reverse primers. Any commercially available kits for cDNA synthesis can be used.
  • the nucleic acids are used in the construction of expression vector(s).
  • the expression vector(s) are transfected into a suitable host. Non-limiting examples include E. coli, yeast, insect cell, and mammalian systems, such as a Chinese hamster ovary cell line. Antibody production can be induced by standard method known in the art.
  • An antibody that binds a RBC antigen can be prepared by immunizing a suitable subject with an appropriate RBC-specific antigen which can be purified from human erythrocytes.
  • the antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide.
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256:495-497), the human B cell hybridoma technique by Kozbor et al. (1983, Immunol. Today 4:72), the EBV- hybridoma technique by Cole et al. (1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques.
  • standard techniques such as the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256:495-497), the human B cell hybridoma technique by Kozbor et al. (1983, Immunol. Today 4:72), the EBV- hybridoma technique by Cole et al. (1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques.
  • Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide of interest, e.g., using a standard ELISA assay.
  • Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.
  • the monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., 1975, Nature, 256:495, or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567).
  • the term "monoclonal antibody” as used herein also indicates that the antibody is an immunoglobulin.
  • a mouse or other appropriate host animal such as a hamster
  • a hamster is immunized as hereinabove described to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization (see, e.g., U.S. Patent No. 5,914,112, which is incorporated herein by reference in its entirety.)
  • lymphocytes may be immunized in vitro. Lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1986).
  • a suitable fusing agent such as polyethylene glycol
  • the hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
  • HGPRT hypoxanthine guanine phosphoribosyl transferase
  • Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
  • preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the SaIk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 cells available from the American Type Culture Collection, Rockville, Md. USA.
  • Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, 1984, J.
  • Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen.
  • the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immuno-absorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immuno-absorbent assay
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al., 1980, Anal. Biochem., 107:220.
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI- 1640 medium.
  • the hybridoma cells may be grown in vivo as ascites tumors in an animal.
  • the monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • a monoclonal antibody directed against an appropriate RBC-specific antigen can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the antigen.
  • Kits for generating and screening phage display libraries are commercially available (e.g., Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene antigen SurfZAPTM Phage Display Kit, Catalog No. 240612).
  • examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S. Patent Nos.
  • chimeric antibodies In addition, techniques developed for the production of "chimeric antibodies" (Morrison, et al., 1984, Proc. Natl. Acad. ScL, 81, 6851-6855; Neuberger, et al., 1984, Nature 312, 604-608; Takeda, et al., 1985, Nature, 314, 452-454) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region.
  • Humanized antibodies are antibody molecules from non-human species having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule, (see e.g., U.S. Patent No. 5,585,089, which is incorporated herein by reference in its entirety.)
  • CDRs complementarity determining regions
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT Publication No. WO 87/02671; European Patent Application 184,187; European Patent Application 171,496; European Patent Application 173,494; PCT Publication No. WO 86/01533; U.S. Patent No.
  • Complementarity determining region (CDR) grafting is another method of humanizing antibodies. It involves reshaping murine antibodies in order to transfer full antigen specificity and binding affinity to a human framework (Winter et al. U.S. Patent No. 5,225,539). CDR-grafted antibodies have been successfully constructed against various antigens, for example, antibodies against JL-2 receptor as described in Queen et aL, 1989 (Proc. Natl. Acad. Sci. USA 86:10029); antibodies against cell surface receptors-CAMPATH as described in Riechmann et aL (1988, Nature, 332:323; antibodies against hepatitis B in Cole et al. (1991, Proc. Natl. Acad. Sci.
  • CDR-grafted antibodies are generated in which the CDRs of the murine monoclonal antibody are grafted into a human antibody. Following grafting, most antibodies benefit from additional amino acid changes in the framework region to maintain affinity, presumably because framework residues are necessary to maintain CDR conformation, and some framework residues have been demonstrated to be part of the antigen binding site. However, in order to preserve the framework region so as not to introduce any antigenic site, the sequence is compared with established germline sequences followed by computer modeling.
  • Fully human antibodies are particularly desirable for administration to human patients.
  • Such antibodies can be produced using transgenic mice which are incapable of expressing endogenous immunoglobulin heavy and light chain genes, but which can express human heavy and light chain genes.
  • the transgenic mice are immunized in the normal fashion with an appropriate RBC-specif ⁇ c antigen.
  • Monoclonal antibodies directed against the RBC-specif ⁇ c antigen can be obtained using conventional hybridoma technology.
  • the human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation.
  • Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93).
  • this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies see e.g., U.S. Patent 5,625,126; U.S. Patent 5,633,425; U.S.
  • companies such as Abgenix, Inc. (Freemont, CA; see, for example, U.S. Patent No. 5,985,615) and Medarex, Inc. (Princeton, NJ), can be engaged to provide human antibodies directed against the RBC-specif ⁇ c antigen using technology similar to that described above.
  • Completely human antibodies which recognize and bind a selected epitope can be generated using a technique referred to as "guided selection.”
  • a selected non-human monoclonal antibody e.g., a mouse antibody
  • is used to guide the selection of a completely human antibody recognizing the same epitope Jespers et al., 1994, Bio/technology 12:899-903.
  • a pre-existing anit-CRl antibody including but not limited to H4, H9, H47, H48, 7G9, HB8592, 3D9, 57F, and 1B4 (see, e.g., Talyor et al., U.S. Patent No. 5,487,890, which is incorporated herein by reference in its entirety), can also be used.
  • a hybridoma cell line secreting a high-affinity anti-CRl monoclonal antibody e.g., 7G9 (murine IgG ⁇ a, kappa)
  • MCB master cell bank
  • the master cell bank is tested for mouse antibody production, mycoplasma and sterility.
  • the RBC binding portion is then produced and purified from ascites fluid.
  • the anti-CRl monoclonal antibody used for the production of the RBC binding conjugates is produced in vitro (hollow-fiber bioreactor) and purified under cGMP.
  • Anti-CRl monoclonal antibodies without an effector domain can be generated by treating an appropriate antibody with an enzyme such as pepsin or papain.
  • the drug portion of the RBC binding conjugate of the invention can be produced by various methods known in the art.
  • the drug portion can be modified such that it can be attached to a selected residue of an RBC binding portion.
  • a residue is selected so that the antigen-binding affinity is not compromised after the fragment is covalently-conjugated to the RBC binding portion. More preferably, such a residue is on the surface of the RBC binding portion.
  • a cysteine residue is engineered into an appropriate location in a drug portion to allow site-specific attachment of the drug portion to an RBC binding portion (see, e.g., Lyons et al., Protein Engineering 3:703-708, which is incorporated herein in its entirety).
  • cysteine residue is introduced as well as the method that can be used to generate such an engineered drug portion.
  • the cysteine is introduced to the C-terminus of the drug portion.
  • the drug portion is modified such that it can be attached at a selected residue to an RBC binding portion.
  • a residue is selected so that the activity of the drug is not compromised after it is covalently- conjugated to the RBC binding portion. More preferably, such a residue is away from the active site of the drug portion.
  • a skilled person in the art will be able to determine the residue to which the RBC binding portion is attached as well as the method that can be used to produce such an attachment.
  • the drug portion containing a cysteine residue is produced by a host cell in such a manner that a cysteinyl free thiol is maintained (see, e.g., Carter, U.S. Patent No. 5,648,237, which is incorporated herein in its entirety).
  • the drug portion containing cysteinyl free thiol (also referred to as "drug-cys-SH”) can then be used to produce the RBC binding conjugate of the invention directly with an appropriate RBC binding portion or an appropriately derivatized RBC binding portion which can react with the free thiol to form a covalent bond.
  • the RBC binding portion can be a thiolated RBC binding portion, e.g., an RBC binding portion derivatized with N-succinimidyl-S-acetyl- thioacetate (SATA), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP).
  • SATA N-succinimidyl-S-acetyl- thioacetate
  • SPDP N-succinimidyl-3-(2-pyridyldithio)propionate
  • the drug-cys-SH can be covalently-conjugated with the thiolated RBC binding portion via a disulfide bond.
  • the drug portion contains a recombinantly introduced glycosylation site.
  • a drug portion can be produced use the method described in WO92/16555, which is incorporated herein by reference in its entirety.
  • the RBC binding conjugate of the present invention can be a covalent conjugate of one or more polypeptide drug portions, e.g., enzymes, with a polypeptide, e.g., an antibody, that binds the red blood cell.
  • a conjugate can be produced by any standard chemical conjugating methods known in the art, such methods can employ either maleimide chemistry, biotin chemistry, or hydrazide chemistry.
  • a conjugating method employing a bifunctional linker is used.
  • cross-linking agents including but not limited to, protein A, glutaraldehyde, carbodiimide, N-succinimidyl-S-acetyl-thio acetate (SATA),
  • N-succinirnidyl-3-(2-pyridyldithio)propionate SPDP
  • sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate sSMCC
  • succinimidyl 6- hydrazinonicotinate acetone hydrazone SANH
  • succinimidyl 4-formyl benzoate a conjugating method employing a bifunctional "water- soluble polymer linker, e.g., a poly(ethylene glycol) linker, is used.
  • the conjugate of the invention can also be prepared by recombinant methods or by a method utilizing protein trans-splicing.
  • SATA is used to derivatize the drug portion.
  • a skilled person in the art will be able to determine the concentrations of the drug portion and SATA.
  • the following protocol is used.
  • a solution of SATA in DMSO is prepared.
  • the drug portion is dialyzed against PBSE buffer.
  • the coupling reaction is initiated by combining the drug portion and SATA at a molar ratio of about 1 :6.
  • the reactants are mixed by inversion and incubated at room temperature for a desired period of time with mixing.
  • a hydroxylamine HCl solution is prepared by adding hydroxyamine and EDTA to MES.
  • the Hydroxylamine HCl solution is added to the reaction mixture from the SATA coupling step at an appropriate molar ratio, e.g., a molar ratio of about 2000:1, and incubated for a desired period of time at room temperature under argon atmosphere.
  • the reaction mixture is then desalted by chromatography, e.g., using an Amersham Hi-Prep desalting column in MES buffer.
  • the SATA derivatized drag portion can then be used with an appropriately derivatized RBC binding portion, e.g., a maleimide derivatized RBC binding portion, to produce the RBC binding conjugate of the invention.
  • the drug portion containing a cysteine residue is produced by a host cell in such a manner that a free thiol is maintained (see, e.g., Carter, U.S. Patent No. 5,648,237, which is incorporated herein in its entirety).
  • the drug portion containing a free thiol is secreted by the host cell.
  • the drug portion containing the free thiol can then be recovered and used with an appropriately derivatized RBC binding portion, e.g., a maleimide derivatized RBC binding portion, to produce the RBC binding conjugate of the invention.
  • the RBC binding portion is derivatized with a maleimide using any method known in the art.
  • a skilled person in the art will be able to determine the concentrations of the RBC binding portion and maleimide to achieve a desired number of cross-Unking sites on the RBC binding portion.
  • the antibody is derivatized with maleimide as follows: a fresh stock solution of sSMCC Conjugation solution is prepared in PBSE buffer; the antibody is dialyzed exhaustively against PBSE buffer; the coupling reaction is initiated by combining the antibody and sSMCC at a molar ratio of about 1 :6; the reactants are mixed by inversion and incubated at room temperature for 60 min with mixing; and the sSMCC-antibody is recovered by size exclusion chromatography using FPLC with two Pharmacia 26/10 Desalting Columns in series (cat#17-5087-01). The column is preferably pre-washed with distilled water followed by PBSE buffer according to the manufacturer's instructions before loaded with the reaction mixture.
  • the maleimide modified antibody is eluted in the void volume with PBSE buffer and should be used within 15 min.
  • the maleimide derivatized RBC binding portion can then be allowed to react with an appropriately drug portion, e.g., a SATA derivatized drug portion, to produce the RBC binding conjugate of the invention.
  • the RBC binding portion is thiolated, e.g., derivatized with N-succinimidyl-S-acetyl-thioacetate (SATA),
  • N-succinimidyl-3-(2-pyridyldithio)propionate SPDP
  • the thiolated RBC binding portion is then allowed to react with an appropriately drug portion, e.g., a SATA derivatized drug portion, to produce the RBC binding conjugate of the invention.
  • the derivatized RBC binding portion e.g., antibody-maleimide, antibody-PEG- maleimide, or antibody-SH
  • the drug portion containing a free thiol can be combined at a desired molar ratio of derivatized RBC binding portion: drug portion.
  • a skilled person in the art will be able to determine the molar ratio of the derivatized RBC binding portion and drug portion to achieve a desired number of drug portions to each RBC binding portion.
  • the antibody-maleimide and the drug-SH are combined at a molar ratio of about 2:1 (derivatized RBC binding portion:drug portion).
  • the derivatized RBC binding portion and the drug-SH are combined at a molar ratio of about 1 : 1 (derivatized RBC binding portion.drug portion).
  • 1, 2, 3, 4, 5 or 6 drug portions are conjugated to each RBC binding portion.
  • the RBC binding portion and the drug portion can also be covalently conjugated via a suitable water-soluble polymer (see, e.g., WO92/16555, which is incorporated herein by reference in its entirety).
  • Water-soluble polymers that can be used in the present invention include but not limited to polyalkylene oxides, polyoxyethylenated polyols, polyacrylamides, polyvinyl pyrrolidone, polyvinyl alcohol, dextran, and other carbohydrate-based polymers.
  • the molecular weight of the polymer can be selected based upon the end use of the conjugate by an ordinary skilled person in the art. In general, the useful range of molecular weight is between about 600 and about 100,000 Daltons, and preferably between about 1,000 and about 40,000 Daltons.
  • the RBC binding portion and the drug portion in the RBC binding conjugates of the invention can be conjugated via a water-soluble polymer linker at desired residues using methods known in the art, including but are not limited to lysine (reactive through its e-NH2); bistidine, tryptophan, or cysteine (reactive through its sulfhydryl SH; See, e.g., Goodson et al, 1990, Biotechnology 8:343); aspartic acids (reactive through their carboxyl functionalities); arginine, serine, or threonine (reactive through its hydroxyl OH); or glutamic acid (reactive through its carboxyl functionalities).
  • the RBC binding portion and the drug portion in the RBC binding conjugates are glycopolypeptides, they can also be attached to PEG linkers at the saccharide units (see, e.g., WO92/16555).
  • a bifunctional polymer is used to derivatize the RBC binding portion or the drug portion using one of the functional groups of the polymers.
  • the derivatized RBC binding portion or the derivatized drug portion is then conjugated with a suitably derivatized counterpart portion via the remaining functional group of the linker polymer.
  • a bifunctional polymer is reacted with both the RBC binding portion and the drug portion in one reaction. In the latter embodiment, it is preferable that the two functional groups of the polymer react to different types of residues of the RBC binding portion and the drug portion so that production of undesirable homodimers is minimized.
  • the polymer unit is attached c ⁇ valently to the polypeptide or glycopolypeptide RBC binding portions or the polypeptide or glycopolypeptide drug portions (hereinafter referred simply as the "polypeptide” or the “glycopolypeptide") by reacting an acyl hydrazine derivative of the polymer with the polypeptide or glycopolypeptide having a reactive carbonyl group or an activated peptide carboxylic acid group.
  • a reactive carbonyl group refers to either a ketone or aldehyde group, excluding other carboxyl-containing groups such as amides.
  • the polymers are attached to the polypeptide or glycopolypeptide by reacting with aldehyde groups, because aldehyde groups are more reactive than ketones.
  • the carbonyl group can be generated either on a peptide or a saccharide unit.
  • Dixon (Dixon, 1984, J. Protein Chem. 3:99) describes several methods to generate reactive carbonyl groups on the N-terminus of a polypeptide molecule, which is incorporated herein by reference in its entirety.
  • carbonyl groups can be generated on peptides by reacting a polypeptide or glycopolypeptide with a suitable heterobifunctional reagent such as a reactive ester of formyl benzoic acid, disclosed by King et al., 1986, Biochemistry 25: 5774, which is incorporated herein by reference in its entirety.
  • carbonyl groups can be generated on saccharide units of glycopolypeptides by oxidizing vicinal diols of carbohydrate moieties of glycopolypeptides with excess periodate or with an enzyme, e.g. galactose oxidase.
  • the polymer acyl hydrazine reacts with the reactive carbonyl group on the polypeptide or glycopolypeptide to form a hydrazone linkage between the polymer and the polypeptide or glycopolypeptide (see, e.g., WO 92/16555).
  • the hydrazone can be reduced to a more stable alkyl hydrazide by using a suitable agent, e.g., NaBHU or NaCNBHa.
  • R represents the water-soluble polymers
  • X is a molecular moiety comprising a functional terminal group on the polymer or a polypeptide linked via a functional group
  • Z is O, NH, S or a lower alkyl group containing up to ten carbon atoms and either or both of Rl and R2 are independently selected from oxidized carbohydrate moieties of glycopolypeptides and peptide units of polypeptides and glycopolypeptides on which, reactive carbonyl groups have been generated:
  • the activated peptide carboxylic acid group is derived either from a C-terminus carboxylic acid group or a carboxylic acid group of aspartic or glutamic acid residues.
  • Activated carboxylic acid groups are carboxylic acid groups substituted with a suitable leaving group capable of being displaced by the polymer acyl hydrazine.
  • the polymer acyl hydrazine reacts with the activated peptide carboxylic acid group to form a diacylhydrazine linkage between the polymer and the polypeptide or glycopolypeptide.
  • the reaction of polymer acyl hydrazine derivatives with activated peptide carboxylic acid groups of polypeptides and glycopolypeptides to form diacylhydrazides is illustrated by the reaction sequence of Scheme 2: Scheme 2
  • R again represents the water-soluble polymer
  • X is a molecular moiety comprising a functional terminal group on the polymer or a polypeptide linked via a functional group
  • Z is O, NH, S or a lower alkyl group containing up to ten carbon atoms.
  • R3 represents a polypeptide containing aspartic acid, glutamic acid or a C-terminus carboxylic acid residues.
  • R4 represent a leaving group substituted on the peptide carboxylic acid when the carboxylic acid group is activated. Examples of suitable leaving groups are disclosed by Bodanszky, Principles of Peptide Synthesis (Springer- Verlag, New York, 1984), which is incorporated herein by reference in its entirety.
  • Such leaving groups include, but are not limited to, imidazolyL, triazolyl, N- hydroxysuccinimidyl, N-hydroxynorbornenedicarboximidyl and phenolic leaving groups, and are substituted onto the peptide carboxylic acid group by reacting the polypeptide or glycopolypeptide in the presence of an activating reagent with the corresponding imidazole, triazole N-hydroxysuccinimide, N-hydroxynorbornene dicarboximide and phenolic compounds.
  • Suitable activating reagents are also well-known and disclosed by Bodanszky, Principles of Peptide Synthesis (Springer- Verlag, New York, 1984), which is incorporated herein by reference in its entirety.
  • activating reagents include, but are not limited to, water-soluble carbod ⁇ mides such as N-ethyl-N'-(3- dimethylaminopropyl) carbod ⁇ mide (EDC) and N-cyclohexyl-N'-(2-morphoh ' noethyl) carbod ⁇ mide, p-toluene sulfonate, 5-substituted isoxazolium salts, such as Woodward's Reagent K.
  • the acyl hydrazine polymer derivatives used in Schemes 1 and 2 are shown with the general structure (I):
  • X can be a hydroxyl group, in which case the polymer has two labile groups per polymer moiety capable of reacting to form a derivative that can be covalently linked with a polypeptide or glycopolypeptide.
  • X can therefore also be a group into which the terminal hydroxyl group may be converted, including the reactive derivatives disclosed in U.S. Patent Nos. 4,179,337 and 4,847,325, both of which are incorporated herein by reference in their entireties, as well as the acyl hydrazine derivatives described in WO 92/16555, which is incorporated herein by reference in its entirety.
  • heterobifunctional polymers can be prepared by methods known to those skilled in the art, including the methods disclosed supra with reference to the preparation of acyl hydrazine derivatives, as well as the methods disclosed by Zalipsky et al., 1986, Polvm. Prior. 27(1):1, and Zalipsky et al., 1990, J. Bioact. Comsat. Porvm. 5:227, both of which are incorporated herein by reference in their entireties.
  • AA represents an amino acid or a peptide sequence.
  • AA can be a peptide sequence of any of the common amino acids, or at least one amino acid residue.
  • AA being one amino acid residue, it is preferable that it is a residue that does not appear naturally in proteins. Examples of such unusual residues include, but are not limited to, ⁇ - or ⁇ -amino butyric acid, norleucine, homoserine, ⁇ -alanine, ⁇ -caproic acid, and the like.
  • the acyl hydrazine derivative is prepared by reacting the terminal -OH group of methoxylated PEG (mPEGlOH) with phosgene to form mPEG- chloroformate as described in U.S. Patent No. 5,122,614, which is incorporated herein by reference in its entirety.
  • the reaction is carried out in organic solvents in which the reactants are soluble, such as methylene chloride, and will run to completion overnight at room temperature.
  • the solvents and excess phosgene are removed and the residue of polymeric chloro formate is then reacted with an excess of hydrazine.
  • This reaction can be carried out under mild conditions and typically runs to completion at room temperature and the resulting product can be readily converted to a hydrazide by hydrazinolysis.
  • the acyl hydrazine polymer derivative containing a peptide sequence is then recovered and purified by a method known in the art.
  • the acyl hydrazine polymer derivative containing a peptide sequence or an amino acid can be prepared by reacting the peptide sequence with a succinimidyl carbonate active ester of the polymer, as disclosed in U.S. Patent No. 5,122,614, or by directly reacting isocyanate derivatives of an amino acid with the terminal hydroxyl group of the polymer as disclosed by Zalipsky et al., 1987, Int. J Peptide Protein Res. 30:740, both of which are incorporated herein by reference in their entireties. Both reactions can be carried out under mild conditions, running to completion at room temperature in organic solvents in which the polymer is soluble, such as methylene chloride.
  • the conjugation of a polypeptide or glycopolypeptide with a water- soluble polymer first involves either oxidizing carbohydrate moieties of the glycopolypeptide or activating carboxylic acid groups of peptide moieties of the polypeptides or glycopolypeptides.
  • the carbohydrate moieties can be oxidized by reacting the glycopolypeptide in aqueous solution with sodium periodate or with an enzyme, e.g., galactose oxidase or a combination of neuraminidase and galactose oxidase as disclosed by Solomon et al., 1990, J. Cbromatoaraphy 510:321-9.
  • the reaction runs rapidly to completion at room temperature.
  • the reaction medium is preferably buffered, depending upon the requirements of the polypeptide or glycopolypeptide.
  • the oxidized glycopolypeptide is then recovered and separated from the excess periodate by column chromatography.
  • Polypeptides or glycopolypeptides having peptide units on which reactive carbonyl groups have been generated may be directly reacted with the acyl hydrazine polymer derivatives in an aqueous reaction medium.
  • This reaction medium may also be buffered, depending upon the pH requirements of the polypeptide or glycopolypeptide and the optimum pH for the reaction, which pH is generally between about 5.0 and about 7.0 and preferably about 6.0.
  • the optimum reaction media pH for the stability of particular polypeptides or glycopolypeptides and for reaction efficiency, and the buffer in which this can be achieved, is readily determined by those of ordinary skill in the art.
  • mild conditions refer to conditions in which the temperatures are in the range between about 4 and about 37°C.
  • the reactions will run somewhat faster to completion at higher temperatures, as long as the temperature of the reaction medium does not exceed the temperature at which the polypeptides or glycopolypeptides begin to denature.
  • certain polypeptides and glycopolypeptides will require reaction with the polymer acyl hydrazine derivatives at reduced temperatures to minimize loss of activity and/or prevent denaturing.
  • the reduced temperature required by particular polypeptides and glycopolypeptides is preferably no lower than 4°C and more preferably no lower than 0 0 C. Under such conditions, the reaction will still take place, although longer reaction times may be necessary.
  • the reaction medium may be optionally buffered, depending upon the requirements of the polypeptide or the glycopolypeptide, and the optimum pH at which the reaction takes place.
  • the conjugated product is recovered and purified by diafiltration, column chromatography or the like.
  • the degree of polymer conjugation of the polypeptide or glycopolypeptide can then be determined by amino acid analysis.
  • the other end of the linker can be a functional group that covalently reacts with the protein, such as N-hydroxysuccinimide (NHS).
  • NHS-PEG-aryl-aldehyde for conjugating to SANH (NHS-4-hydrazino- nictoinamde acetone hydrazone); NHS-PEG-arylhydrazide for conjugating to SFB (succinimidyl fo ⁇ nylbenzoate); and NHS-PEG-aryaldehyde for conjugating to NHS- PEG-arylhydrazide.
  • SANH NHS-4-hydrazino- nictoinamde acetone hydrazone
  • NHS-PEG-arylhydrazide for conjugating to SFB (succinimidyl fo ⁇ nylbenzoate)
  • NHS-PEG-aryaldehyde for conjugating to NHS- PEG-arylhydrazide.
  • each part of the linker is used to derivatize one of the proteins on
  • hydrazide linker systems form unstable hydrazone bonds that require toxic cyanoborohydride for a reduction reaction;
  • the PAH linkers have a polymeric PEG spacer that extends into the solvent and facilitates the conjugation reaction, while other hydrazide linkers have low reactivity due to their short spacers;
  • PEGylated protein evades the immune reaction;
  • PEGylated proteins have improved pharmacokinetic profiles;
  • the PEG spacer facilitates the enzymatic action of the conjugate by the RBC-bound conjugate; and (6) PEG may increase the solubility of the compounds.
  • the polymer is linked to the RBC binding portion or the drug portion via the functional group X.
  • X can be any functional group known in the art, e.g., a maleimide or a N-Hydroxy-Succinimidyl (NHS) group.
  • the derivatized RBC binding portion or drug portion is then reacted with a counterpart portion or a derivatized counterpart portion to form a hydrazide/aldehyde linkage.
  • X is an NHS group.
  • the polymer is first reacted with the RBC binding portion via the NHS chemistry.
  • the enzyme drug portion is derivatized to have an aldehyde group.
  • the RBC binding portion and the drug portion are then conjugated via a hydrozide/aldehyde linkage.
  • the RBC binding portion and the drug portion are covalently conjugated using polyethylene glycol (PEG) or a PEG copolymer (see WO . 2004/0244889).
  • PEG polyethylene glycol
  • Soluble block copolymers of PEG with polypropylene glycol or polypropylene oxide can also be used in the present invention.
  • the PEG moiety can have a molecular weight in the range of 200 to 40,000 Daltons.
  • the PEG moiety has a molecular weight in the range of 500 to 8000 Daltons.
  • the RBC binding conjugates having PEG linkers are produced by a method described in US Patent No. 4,179,337, which is incorporated herein by reference in its entirety.
  • Other methods of attaching a PEG linker to a protein can also be used (see, e.g., US Patent No. 5,122,614; Veronese et al. 1985, Applied Biochem, and Biotech,, 11: 141-152; Katre ef ⁇ /. US Patent No. 4,766,106 and 4,917,888; Roberts MJ. etal., 2002 Advanced Drug Delivery Reviews, 54: 459-476; U.S. 5,766,897; U.S. 6,433,158 Bl; U.S. 5,849,860; all of which are incorporated herein by reference in their entirety).
  • a polymer linker containing an aryl aldehyde functional group is used to derivatize the RBC binding portion or the drug portion.
  • the counterpart portion is derivatized with a suitable cross-linker molecule to contain a hydrazide.
  • the two portions are then reacted to conjugate via a aldehyde/hydrozide linkage.
  • the RBC binding portion is derivatized with N- Hydroxy-Succinimidyl Polyethylene Glycol-Benzaldehyde (PBA) (see, WO 2004/0244889 and Example 6.2.).
  • the polypeptide drug portion is derivatized with a bifunctional hydrozone, e.g., succinimidyl C64-hydrazino-nictoinamde acetone hydrazone.
  • a bifunctional hydrozone e.g., succinimidyl C64-hydrazino-nictoinamde acetone hydrazone.
  • the derivatized RBC binding portion and the derivatized drug portion are then conjugated via a hydrozide/aldehyde linkage.
  • heterofunctional PEG linkers are used to produce the RBC binding conjugates.
  • Heterofunctional PEG linkers have the general formula X- PEG-Y, wherein X and Y represent derivatization or functional groups (e.g., activated functional groups).
  • a "functional group”, as used herein, refers to a group of covalently attached atoms, that are either electrophillically or nucleophillically activated and can derivatize another molecule through a covalent linkage.
  • functional groups include but are not limited to, COOH, -COOR, where R is lower alkyl or phenyl (carboxylic ester), -COZ, wherein Z is a halide, -CHO (aldehyde), -C(O)R (ketone), - SO 2 Z (wherein Z is a halide or CF 3 ), -SO 2 NHZ (Z is halide), -SO 2 NH 2 , -maleimide, - amino, -alkyl halide, -alkyl-Z (where Z is mesylate, triflate or tosylate), -alkyl isocyanate, -alkyl isothiocyanate, -alkyl amine, -alkyl-OH, -alkyl-SH, -alkysulfone, - alkylsulfonamide, -alkyl aldehyde, -alkyl ketone, -alkyl
  • the X and Y activated functional groups of the heterofunctional PEG linker are identical, and the X and Y activated functional groups are directed to modify the same amino acid type on the RBC binding portion and the drug portion of the RBC binding conjugate (e.g., an anti-CRl antibody and a tPA). In another embodiment, the X and Y activated functional groups are not the same and are directed to modifying different amino acid types of the RBC binding portion and the drug portion of the RBC binding conjugate (e.g., an anti-CRl antibody and tPA).
  • the amino acids of the RBC binding portion or the drug portion which are modified with PEG linkers on the surface of the RBC binding portion or the drug portion.
  • the N-terminal amino group See e.g., Kinstler et al, Pharm. Res. 13 : 1996) or the C-terminal carboxylic acid of the RBC binding portion or the drug portion are derivatized using PEG linkers. Conditions suitable for reaction between PEG linkers and amino acid residues within the RBC binding portion or the drug portion are known to those skilled in the art.
  • the X or Y activating functional groups of the heterofunctional PEG linkers used in cross-Unking the RBC binding portion and the drug portion of the invention are electrophillically activated by methods known in the art. At least one of the hydroxyl groups on the PEG linker is activated with a functional group (X or Y) susceptible to nucleopbilic attack by the nitrogen of an amino group on a first or second recognition binding moiety.
  • electrophillically activated PEG linkers are used to modify amine residues of a first or second recognition binding moiety.
  • the amine conjugation of PEG linkers are well known in the art, in which electrophillically activated PEG linkers target nucleophilic amine groups.
  • Examples of functional groups of PEG linkers that can be used for the modification of amine residues of a RBC binding portion and/or a drug portion include but are not limited to, -PEG dichlorotriazene, -PEG tresylate, -PEG succinimidyl carbonate, -PEG benzotriazole carbonate, -PEG p-nitrophenyl carbonate, -PEG trichlorophenyl carbonate, -PEG carbonylimidazole, or -PEG succinimidyl succinate.
  • electrohilically activated PEGs used in accordance of the invention are -PEG succinimidyl succinate (-PEG-SS), succinimide of PEG propionic acid (-PEG-SPA), or succinimide of PEG Butanoate Acid (-PEG-SBA).
  • PEG linkers that can be used for the modification of amine residues within a RBC binding conjugate of the invention include but are not limited to, -PEG2-H- hydroxysuccinimide (-PEG2-NHS), -PEG-Benzotriazole carbonate (-PEG-BTC), -PEG- Propionaldehyde (-PEG-ALD), -PEG-Acetaldehyde diethyl acetal (-PEG- ACET), or - PEG2-Aldehyde (-PEG2-ALD).
  • the X or Y activating groups of the heterofunctional PEG linkers used in producing the RBC binding conjugates are Lysine-active PEGs.
  • the most preferred PEG derivative for lysine modification are N-hydroxylsuccinimide ("NHS") active esters such as PEG succinimidyl succinate (-PEG-SS) and succinimidyl propionate (-PEG-SPA).
  • NHS N-hydroxylsuccinimide
  • -PEG-SS PEG succinimidyl succinate
  • -PEG-SPA succinimidyl propionate
  • Equal masses of lysine-active PEG (MW, 5000) and a first or second recognition binding moiety of the invention (i.e., anti-CRl antibody) to be derivatized are mixed at pH 8-9.5, at room temperature for 30 minutes, or a time sufficient for derivatization to take place.
  • a molar ratio of PEG (MW 5000) to protein amino groups of 1-5 to 1 is used.
  • the X or Y activating functional groups of the heterofunctional PEG linkers used in producing the RBC binding portion and/or the drug portion are used for modification of cysteine residues in a polypeptide.
  • Examples of functional groups in bifunctional PEG linkers that can be used for the modification of cysteine residues in a RBC binding conjugate of the invention include but are not limited to, -PEG2-forked maleimide, -PEG-forked maleimide, -PEG-maleimide, or -PEG2 maleimide.
  • Methods for attaching PEG linkers to cysteine residues are disclosed in US Patent No. 5,766,897 which is incorporated herein by reference in its entirety.
  • site-specific derivitization of a cysteine residue using a PEG linker can be achieved using the methods and compositions of the invention by engineering specific cysteine mutants by site-directed mutagenesis methods known in the art (Kunkel et al, 1988, Nucleic Acids and Molecular Biology, Eckstein, F. Lilley, eds., Springer- Verlag, Berling and Heidelberg, vol. 2 p.124).
  • the RBC binding conjugates of the invention are conjugated using Sulfhydryl-selective PEGs.
  • the most preferred PEG linkers for sulfhydryl modification are vinylsulfone, iodoacetamide, and maleimide.
  • Examples of other hetereofunctional PEG linkers that can be used in the invention include but are not limited to NHS-vinylsulfone and NHS-Maleimide (NHS- PEG-VS and NHS-PEG-Maleimide, respectively), bis-hydrazide-PEG, bis-hydrazine- PEG, and aldehyde-PEG-NHS.
  • the heterofunctional PEG linker is a compound of Formula (III) as follows (see WO 2004/0244889):
  • R is phenyl, naphthyl, or aromatic heterocycle, any of which is substituted with at least one -C(O)H or -NH-NH 2 group.
  • Aromatic heterocycle refers to a 5- to 10-membered monocyclic or bicyclic aromatic carbocycle in which 1-4 of the ring carbon atoms have been independently replaced "with a N, O or S atom.
  • aromatic heterocycle group include, but are not limited to, pyrrolyl, imidazolyl, benzimidazolyl, tetrazolyL, indolyl, isoquinolinyl, quinolinyl, quinazolinyl, purinyl, isoxazolyl, benzisoxazolyl, furanyl, furazanyl, pyridyl, oxazolyl, benzoxazolyl, thiazolyl, benzthiazolyl and thiophenyl.
  • R is phenyl
  • R is pyridyl
  • R is
  • R is
  • the RBC binding portion that binds a C3b-]ike receptor i.e., an anti-CRl antibody, e.g., an anti-CRl monoclonal antibody without a functional Fc domain
  • a C3b-]ike receptor i.e., an anti-CRl antibody, e.g., an anti-CRl monoclonal antibody without a functional Fc domain
  • the protocol for NHS-PEG-maleimide can be as follows: The anti- CRl antibody is derivatized with NHS-PEG-maleimide at a molar ratio of 6: 1; 6X NHS- PEG-maleimide: IX anti-CR antibody, such that the reaction proceeds at room temperature for two hours at gentle inversion every 15-30 minutes, wherein the anti- CRl antibody is derivatized at one or more sites with NHS-PEG-maleimide.
  • the resulting product from the derivitization is then desalted by chromatography using standard procedures known in the art (e.g., using an Amersham Hi-Prep 26/10 desalting column in MES buffer).
  • the RBC binding portion that binds a C3b- like receptor i.e., an anti-CRl antibody, e.g., an anti-CRl monoclonal antibody without a functional Fc domain
  • a C3b- like receptor i.e., an anti-CRl antibody, e.g., an anti-CRl monoclonal antibody without a functional Fc domain
  • Modification using NHS-PEG-benzaldehyde may have several advantages relative to other modification procedures such as those involving maleimide chemistry.
  • molecules, e.g., antibodies, modified with NHS-PEG-benzaldehyde tend to be stable over an extended period of time, e.g., at least one month, because the hydrazone or aldehyde moiety is stable under the pH range where the antibody is typically stored. Therefore, the antibody derivatization reaction can be carried out well in advance of the conjugation reaction. Modification using NHS-PEG-benzaldehyde may thus be preferred for commercial production, because the production schedule can be more flexible and the unconjugated monomeric fraction can be recycled.
  • Another benefit of modifying antibodies with NHS-PEG-benzaldehyde is that the hydrazine or aldehyde chemistry will not lead to bond formation with other functional groups in the antibody; any weak bond that could form between the amino group and the aldehyde is hydrolyzed in the aqueous buffer under physiological conditions.
  • the derivatized antibodies might react with the free sulfhydryl group on the antibody, leading to an undesired modification.
  • Yet another particular benefit of the NHS-PEG-benzaldehyde linker of the invention is that it requires no reducing agent for a stable bond formation over the pH range where antibodies are typically maintained in the stable form.
  • sulfhydryl modified proteins may form homodimers, there is no homodimer formation of the antibody using the hydrazone linker.
  • Yet another benefit of using the hydrazine chemistry is that the reaction kinetics of hydrazine/carbonyl linkage is fast and can be carried out in a condition where the antibody can be maintained in the active form.
  • the RBC binding portion or the drug portion can be derivatized with PEG linkers using any protocol known to those skilled in the art. It will be apparent to one skilled in the art that the molar ratio of the PEG linker used in derivatizing the RBC binding portion or the drug portion depends on the molecular weight of the PEG linker used and the molecular weight of the molecule being derivatized. One skilled in the art can determine the molar ratio of the PEG linker to be used in the derivitization of the RBC binding portion or the drug portion using routine experimentation.
  • the molar ratio of the NHS-PEG-maleimide to the RBC binding portion orthe drug portion is 3:1, 4:1, 5:1, 6:1, or 8:1.
  • Linear PEG linkers are the preferred linking reagents for use in the invention. In some embodiments, other types of linking reagents may also be used. Examples of additional linking reagents include but are not limited to, modified PEG linkers, branched PEG linkers (e.g., PEG2), linear forked PEG linkers, branched forked PEG linkers, or cross-linked PEG linkers.
  • the RBC binding portion and/or the drug portion can be thiolated using reagents and methods known in the art, in order to react with PEG derivatives directed at sulfhydryl groups.
  • amines of the RBC binding portion and/or the drug portion can be indirectly thiolated by reaction with succinimidyl 3-(2-pyridyldithio)propionate (“SPDP"), followed by reduction with DTT or tris-(2-carboxyethyl) phospohine (“TCEP").
  • SPDP succinimidyl 3-(2-pyridyldithio)propionate
  • TCEP tris-(2-carboxyethyl) phospohine
  • Amines can also be thiolated by reaction with succinimidyl acetylthioacetate ("SATA") followed by removal of the acetyl group with 5OmM hydroxylamine or hydrazine at or near neutral pH. Additionally, thiols can be incorporated at carboxylic acid groups by an EDAC mediated reaction with cystamine followed by reduction of the disulfide with DTT or TCEP. Other techniques for thiolation of the RBC binding portion or the drug portion are well known in the art and can be used in the methods of the invention.
  • SATA succinimidyl acetylthioacetate
  • thiols can be incorporated at carboxylic acid groups by an EDAC mediated reaction with cystamine followed by reduction of the disulfide with DTT or TCEP.
  • Other techniques for thiolation of the RBC binding portion or the drug portion are well known in the art and can be used in the methods of the invention.
  • the RBC binding portion and/or the drug portion can be modified using hydrazine or aldehyde amine modification reagents for example with, "SANH”; succinimidyl 6-hydrazinonicotinate acetone hydrazone or "SFB”; succinimydyl 4- formylbenzoate.
  • hydrazine or aldehyde amine modification reagents for example with, "SANH”; succinimidyl 6-hydrazinonicotinate acetone hydrazone or "SFB”; succinimydyl 4- formylbenzoate.
  • covalent conjugation of the RBC binding portion and the drug portion of the RBC binding conjugate of the invention are carried out in a site- directed manner.
  • PEG linker can be conjugated site-specifically to oxidized carbohydrate residues in the RBC binding portion or the drug portion.
  • Methods to oxidize carbohydrates are well known in the art, and include but are not limited to enzymatic oxidation (e.g. glucose oxidase) or chemical oxidation (e.g., periodate). Oxidation of carbohydrate residues generates multiple reactive aldehyde groups which can be conjugated with PEG linkers that have for example, an amine or a hydrazide functional group.
  • a glycosylation site is first introduced into the RBC binding portion and/or the drug portion using a recombinant method (see, e.g., WO 92/16555, which is incorporated herein by reference in its entirety).
  • PEG linker can also be conjugated site-specifically to a residue having a free thiol.
  • the RBC binding portion and/or the drug portion containing a cysteine residue is produced by a host cell in such a manner that a free thiol is maintained (see, e.g., Carter, U.S. Patent No. 5,648,237, which is incorporated herein in its entirety).
  • the polypeptide containing a free thiol is secreted by the host cell.
  • the RBC binding portion and/or the drug portion containing the free thiol can then be recovered and used with an appropriately derivatized PEG, e.g., a maleimide derivatized PEG, to produce the RBC binding portion and/or the drug portion.
  • an appropriately derivatized PEG e.g., a maleimide derivatized PEG
  • the RBC binding portion is conjugated to the N-terminus of an N-intein of a suitable split intein to produce an N- intein RBC binding portion fragment, whereas the drug portion is conjugated to the C- terminus of the C-intein of the split intein to produce a C-intein drug portion fragment.
  • the N-intein RBC binding portion fragment and the C-intein drug portion fragment are then brought together such that they reconstitute and undergo trans-splicing to produce the RBC binding conjugate.
  • the RBC binding conjugate produce by protein trans-splicing can contain a single drug portion conjugated to the RBC binding portion.
  • split inteins can be used for the production of the RBC binding conjugates of the present invention.
  • naturally occurring split inteins are used for the production of the RBC binding conjugates.
  • engineered split intein based on naturally occurring non-split inteins are used for the production of the RBC binding conjugates.
  • a split intein can be modified by adding, deleting, and/or mutating one or more amino acid residues to the N-intein and/or the C-intein such that the modification improves or enhances the intein's proficiency in trans-splicing and/or permits control of trans- splicing processes.
  • a Cys residue can be included at the carboxy terminus of a C-intein so that the requirement that the molecular moiety conjugated to the C-intein must start with a Cys is alleviated.
  • one or more native proximal extein residues are added to the N- and/or C- intein to facilitate trans-splicing in a foreign extein content.
  • the N-intein the RBC binding portion fragment is preferably produced by fusing an appropriate antigen recognition moiety that binds a C3b-lilce receptor to the N- terminus of the N-intein of a suitable split intein.
  • the C- terminus of the heavy chain of an anti-CRl mAb is fused to the N-terminus of the N- intein of a split intein.
  • the C-intein the drug portion fragment is preferably produced by fusing the drug portion to the C-terminus of the C-intein of a suitable split intein.
  • the amino acid residue immediately at the C-terminal side of the splice junction of the C- intein is a cysteine, serine, or threonine.
  • the RBC binding conjugate is produced by mixing the N-intein RBC binding portion fragment and the C-intein drug portion fragment in vitro so that the fragments reconstitute and undergo trans-splicing.
  • the RBC binding conjugates used in the present invention can also be produced recombinantly, where nucleotide sequences which encode antibody variable domains with binding specificity to an RBC surface receptor or antigen are fused to nucleotide sequences which encode a polypeptide drug molecule, see e.g., WO 01/80883, which is incorporated herein by reference in its entirety.
  • the nucleic acid encoding a polypeptide drug portion is fused to the nucleic acid encoding an antibody variable domain with binding specificity to an RBC surface receptor or antigen to obtain a fusion nucleic acid encoding a single polypeptide RBC binding conjugate.
  • the nucleic acid is then expressed in a suitable host to produce the RBC binding conjugate.
  • RBC binding conjugates produced by a method such as described supra are then preferably purified.
  • RBC binding conjugates can be purified by any method known to one skilled in the art using molecular size or specific binding affinity or a combination thereof.
  • the RBC binding conjugates can be purified by ion exchange chromatography using columns suitable for isolation of the RBC binding conjugates of the invention including DEAE, Hydroxylapatite, Calcium Phosphate (see generally Current Protocols in Immunology, 1994, John Wiley & Sons, Inc., New York, NY).
  • RBC binding conjugates are purified by three-step successive affinity chromatography (Corvalan and Smith, 1987, Cancer Immunol. Immunother., 24: 127-132): the first column is made of protein A bound to a solid matrix, wherein the Fc portion of the antibody binds protein A, and wherein the antibodies bind the column; followed by a second column that utilizes C3b-like receptor bound to a solid matrix which assays for C3b-like receptor binding via the RBC binding portion portion of the RBC binding conjugate; and followed by a third column that utilizes specific binding of an antigenic molecule of interest which binds the antigen recognition portion of the RBC binding conjugate.
  • the RBC binding conjugates can also be purified by a combination of size exclusion HPLC and affinity chromatography.
  • the appropriate fraction eluted from size exclusion HPLC is further purified using a column containing an antigenic molecule specific to the antigen recognition portion of the RBC binding conjugate.
  • the RBC binding conjugates can be characterized by various methods known in the art.
  • the yield of RBC binding conjugate can be characterized based on the protein concentration.
  • the protein concentration is determined using a Lowry assay.
  • the RBC binding conjugate produced by the method of the present invention has a protein concentration of at least 0.100 mg/ml, more preferably at least 2.0 mg/ml, still more preferably at least 5.0 mg/ml, most preferably at least 10.0 mg/ml.
  • the concentration of the RBC binding conjugates is determined by measuring UV absorbance. The concentration is determined as the absorbance at 280nm.
  • the RBC binding conjugate produced by the method of the present invention has an absorbance at 280nm of at least 0.14.
  • the RBC binding conjugate of the invention can also be characterized using any other standard method known in the art.
  • high-performance size exclusion chromatography (HPLC-SEC) assay is used to determined the content of contamination by free IgG proteins.
  • the RBC binding conjugate composition produced by the method of the present invention has a contaminated IgG concentration of less than 6.0 mg/ml, more preferably less than 2.0 mg/ml, still more preferably less than 0.5 mg/ml, most preferably less than 0.03 mg/ml.
  • the RBC binding conjugates can be characterized by using SDS- PAGE to determine the molecular weight of the RBC binding conjugate.
  • the RBC binding conjugate can also be characterized based on the functional activity of the RBC binding conjugates.
  • the anti-CRl binding activity is determined using ELISA with immobilized CRl receptor molecules (attached to a solid phase, e.g., a microtiter plate) (see Porter et al., U.S. provisional application No. 60/380,211, which is incorporated herein by reference in its entirety).
  • the assay is also referred to as a CRl/Antibody assay or CAA, and can be used generally to measure any RBC binding portion, or HP or AHP containing an RBC binding portion.
  • ELISA/CR1 plates are prepared by incubating ELISA plates, e.g., high binding flat bottom ELISA plates (Costar EIA/RIA strip plate 2592) with a suitable amount of a bicarbonate solution of CRl receptors.
  • concentration of the bicarbonate solution of CRl receptors is 0.2 ug/ml prepared from 5 mg/ml sCRl receptors stock (Avant Technology Inc.) and a carbonate-bicarbonate buffer (pH 9.6, Sigma C-3041).
  • 100 ul CRl-bicarbonate solution is dispensed into each well of the ELISA plates and the plates are incubated at 4°C overnight.
  • the plates are then preferably washed using, e.g., a wash buffer (PBS, 0.1% Tween-20, 0.05% 2-Chloroacetamide).
  • a SuperBlock Blocking Buffer in PBS (Pierce) is added to the plates for about 30-60 min at room temperature after the wash.
  • the plates can then be dried and stored at 4°C .
  • the titration of anti-CRl Abs or RBC binding conjugates can be carried out using a CRl binding protein, e.g., human anti-CRl IgG, as the calibrator.
  • the calibrator a human anti-CRl IgG having a concentration of 0.2 or 0.6 mg/ml.
  • the titration of the purified composition of RBC binding conjugates of the invention is carried out using PBS, 0.25% BSA, 0.1% Tween-20 as the diluent buffer, PBS, 0.1% Tween-20, 0.05% 2-Chloroacetamide as the wash buffer, TMB-Liquid Substrate System for ELISA (3,3',5,5'-Tetramethyl-Benzidine) and 2N H 2 SO 4 as the stop solution.
  • the RBC binding conjugate composition produced by the method of the present invention has an CAA titer of at least 0.10 mg/ml, more preferably at least 0.20 mg/ml, still more preferably at least 0.30 mg/ml, and most preferably at least 0.50 mg/ml.
  • a specific anti-CRl activity is determined.
  • the specific anti-CRl activity is a ratio of CAA and Lowry.
  • the specific amino acids that have been modified with a linker can be determined.
  • the method can measure the loss of the specific amino acids in unmodified form due to the modification.
  • unmodified lysine groups can be determined using the "Habeeb Method" where unmodified lysine groups react with trinitrobenzenesulfonic acid followed by UV measurement (Habeeb, 1966 Anal Biochem. 14:328; Karr et al, 1986, J. Chrom. 354:269; Abuchowski et al, 1977 J. Biol.
  • Another approach is reaction with Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acids) (See Grassetti et al, 1967 Biochem. Biophys. 119:41; Riddles et al, 1979, Anal. Bioch 94:75).
  • the RBC binding conjugates of the present invention can be used in treating or preventing a disease or disorder associated with the presence of a pathogenic substance in the blood.
  • the pathogenic substance can be any substance that is present in the circulation and that is injurious to or undesirable in the subject to be treated, including but not limited to physiologically produced substances, e.g., clots, and foreign pathogenic substances, e.g., toxins and microorganisms.
  • the RBC binding conjugates of the present invention convert or degrade such pathogenic substances into nonpathogenic substances, thereby treating or preventing the disease or disorder.
  • the RBC binding conjugates of the present invention can react with such pathogenic substances directly.
  • the RBC binding conjugates of the present invention can also catalyze reactions of such pathogenic substances with other substances.
  • the RBC binding conjugates of the present invention can also be used in a combination therapy in which one or more other therapeutic agents are also administered. In a preferred embodiment, the RBC binding conjugates of the present invention are used as a means for delivery
  • the RBC binding conjugates of the present invention can be used as a means for delivery of anticoagulants or thrombolytics. Such RBC binding conjugates can be used to treat or prevent disease conditions associated with the formation of clots in the blood of a subject.
  • the drug portion of the RBC binding conjugate comprises a streptokinase, staphylokinase, tissue-type plasminogen activator or tPA, or urokinase, which act to dissolve intravascular clots by activating the protease plasmin to digest fibrin.
  • the RBC binding conjugates of the present invention can be used in treating or preventing a disease or disorder associated with the deficiency of a substance in a subject.
  • the RBC binding conjugates of the present invention can be used as a means for delivery of enzymes in an enzyme replacement therapy.
  • Such RBC binding conjugates can be used to treat or prevent disease conditions associated with enzyme deficiency in a subject, e.g., various metabolic diseases.
  • the invention provides a conjugate comprising ⁇ -ghicocerebrosidase or an analogue covalently-conjugated to an anti-CRl or anti-glycophorin A antibody for treating Gaucher disease.
  • the invention provides a conjugate comprising an ⁇ -galactosidase A or an analogue covalently-conjugated to an anti-CRl or anti-glycophorin A antibody for treating Fabry disease.
  • the RBC binding conjugates of the present invention can also be used as a means for delivery of an anti-cancer agent.
  • Such RBC binding conjugates can be used to treat or prevent cancers in a subject.
  • the anti-cancer agent can be an agent that degrades small molecules for which cancer cells need.
  • the anti-cancer agent can also degrade macromolecules such as membrane polysaccharides, structural and functional protein, or nucleic acids.
  • the invention provides a conjugate comprising L-asparaginase or an analogue covalently-conjugated to an anti-CRl or anti-glycophorin A antibody for preventing and treating various types of cancers, including but not limited to, acute lymphocytic leukaemia.
  • the cancers cells of these types of cancers are deficient in their ability to synthesize the nonessential amino acid L-asparagi ⁇ e and must extract the amino acid from the body.
  • most normal cells can produce their own L-asparagine.
  • the RBC binding conjugate can be used to degrade L-asparagine in the blood stream, and thereby prevent the growth of cancer cells.
  • enzymes that can be used in treating or preventing cancers include but are not limited to L-glutaminase-L-asparaginase, L-methioninase, L- phenylalanine ammonialyase, L-arginase, L-tyrosinase, L-serine dehydratase, L- threonine deaminase, indolyl-3-alkane hydroxylase, neuraminidase, ribonuclease, various proteases, pepsin, various carboxypeptidases that are capable of hydrolyzing the L-glutamyl moiety of folic acid, or any analogue thereof.
  • Such a therapy can also be used in conjunction with other therapies that target a specific cancer.
  • the RBC binding conjugates of the present invention can also be used as a means for delivery of an anti-infectious agent.
  • Such RBC binding conjugates can be used to treat or prevent infectious diseases in a subject, e.g., various bacterial or viral infections.
  • the invention provides a conjugate comprising a lysozyme, e.g., lysostaphin, or an analogue covalently-conjugated to an anti-CRl or anti-glycophorin A antibody for treating bacterial infections, e.g., infections caused by Staphylococcus aureus. Lysostaphin kills bacteria by cleaving the glycoprotein of the bacterial wall and resulting in lyses of the bacterial cells.
  • Lysostaphin is capable of destroying bacteria whether they are active or resting and is thus capable of killing large numbers of microorganisms. It is particularly useful in instances where an initial and rapid reduction in bacterial count is necessary.
  • Such an RBC binding conjugate can be administered in combination with standard antibiotics.
  • the RBC binding conjugates of the present invention can also be used as a means for delivery of an antidote.
  • Such RBC binding conjugates can be used to treat an overdose of certain substance in a subject, e.g., an overdose of methotrexate.
  • the invention provides a conjugate comprising a carboxypeptidase Gl or an analogue covalently-conjugated to an anti-CRl or anti- glycophorin A antibody for treating methotrexate overdose.
  • the preferred subject for administration of a RBC binding conjugate of the invention, for therapeutic or prophylactic purposes is a mammal including but is not limited to non-human animals (e.g., horses, cows, pigs, dogs, cats, sheep, goats, mice, rats, etc.), and in a preferred embodiment, is a human or non-human primate.
  • non-human animals e.g., horses, cows, pigs, dogs, cats, sheep, goats, mice, rats, etc.
  • RBC binding conjugates can be combined into a "cocktail" of RBC binding conjugates.
  • Such cocktail of RBC binding conjugates can include RBC binding conjugates each having an RBC binding portion conjugated to any one of several desired drug portions.
  • the RBC binding conjugate cocktail can comprise a plurality of different RBC binding conjugates, wherein each different RBC binding conjugate in the plurality contains a different drug portion that targets a different pathogenic substance.
  • Such RBC binding conjugate cocktails are useful as personalized medicine tailored according to the need of individual patients.
  • a cocktail of RBC binding conjugates can include RBC binding conjugates each having a different RBC binding portion which binds a different blood cell antigen conjugated to a desired drug portion.
  • Such RBC binding conjugate cocktails can be used to increase the drug load on each red blood cell.
  • the RBC binding conjugates of the present invention can also be used as a means for delivery of a diagnostic agent.
  • Such RBC binding conjugates can be used to image internal tissues or organs and blood vessels in a subject.
  • diagnostic agents include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. See generally U.S. Patent No. 4,741,900 for metal ions which can be conjugated to proteins for use as diagnostics.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase.
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin.
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin.
  • luminescent material include luminol.
  • bioluminescent materials include luciferase, luciferin, and aequorin.
  • radioactive material examples include I, ⁇ , In or Tc. IV. DOSEAGE OF RBC BINDING CONJUGATES
  • the dose can be determined by a physician upon conducting routine tests. Prior to administration to humans, the efficacy is preferably shown in animal models. Any animal model for a blood borne disease known in the art can be used.
  • the dose of the RBC binding conjugate can be determined based on the red blood cell concentration and the number of target surface antigen, e.g., C3b-like receptor epitope sites, bound by the RBC binding portion per red blood cell.
  • a therapeutically effective amount of RBC binding conjugate (i.e., an effective dosage) may range from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 0.1 to 10 mg/kg body weight.
  • treatment of a subject with a therapeutically effective amount of a RBC binding conjugate can include a single treatment or, preferably, can include a series of treatments.
  • a subject is treated with a RBC binding conjugate in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks.
  • the effective dosage of a RBC binding conjugate, used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein.
  • RBC binding conjugate agents depends upon a number of factors within the ken of the ordinarily skilled physician, veterinarian, or researcher.
  • the dose(s) of the RBC binding conjugate will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the RBC binding conjugate to have upon a pathogenic antigenic molecule or autoantibody.
  • appropriate doses of RBC binding conjugates depend upon the potency of the RBC binding conjugate with respect to the biological activity it is to carry out. Such appropriate doses may be determined using a suitable assay.
  • RBC binding conjugates When one or more of these RBC binding conjugates is to be administered to an animal (e.g., a human) in order to cause the degradation of a pathogenic substance, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained.
  • a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained.
  • the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the RBC binding conjugate employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the concentration of pathogenic substance to be cleared.
  • compositions suitable for administration can be incorporated into pharmaceutical compositions suitable for administration.
  • Such compositions typically comprise RBC binding conjugate and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the RBC binding conjugate, use thereof in the compositions is contemplated. Supplementary RBC binding conjugates can also be incorporated into the compositions.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • the preferred route of administration is intravenous.
  • Other examples of routes of administration include parenteral, intradermal, subcutaneous, transdermal (topical), and transmucosal.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF; Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that the viscosity is low and the RBC binding conjugate is injectable. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanoL, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the RBC binding conjugate (e.g., one or more RBC binding conjugates) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the RBC binding conjugate into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the RBC binding conjugates are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811 which is incorporated herein by reference in its entirety.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of RBC binding conjugate calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the RBC binding conjugate and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such a RBC binding conjugate for the treatment of individuals.
  • compositions can be included in a kit, in a container, pack, or dispenser together with instructions for administration.
  • the RBC binding conjugate is prebound to red blood cells of the subject ex vivo, prior to administration.
  • red blood cells are collected from the individual to be treated (or alternatively red blood cells from a non- autologous donor of the compatible blood type are collected) and incubated with an appropriate dose of the therapeutic RBC binding conjugate for a sufficient time so as to allow the conjugate to bind the antigen on the surface of the red blood cells.
  • the red blood cell/RBC binding conjugate mixture is then administered to the subject to be treated in an appropriate dose (see, for example, Taylor et al., U.S. Patent No. 5,487,890).
  • the invention provides a method of treating a mammal having an undesirable condition associated with the presence of a pathogenic substance, comprising the step of administering a red blood cell/RBC binding conjugate complex to the subject in a therapeutically effective amount, the complex consisting essentially of a red blood cell bound to one or more RBC binding conjugates.
  • the method alternatively comprises a method of treating a mammal having an undesirable condition associated with the presence of a pathogenic substance comprising the steps of (a) contacting a RBC binding conjugate with red blood cells to form a red blood cell/RBC binding conjugate complex; and (b) administering the red blood cell/RBC binding conjugate complex to the mammal in a therapeutically effective amount.
  • the invention also provides a method of making a red blood cell/RBC binding conjugate complex comprising contacting a RBC binding conjugate with red blood cells under conditions conducive to binding, such that a complex consisting essentially of a red blood cell bound to one or more RBC binding conjugates forms.
  • the RBC binding conjugate such as a RBC binding conjugate
  • the RBC binding conjugate is prebound to red blood cells in vitro as described above, using a blend of at least two different RBC binding conjugates that bind different surface antigens on the red blood cells, e.g., different and non-overlapping recognition sites on the C3b-like receptor.
  • the number of RBC binding conjugates that can bind to a single red blood cell is increased.
  • kits comprising in one or more containers the RBC binding conjugates of the invention.
  • Kits containing the pharmaceutical compositions of the invention are also provided.
  • Example 6.1 describes the production of the conjugate.
  • Example 6.2 describes the production of the bifunctional polymeric NHS-PEG-benzaldehyde.
  • the following example describes the production of a tPA - RBC binding portion conjugate.
  • the RBC binding portion was an anti-CRl monoclonal antibody 7G9.
  • the antibody was derivatized with the bifunctional polymeric NHS-PEG-benzaldehyde (PBA).
  • Tissue plasminogen activator (tPA) was derivatized with the bifunctional compound succinimidyl C64-hydrazino-nictoinamde acetone hydrazone (Hz) (Solulink). Thirty nmoles of Hz was used to modify 5 nmole of 7G9 in sample-buffer: 0.15M NaCl, 50 mM potassium phosphate, pH 7.4.
  • the conjugation reaction was initiated by mixing the 0.6 mg of tP A-Hz with 0.3 mg of 7G9-PBA (weight ratio of tPA:7G9 was at 2: 1, molar ratio at 4.6: 1) at a total protein concentration of -0.61 mg/ml. The reaction was allowed to proceed for 16 hours at room temperature. The conjugate sample was then purified on a Suprose 6 column (Amersham) which had been equilibrated with 2XPBS: 20 mM phosphate, 0.3M NaCl, pH7.4. The conjugates had molecular weights greater than 220,000 g/mol. The fractions were analyzed by SDS-PAGE ( Figure 1) and the isolated conjugates contained 1-2 tPA molecules for each 7G9 molecule.
  • Carboxy-PEG-benzaldehyde (CPB) was precipitated by adding cold isopropyl alcohol (IPA) to the combined ethereals. The precipitate was then washed with cold IPA then dissolved in 8 ml of DCM. To the resulting solution was added 0.8 ml of 10% of sodium phosphate buffer at pH 5.0, followed by 150 mg of (l-ethyl-3-(3-dimethylamino propyl)carbodiimide (EDC), and 102 mg of N-hydroxysucciiiimide (NHS).
  • IPA isopropyl alcohol
  • EDC l-ethyl-3-(3-dimethylamino propyl)carbodiimide
  • NHS N-hydroxysucciiiimide
  • the residual carboxyl group in the intermediate product was completely converted to the final product by another reaction with EDC and NHS.
  • 50 mg of the intermediate product was dissolved in 2.5 ml of ethyl acetate. 16.11 mg of NHS and 28.475 mg of EDC were added.
  • the reaction mixture was stirred for 1.5 hours under Argon.
  • the reaction mixture was concentrated down to a colorless gumlike material.
  • Two ml of ether was added to allow a precipitate to form.
  • Ether was decanted and the residue was washed with ether for two more times.
  • a solid material (23 mg) was collected as the final product.
  • the compound was analyzed on a thin layer chromatography plate and was obeserved as a distinct spot.

Abstract

The present invention provides methods and compositions for using erythrocyte (RBC) as a carrier for delivery of drugs, e.g., therapeutic enzymes or mammalian serum proteins. The invention involves conjugating the drug to an antibody that binds RBC. When administered to a patient, the RBC binding conjugate binds to RBC, and is delivered to desired locations by the RBC. The invention provides methods of using the RBC binding conjugate for preventing and treating diseases. The invention also provides methods for preparing the RBC binding conjugate.

Description

METHODS AND COMPOSITIONS FOR USING ERYTHROCYTES AS CARRDERS FOR DELIVERY OF DRUGS
RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Patent Application No. 60/774,890, filed on February 17, 2006, tilted "METHODS AND COMPOSITIONS FOR USING ERYTHROCYTES AS CARRIERS FOR DELIVERY OF DRUGS." The entire contents of this application are hereby incorporated herein by reference.
FIELD OF THE INVENTION
The invention relates to methods and compositions for using the erythrocyte, or red blood cell (RBC) as a carrier for delivery of one or more biologically active molecules, e.g., enzymes or mammalian serum proteins. The invention relates to methods of using the RBC binding conjugate for preventing and treating diseases. The invention also relates to methods of preparing the RBC binding conjugate.
BACKGROUND OF THE INVENTION
Primate erythrocytes, or red blood cells (RBCs), play an essential role in the clearance of antigens from the circulatory system. The formation of an immune complex in the circulatory system activates the complement factor C3b in primates and leads to the binding of C3b to the immune complex. The C3b/immune complex then binds to the type 1 complement receptor (CRl), a C3b receptor, expressed on the surface of erythrocytes via the C3b molecule attached to the immune complex. The immune complex is then chaperoned by the erythrocyte to the reticuloendothelial system (RES) in the liver and spleen for neutralization. The RES cells, most notably the fixed-tissue macrophages in the liver called Kupffer cells, recognize the C3b/immune complex and break this complex from the RBC by severing the C3b receptor-RBC junction, producing a liberated erythrocyte and a C3b/immune complex which is then engulfed by the Kupffer cells and is completely destroyed within subcellular organelles of the Kupffer cells.
Taylor et al. discloses a method of utilizing the RBCs for removing pathogens from the circulatory system (U.S. Patent Nos. 5,487,890; 5,470,570; and 5,879,679). Taylor et al. have shown that chemical crosslinking of a first monoclonal antibody (mAb) specific to a primate C3b receptor to a second monoclonal antibody specific to a pathogenic antigenic molecule creates a bispecific heteropolymeric antibody (HP) which offers a mechanism for binding a pathogenic antigenic molecule to a primate's C3b receptor without complement activation. A HP that can be used to remove a pathogenic antigen specific autoantibody from the circulation is also reported. Such a HP, also referred to as an "Antigen-based Heteropolymer" (AHP), contains a CRl specific monoclonal antibody covalently-conjugated to an antigen (see, e.g., U.S. Patent No. 5,879,679; Lindorfer, et al., 2001, Immunol i?ev.l83: 10-24; Lindorfer, et al., 2001, J Immunol Methods 24%: 125-138; Ferguson, et . al., 1995, Arthritis Rheum 38: 190-200). In addition to HP and AHP produced by cross-linking, bispecific molecules that have a first antigen recognition domain which binds a C3b-like receptor, e.g., a complement receptor 1 (CRl), and a second antigen recognition domain which binds an antigen can also be produced by methods that do not involve chemical cross-linking (see, e.g., PCT publication WO 02/46208; and PCT publication WO 01/80883). PCT publication WO 01/80833 describes bispecific antibodies produced by methods involving fusion of hybridoma cell lines, recombinant techniques, and in vitro reconstitution of heavy and light chains obtained from appropriate monoclonal antibodies. PCT publication WO 02/46208 describes bispecific molecules produced by protein trans-splicing. PCT publication WO 2004/024889 describes bispecific molecules comprising a polyethylene glycol aldehyde/hydrazide linkage.
Red blood cells (RB Cs) have also been used as carriers for drugs and biomolecules. RBCs have been used as carriers for drugs loaded into the inner volume of RBCs (Poznansky et al., 1984, Pharmacol". Rev. 36:277-324; Kirch et al., 1994, Biotechnol. App. Biochem. 19:331-363; Kinoshita et al., 1978. Nature 272:258-260). Ex vivo loading of drugs to RBCs can also be used, e.g., RBCs can also be obtained from the patient's blood, loaded with drug, and re-injected.
A variety of diseases may be treated by therapeutical agents that are biologically or chemically active in the blood. It is often desirable that such agents remain in the blood for a sufficiently long period of time without degradation and/or loss due to uptake by other tissues. For certain agents, it may also be desirable to limit their tissue uptake to eliminate or reduce harmful side effects. Attaching such agents to red blood cells offers an attractive means to achieve these goals.
For example, many disease conditions are a result of the clogging of blood vessels by intravascular clots, including myocardial infarction, disseminated intravascular coagulation, stroke and pulmonary embolism, as well as less acute conditions, such as deep venous thrombosis and peripheral vascular diseases, e.g., atherosclerosis, Raynaud's disease, and ischaemias. Pulmonary thromboembolism, a condition of high mortality, is often a result of deep venous thrombosis, as pulmonary emboli often result from thrombi in the deep venous system. Disseminated intravascular coagulation may be caused by various conditions, such as severe injuries and infections, tumors, hemolytic transfusion reactions, vasculitis, heatstroke, hemangomias and certain poisoning. For example, in an injury, hemostasis and tissue repair is initiated by the release of thromboplastin from injured cells. The release thromboplastin reacts with factor Vπ in the surrounding plasma to form factor X activator. Prothrombin is converted into thrombin by factor V. Thrombin cleaves fibrinogen to form fibrin monomers and activates factor XEII to form XIIIa. Factor XIIIa causes fibrin monomers to crosslink forming aggregates. Such conditions lead to the activation of the hemostatic mechanisms, which overwhelms the available inhibition mechanisms, e.g., the dilutional effects of the blood flow, antithrombines, antiplasmin and other mechanisms that down- regulate hemostasis, and causes excessive release of thrombin.
Anticoagulant or antithrombotic agents, such as heparin, dicumarol, antithrombin concentrates and hirudin, have been commonly used for the treatment of these diseases or conditions. Fibrinolytics, such as streptokinase, staphylokinase, tissue-type plasminogen activator or tPA, and urokinase, have also been used in the treatment of some of these diseases or conditions, such as myocardial infarction and stroke. These agents act to dissolve intravascular clots by activating plasmin, a protease that digests fibrin. Plasminogen, the inactive precursor of plasmin, is converted to plasmin by cleavage of a single peptide bond. Plasmin itself is a nonspecific protease that digests fibrin clots as well as other plasma proteins, including several coagulation factors.
The application of fibrinolytic agents to dissolve clots formed in other vascular areas such as deep venous areas has been less successful due partly to the rapid elimination and inactivation of the fibrinolytics agents (Plow, et al., 1995, FASEB J. 9:939-945; Narita, et al. 1995, J. Clin. Invest. 96:1164-1168). For example, tPA and urokinase undergo rapid inactivation by a circulating plasminogen activator inhibitor. Plasmin itself is inactivated by a circulating glycoprotein, α-2-antiplasmin (Co lien, 1996, Circulation 93:857-865; Reilly, et al, 1991, Arterioscl. Thromb. 11 :1276-1286). α-2-antiplasmin also inactivates staphylokinase (Collen, et al., 1993, Eur. J. Biochem. 216:307-314). Although therapeutic doses of plasminogen activators can overcome the inhibitory activity of plasminogen activator inhibitor and α-2-antiplasmin, other inhibitors of plasminogen activators, e.g., Cl-inhibitor, α2-macroglobulin, and antitrypsin may also contribute to the decrease in the fibrinolytic response upon treatment with plasminogen activators (Collen, 1996, Circulation 93:857-865). Such inactivation or degradation of plasminogen activators and plasmin reduces the effectiveness of thrombolytic therapy.
Therapies involving infusion of plasminogen activators intravenously for prolonged periods of time have also been investigated. Such therapies have been found to cause side effects such as hemorrhage and tissue proteolysis. The latter was found to be a result of deposition of plasminogen activators in tissues, which leads to plasmin activation in tissues. Activated plasmin degrades the extracellular matrix, and causes vascular remodeling, abnormal elevation of vascular permeability and partial denudation of subendothelium (Plow et al. 1995. FASEB J. 9:939-945; Shreiber et al. 1995. J. Cell. Physiol. 165:107-118).
U.S. Patent No. 6,488,927 and U.S. Patent Publication 2002/0099000 disclose compositions and methods for prevention and treatment of deep vein thrombosis, pulmonary embolism and other diseases or syndromes resulted from uncontrolled formation of intravascular fibrin clots using a composition that comprises a drug, such as a fibrinolytic or anticoagulant drug, e.g., a plasminogen activator, biocompatibly coupled to the red blood cells. The red blood cells serve as carriers of the drug, allowing prolonged circulation and restricted tissue uptake. These references teach a monovalent conjugation of plasminogen activators to biotinylated RBCs via streptavidin (referred to herein as SA/b-RBC). These references also teach crosslinking of plasminogen activators to biotinylated anti-CRl monoclonal antibodies which then couple specifically with red blood cells in whole blood. U.S. Patent No. 5,840,733 teaches compounds comprising chemically reactive intermediates which can react with available reactive functionalities on blood components to form covalent linkages, where the resulting covalently-bound conjugates are found to have thrombin inhibition activity. Specifically, the thrombin inhibitor compounds of the '733 patent are derivatives of the known thrombin inhibitor argatroban, which can be covalently linked to chemically reactive functionalities on various blood components. The '733 patent also teaches methods for inhibiting thrombin activity in vivo comprising administering to the bloodstream of a mammalian host the disclosed compounds.
U.S. Patent No. 5,843,440 teaches bifunctional reagents useful in reducing the biological effect of an undesirable blood-borne agent. The reagents comprise conjugates of a first binding member specific for a blood-borne agent having a detrimental biological activity in a mammalian host, such as a growth factor, coagulation factor, enzyme, toxin, drug of abuse, microbe, autoreactive immune cell, infected or tumorous cell, joined to an second binding member specific for an anchor, where the anchor is a long-lived blood component, including cells, such as a erythrocyte, platelet or endothelial cell and serum proteins, such as albumin, ferritin, or steroid binding proteins. The λ440 patent teaches therapeutic uses of the conjugates for coupling blood born agent to the blood component so as to reduce the biological activity or effective concentration of the agent, modulate the volume of distribution of the agent, target the agent to sites of enhanced immune response, or facilitate agent clearance from the bloodstream.
Polyethylene glycol (PEG) has been used to conjugate proteins (see, e.g., WO 92/16555 and WO 2004/024889). Conjugation of PEG to proteins increases their shelf lives. Comparing to other cross-linkers, PEG linkers are non-immunogenic. To covalently attach PEG to a molecule, the hydroxyl end groups of PEG molecules are first converted into reactive functional groups to generate activated PEG. PEG molecules can be covalently attached to various groups on the surface of a protein. For example, PEG can be attached to amino groups using PEG-succinimide derivatives (see, e.g., U.S. Patent No. 4,179,337). PEG can also be attached to sulfhydryl groups using PEG-maleimide derivatives (see, U.S. Patent No. 4,179,337). However, the amino groups of many proteins are often associated with moieties responsible for the biological ■ activity of the proteins. Modification of amino groups may render these proteins biologically inactive. Similarly, sulfhydryl groups are also often associated moieties having biological or enzymatic activities, and are not readily available for modification.
Methods for covalently attaching PEG to other moieties in a protein and glycoprotein without a loss of activity have also been disclosed. U.S. Patent No. 4,847,325 discloses covalently attaching PEG to CSF-I by reacting PEG-amine, PEG- hydrazine or PEG-hydrazide with CSF-I that had been oxidized with periodate to convert vicinal diols in, the sugars to aldehydes. PCT publication WO92/16555 discloses a method of producing a biologically active macromolecular conjugate comprising a biologically active polypeptide or glycopolypeptide covalently attached to one or more PEG molecules at a reactive carbonyl or carboxylic acid group of a peptide moiety on the polypeptide or glycopolypeptide by a linkage containing a hydrazide or hydrazone functional group. The linkage is formed by reacting an acyl hydrazine derivative of the PEG with a polypeptide or glycopolypeptide having an activated carboxylic acid group or a reactive carbonyl group. Hydrazides readily form relatively stable hydrazone linkages by condensation with aldehydes and ketones (Andresz, et al., 1978, Makromol. Chem. 179:301). This property has also been used for modification of glycoproteins through oxidized oligosaccharide moieties (Wilchek et al., 1987, Meth. Enzymol. 138:429). Activated PEG-hydrazide reacts with an aldehyde group, which is normally absent on the polypeptide chain of a protein, but can be generated in a protein containing carbohydrate moieties by oxidation of the sugar ring. Methods for activation of immunoconjugates are also described in SeIa et al., 1987, Immuno conjugates, Vogel ed., Oxford University Press.
U.S. Patent No. 6,743,908 discloses a method of generating polypeptides having introduced glycosylation sites. This patent also discloses site-specific attachment of PEGs to such inserted glycosylation sites.
Discussion or citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention.
SUMMARY OF THE INVENTION
The invention provides a red blood cell (RBC) binding conjugate comprising a monoclonal antibody or a fragment thereof covalently conjugated to a mammalian serum protein or to an enzyme, wherein the monoclonal antibody or fragment thereof (i) binds a C3b-like receptor on a red blood cell, and (ii) lacks a functional effector domain. The monoclonal antibody can be a murine monoclonal antibody, a humanized monoclonal antibody, or a human monoclonal antibody. In a preferred embodiment, the monoclonal antibody or fragment binds a CRl receptor on a red blood cell. For example, the monoclonal antibody can be selected from the group consisting of anti- CRl antibodies H4, H9, H47, H48, 7G9, HB8592, 3D9, 57F, and 1B4.
In a preferred embodiment, the monoclonal antibody or fragment thereof is selected from the group consisting of an Fab, an Fab', an (Fab^, and an Fv fragment of an immunoglobulin molecule that binds the C3b-like receptor.
In another preferred embodiment, the monoclonal antibody or fragment thereof comprises a monoclonal antibody in which the effector domain is inactivated. In one embodiment, the effector domain of the monoclonal antibody comprises one or more mutations such that the effector domain loses its effector function.
Any suitable mammalian serum protein or enzyme can be conjugated to the monoclonal antibody or a fragment to generate the RBC binding conjugate of the present invention. In one embodiment, the mammalian serum protein is a human serum protein. In another embodiment, the mammalian serum protein comprises a mammalian serum enzyme or a functional fragment thereof.
In one embodiment, the mammalian serum protein is selected from the group consisting of a tissue-type plasminogen activator, a receptor of a tissue-type plasminogen activator, a streptokinase, a staphylokinase, a urokinase, and Factor VOX The invention also provides a method for treating associated with the formation of clots in its circulation, comprising the step of administering to the mammal a therapeutically effective amount of a RBC binding conjugate which contains such a mammalian serum protein.
In another embodiment, the mammalian serum protein is β-glucocerebrosidase. The invention also provides a method of treating a patient having Gaucher disease, comprising the step of administering to the patient a therapeutically effective amount of the RBC binding conjugate in which the mammalian serum protein is β- glucocerebrosidase.
In still another embodiment, the mammalian serum protein is α-galactosidase A. The invention also provides a method of treating a mammal having Fabry disease, comprising the step of administering to the patient a therapeutically effective amount of the RBC binding conjugate in which the mammalian serum protein is α-galactosidase A.
In still another embodiment, the mammalian serum protein is a cytokine. The cytokine can be selected from the group consisting of IFN-α, IFN-β, EFN-γ, JL-2, IL-3, IL-4, IL-5, IL-6, BL-7, IL-8, IL-9, IL-IO, IL-12 and IL-15. The invention also provides a method of treating a mammal having cancer or a bacterial or viral infection, comprising the step of administering to the mammal a therapeutically effective amount of the RBC binding conjugate in which the mammalian serum protein is such a cytokine.
In still another embodiment, the mammalian serum protein is a peptide hormone. The peptide hormone can be selected from the group consisting of antimullerian hormone (AMH), adiponectin, adrenocorticotropic hormone (ACTH), angiotensinogen and angiotensin, antidiuretic hormone (ADH), atrial-natriuretic peptide (ANP), calcitonin, cholecystokinin (CCK), corticotropin-releasing hormone (CRH), erythropoietin (EPO), follicle stimulating hormone (FSH), gastrin, glucagon, gonadotropin-releasing hormone (GnRH), growth hormone-releasing hormone (GHRH), human chorionic gonadotropin (hCG), growth hormone (GH), insulin, insulin-like growth factor (IGF), leptin, luteinizing hormone (LH), melanocyte stimulating hormone (MSH or α-MSH), neuropeptide Y, oxytocin, parathyroid hormone (PTH), prolactin (PRL), relaxin, renin, secretin, somatostatin, thrombopoietin, thyroid-stimulating hormone (TSH), and thyrotropin-releasing hormone (TRH). The invention also provides a method for hormone replacement therapy in a mammal, comprising the step of administering to the mammal a therapeutically effective amount of the RBC binding conjugate in which the mammalian serum protein is such a peptide hormone.
In one embodiment, the enzyme is selected from the group consisting of L- asparagine, L-glutaminase-L-asparaginase, L-methioninase, L-phenylalanϊne ammonialyase, L-arginase, L-tyrosinase, L-serine dehydratase, L-threonine deaminase, indolyl-3-alkane hydroxylase, neuraminidase, ribonuclease, a protease, pepsin, and a carboxypeptidase. The invention provides a method of treating a mammal having a cancer, comprising the step of administering to the mammal a therapeutically effective amount of the RBC binding conjugate which contains such an enzyme.
In another embodiment, the enzyme is lysostaphin. The invention also provides a method of treating a mammal having a bacterial infection, comprising the step of administering to the mammal a therapeutically effective amount of the RBC binding conjugate in which the enzyme is lysostaphin.
In a preferred embodiment, the mammalian serum protein or the enzyme is covalently conjugated at a selected residue to the monoclonal antibody or fragment thereof. In one embodiment, the selected residue is selected from the group consisting of a cysteine residue, a residue comprising a reactive carbonyl or carboxylic acid group when oxidized, and a lysine residue.
In preferred embodiments, the RBC binding conjugate preserves at least 5%, 15%, 25%, 50%, 90%, or 99% of the biological activity of the mammalian serum protein or the enzyme when unconjugated.
In other preferred embodiments, the RBC binding conjugate further comprises a polymer linker, wherein the mammalian serum protein or the enzyme is covalently conjugated to the monoclonal antibody or fragment thereof via the polymer linker. The polymer linker can be selected from the group consisting of polypeptide, polyalkylene oxide, polyoxyethylenated polyol, polyacrylamide, polyvinyl pyrrolidone, polyvinyl alcohol, and dextran. In a preferred embodiment, the polymer linker is a polyethylene glycol (PEG) linker. In another preferred embodiment, the polymer linker is a PEG hydrazide/aldehyde linker.
In still other preferred embodiments, the RBC binding conjugate is a single polypeptide comprising the monoclonal antibody or fragment thereof fused to the mammalian serum protein or the enzyme.
The invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the RBC binding conjugate of the invention and a pharmaceutically acceptable carrier. In one embodiment, RBC binding conjugate in the pharmaceutical composition is purified. The invention also provides a method of producing a red blood cell (RBC) binding conjugate. In one embodiment, the method comprises contacting a monoclonal antibody or fragment thereof with a mammalian serum protein or with an enzyme, wherein the monoclonal antibody or fragment binds a C3b-like receptor on a red blood cell, wherein the monoclonal antibody or fragment or the mammalian serum protein or enzyme is derivatized with a bifunctional polymer linker such that the monoclonal antibody or fragment thereof or the mammalian serum protein or enzyme comprises a reactive hydrazide group, and wherein (i) the mammalian serum protein or enzyme, if the monoclonal antibody or fragment is derivatized, or (ii) the monoclonal antibody or fragment thereof, if the mammalian serum protein or enzyme is derivatized, comprises a reactive carbonyl or carboxylic acid group, under conditions conducive for reaction between the reactive hydrazide group and the reactive carbonyl or carboxylic acid group, thereby producing the RBC binding conjugate. In another embodiment, the method comprises contacting a monoclonal antibody or fragment thereof with a mammalian serum protein or with an enzyme, wherein the monoclonal antibody or fragment binds a C3b-like receptor on a red blood cell, wherein the monoclonal antibody or fragment thereof or the mammalian serum protein or enzyme is derivatized with a bifunctional polymer linker such that the monoclonal antibody or fragment thereof or the mammalian serum protein or enzyme comprises a reactive carbonyl or carboxylic acid group, and wherein (i) the mammalian serum protein or enzyme, if the monoclonal antibody or fragment thereof is derivatized, or (ii) the monoclonal antibody or fragment thereof, if the mammalian serum protein or enzyme is derivatized, comprises a reactive hydrazide group, under conditions conducive for reaction between the hydrazide group and the reactive carbonyl or carboxylic acid group, thereby producing the RBC binding conjugate. In one embodiment, at least one of the reactive hydrazide group and the reactive carbonyl or carboxylic acid group is attached to an aromatic ring. In a preferred embodiment, the polymer is polyethylene glycol (PEG). In another preferred embodiment, the bifunctional polymer linker is N-hydroxy-succinimidyl polyethylene glycol-benzaldehyde. In one embodiment, the reactive hydrozide group is introduced to the monoclonal antibody or fragment thereof or the mammalian serum protein or enzyme by derivatizing the monoclonal antibody or fragment thereof or the mammalian serum protein with succinimidyl C6 4-hydrazino-nictoinamde acetone hydrazone. In another embodiment, the method further comprises producing the derivatized monoclonal antibody or fragment thereof or the derivatized mammalian serum protein or enzyme.
The invention also provides a red blood cell (RBC)ZRBC binding conjugate that consists essentially of a red blood cell bound to one or more RBC binding conjugate of the invention.
The invention also provides a red blood cell (RBC) binding conjugate comprising a monoclonal antibody or a fragment thereof covalently conjugated to a hormone selected from the group consisting of an amine-derived hormone, a steroid hormone, or sterol hormone, wherein the monoclonal antibody or fragment thereof (i) binds a C3b-like receptor on a red blood cell, and (ii) lacks a functional effector domain. The amine-derived hormone can be any one. from the group consisting of catecholamine, epinephrine, dopamine, norepinephrine, melatonin, serotonin, thyroxine and triiodothyronine. The steroid hormone can be any one from the group consisting of glucocorticoid, mineralocorticoid, androgen, estrogen, and progestagen. The androgen can be any one from the group consisting of testosterone, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), androstenedione, and dihydrotestosterone (DHT). The sterol hormone can be a Vitamin D derivative.
The invention also provides a red blood cell (RBC) binding conjugate comprising a monoclonal antibody or a fragment thereof covalently conjugated to a molecule selected from the group consisting of a DNA damaging agent, an antimetabolite, and an anti-mitotic agent, wherein the monoclonal antibody or fragment thereof (i) binds a C3b-like receptor on a red blood cell, and (ii) lacks a functional effector domain. In one embodiment, the DNA damaging agent is selected from the group consisting of camptothecin, topotecan, doxorubicin, etoposide phosphate, teniposide, sobuzoxane, anthracycline antibiotic, mitomycin antibiotic, cisplatin, busulfan, cyclophosphamide, bleomycin, and tamoxifen. In another embodiment, the anti-metabolite is selected from the group consisting of cytosine, arabinoside, fioxuridine, 5-fluorouracil (5-FU), mercaptopurine, gemcitabine, hydroxyurea (HU), and methotrexate (MTX). In still another embodiment, the anti-mitotic agent is selected from the group consisting of vinblastine, vincristine, and paclitaxel (Taxol). BRIEF DESCRIPTION OF FIGURES
FIG. 1 Conjugate of tPA and 7G9 using PEG(5K)-aldehyde-hydrazino linker. The sample was analyzed with SDS-PAGE with a 3-8% Tris-acetate gradient gel.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides methods and compositions for using erythrocytes (red blood cells, RBCs) as carriers for delivery of therapeutic molecules, e.g., therapeutic enzymes or mammalian serum proteins, or diagnostic agents. The invention involves conjugating the drug or the diagnostic agent to an antigen recognition portion that binds RBC, e.g., anti-CRl antibody or anti-glycophorin A antibody. When administered to a patient, the conjugate binds to RBC, and is delivered to desired locations by the RBC.
In a preferred embodiment, the RBC binding portion and the enzymatic portion in the RBC binding conjugate are linked by a polymer/aldehyde/hydrazide linkage. The polymer/aldehyde/hydrazide linker preferably comprises a water soluble, nonimmunogenic polymer. In a most preferred embodiment, the polymer is a polyethylene glycol (PEG). The RBC binding conjugate can be produced using a bifunctional linker molecule that comprises either a hydrazide functional group or an aldehyde functional group at one end. The functional group at the other end can be any functional group that can attach to a protein, such as an N-hydroxysuccinimide (NHS). Preferably, the hydrazide or aldehyde has an aromatic ring attached next to it. The linker molecule is attached to the RBC binding portion or the enzymatic portion via the second functional group. The hydrazide functional group or aldehyde functional group is then allowed to react with an appropriate counterpart portion to form a hydrazide/aldehyde linkage.
The RBC binding conjugates of the present invention can be used as a means for delivery of many types of therapeutic molecules. In one embodiment, the RBC binding conjugate is used to deliver an anticoagulant or thrombolytic, e.g., a streptokinase, staphylokinase, tissue-type plasminogen activator or tPA, or urokinase, for treating or preventing disease conditions associated with the formation of clots in the blood of a subject. In another embodiment, the RBC binding conjugates of the present invention are used to deliver enzymes in an enzyme replacement therapy, e.g., β-glucocerebrosidase or α-galactosidase A, for treating disease conditions associated with enzyme deficiency in a subject, e.g., various metabolic diseases, such as Gaucher disease and Fabry disease.
In another embodiment, the RBC binding conjugates of the present invention are used to deliver an anti-cancer agent, e.g., L-asparaginase, for treating or preventing cancers in a subject.
In still another embodiment, the RBC binding conjugates of the present invention are used to deliver an anti-infectious agent, e.g., lysostaphin, for treating or preventing infectious diseases in a subject, e.g., various bacterial or viral infections.
In still another embodiment, the RBC binding conjugates of the present invention are used to deliver an antidote, e.g., carboxypeptidase Gl for treating an overdose of certain substance in a subject, e.g., an overdose of methotrexate.
I. RBC BINDING CONJUGATES
The RBC binding conjugate of the present invention is a molecule comprising a RBC binding portion that binds a red blood cell, e.g., via a receptor on the red blood cells, and a drug portion, e.g., an biologically active enzymatic portion. The red blood cell membrane possesses a large number of surface antigens. For example, the more abundant human erythrocyte-specific antigens include but not limited to KeIl glycoprotein, Rh glycoprotein, Landsteiner Wiener (LW) glycoprotein, glycophorin A, Band 3, Lutheran glycoprotein, and Duffy (Fy) glycoprotein (see, e.g., Southcott et al., 1999, Blood 93 :4425-35). Other receptors include but not limited to the type 1 complement receptor (CRl receptor). In the present invention, the RBC binding portion may bind to any suitable blood cell surface antigen. In a preferred embodiment, the RBC binding portion binds a CRl receptor. In another embodiment, the RBC binding portion binds glycophorin A.
As used herein, the term "C3b-like receptor" refers to any mammalian circulatory molecule expressed on the surface of a mammalian blood cell, which has an analogous function to a primate C3b receptor, the CRl, in that it binds to a molecule associated with an immune complex, which is then chaperoned by the blood cell to, e.g., a phagocytic cell for clearance.
Glycophorin A is a glycoprotein that spans the plasma membrane of human red blood cell. Glycophorin A is a 131 amino acid protein that spans the membrane once and presents its amino-terminal end at the extra-cellular surface of the red blood cell. Each RBC has some 500,000 copies of the molecule embedded in its plasma membrane. Rh glycoprotein is a hereogenously glycosylated polypeptide that is associated with the Rh polypeptides in the membrane of the red blood cells. The LW antigen is a single spanning glycoprotein.
In the embodiments in which the RBC binding portion of the conjugate binds to the RBC via a CRl receptor, the RBC binding portion can be any antibody or fragment of an antibody that contains a CRl binding domain. However, the RBC binding portion preferably does not comprise a functional antibody effector domain. The effector domain facilitates transfer of the conjugate to the Fc receptors on phagocytic cells for subsequent proteolysis of the conjugate. By deleting or otherwise inactivating an effector domain in the CRl binding portion, the RBC bound drug conjugate remains in the circulation for a longer period of time. In a preferred embodiment, the RBC binding portion is an anti-CRl monoclonal antibody (mAb) with the effector domain partially or entirely removed. In a preferred embodiment, the anti-CRl monoclonal antibody is H4, H9, H47, H48, 7G9, HB8592, 3D9, 57F, or 1B4 (see, e.g., Talyor et al, U.S. Patent No. 5,487,890, which is incorporated herein by reference in its entirety) with the effector domain partially or entirely removed. In another embodiment, the RBC binding portion portion is an anti-CRl antibody, including but is not limited to, an Fab, an Fab', an (Fab')2, or an Fv fragment of an immunoglobulin molecule, or a single-chain variable region fragment (scFv) with specificity for a C3b-like receptor. The RBC binding portion can also be a chimeric antibody, such as but is not limited to a humanized monoclonal antibody in which the complementarity determining regions are mouse, and the framework regions are human thereby decreasing the likelihood of an immune response in human patients treated with the antibody (United States Patent Nos. 4,816,567, 4,816,397, 5,693,762; 5,585,089; 5,565,332 and 5,821,337, each of which is incorporated herein by reference in its entirety). Although, for simplicity, this disclosure often makes reference to an RBC binding portion that binds a C3b-like receptor, it will be understood by a skilled artisan that the disclosure is equally applicable to antibodies that binds other red blood cell receptors.
RBC binding conjugates can also be attached to a red blood cell via antigens other than a C3b-like receptor. Antibodies that bind other RBC-specirϊc antigens, e.g., glycophorin A, are well-known in the art (see, e.g., Southcott et al., 1999, Blood 93:4425-35, which is incorporated herein in its entirety).
In one embodiment, the drug portion comprises a polypeptide or glycopolypeptide that is biologically active. In a preferred embodiment, the drug portion comprises an enzyme. The enzyme can be, but is not limited to, an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase or a ligase, or any analogue thereof.
In specific embodiments, the enzyme can be but is not limited to asparaginase, arginase, arginine deaminase, adenosine deaminase, superoxide dismutase, catalase, chymotrypsin, lipase, uricase, bilirubin oxidase, glucose oxidase, glucosidase, galactosidase, ghicocerebrosidase, or glucuronidase, or any analogue thereof having enzymatic activity.
The drug portion can also comprise a protein selected from among, but not limited to, the following Factor VIH and polypeptide hormones such as insulin, ACTH, glucagon, somatostatin, somatotropins, thymosin, parathyroid hormone, pigmentary hormones, somatomedins, erythropoietin, luteinizing hormone, hypothamic releasing factors, antidiuretic hormones, interleukins, interferons, colony stimulating factors, hemoglobin, prolactin, or anti-angiogenic agents, e.g., angiostatin or endostatin, or any biologically active analogue thereof.
The drug portion can also comprise a glycopolypeptide such as, but are not limited to, immunoglobulins, chorionic gonadotrophin, follicle-stimulating hormone, thyroid-stimulating hormone, ovalbumin, bovine serum albumin (BSA), lectins, tissue plasminogen activator, glycosylated interleukins, glycosilated interferons or glycosilated colony stimulating factors, or any analogue thereof. The drug portion can also comprise an allergen protein or glycoprotein for use as tolerance inducer in reducing allergenicity of such proteins (see, e.g., Sehon et al., 1987, Pharmacol. Toxicol. Proteins 65:205-19).
The drug portion can also comprise a small molecule drug, including but not limited to DNA damaging agents, anti-metabolites, anti-mitotic agents. DNA damaging agents include but are not limited to topoisomerase inhibitor, DNA binding agent. A topoisomerase inhibitor that can be used in conjunction with the invention can be a topoisomerase I (Topo I) inhibitor, a topoisomerase II (Topo II) inhibitor, or a dual topoisomerase I and II inhibitor. A topo I inhibitor can be for example from any of the following classes of compounds: camptothecin analogue (e.g., karenitecin, aminocamptothecin, lurtotecan, topotecan, irinotecan, BAY 56-3722, rubitecan, GI14721, exatecan mesylate), rebeccamycin analogue, PNU 166148, rebeccamycin, TAS-103, camptothecin (e.g., camptothecin polyglutamate, camptothecin sodium), intoplicine, ecteinascidin 743, J- 107088, pibenzimol, camptothecin, topotecan (hycaptamine), irinotecan (irinotecan hydrochloride), belotecan, or an analogue or derivative of any of the foregoing.
Topo π inhibitors include but not limited to anthracycline antibiotics (e.g., carubicin, pirarubicin, daunorubicin citrate liposomal, daunomycin, 4-iodo-4- doxydoxorubicin, doxorubicin, n,n-dibenzyl daunomycin, morpholinodoxorubicin, aclacinomycin antibiotics, duborimycin, menogaril, nogalamycin, zorubicin, epirubicin, marcellomycin, detorubicin, annamycin, 7-cyanoquinocarcinol, deoxydoxorubicin, idarubicin, GPX-100, MEN-10755, valrubicin, KRN55OO), epipodophyllotoxin compound (e.g., podophyllin, teniposide, etoposide, GL331, 2-ethylhydrazide), anthraquinone compound (e.g., ametantrone, bisantrene, mitoxantrone, anthraquinone), ciprofloxacin, acridine carboxamide, amonafide, anthrapyrazole antibiotics (e.g., teloxantrone, sedoxantrone trihydrochloride, piroxantrone, anthrapyrazole, losoxantrone), TAS- 103, fbstriecin, razoxane, XK469R, XK469, chloroquinoxaline sulfonamide, merbarone, intoplicine, elsamitrucin, CI-921, pyrazoloacridine, elliptinium, amsacrine, doxorubicin (Adriamycin), etoposide phosphate (etopofos), teniposide, sobuzoxane, or an analogue or derivative of any of the foregoing.
DNA binding agents include but are not limited to a DNA groove binding agent, e.g., DNA minor groove binding agent; DNA crossϋnking agent; intercalating agent; and DNA adduct forming agent. A DNA minor groove binding agent can be an anthracycline antibiotic, mitomycin antibiotic (e.g., porfϊromycin, KW-2149, mitomycin B, mitomycin A, mitomycin C), chromomycin A3, carzelesin, actinomycin antibiotic (e.g., cactinomycin, dactinomycin, actinomycin Fl), brostallicin, echinomycin, bizelesin, duocarmycin antibiotic (e.g., KW 2189), adozelesin, olivomycin antibiotic, plicamycin, zinostatin, distamycin, MS-247, ecteinascidin 743, amsacrine, antbxamycin, and pibenzimol, or an analogue or derivative of any of the foregoing.
DNA crosslinking agents include but are not limited to antineoplastic alkylating agent, methoxsalen, mitomycin antibiotic, psoralen. An antineoplastic alkylating agent can be a nitrosourea compound (e.g., cystemustine, tauromustine, semustine, PCNU, streptozocin, SarCNU, CGP-6809, carmustine, fotemustine, methylnitrosourea, nimustine, ranimustine, ethylnitrosourea, lomustine, chlorozotocin), mustard agent (e.g., nitrogen mustard compound, such as spiromustine, trofosfamide, chlorambucil, estramustine, 2,2,2-trichlorotriethylamine, prednimustine, novembichin, phenamet, glufosfamide, peptichemio, ifosfamide, defosfamide, nitrogen mustard, phenesterin, mannomustine, cyclophosphamide, melphalan, perfosfamide, mechlorethamine oxide hydrochloride, uracil mustard, bestrabucU, DHEA mustard, tallimustine, mafosfamide, aniline mustard, chlornaphazine; sulfur mustard compound, such as bischloroethylsulfide; mustard prodrug, such as TLK286 and ZD2767), ethylenimine compound (e.g., mitomycin antibiotic, ethylenimine, uredepa, thiotepa, diaziquone, hexamethylene bisacetamide, pentamethylmelamine, altretamine, carzinophilin, triaziquone, meturedepa, benzodepa, carboquone), alkylsulfonate compound (e.g., dimethylbusulfan, Yoshi-864, improsulfan, piposulfan, treosulfan, busulfan, hepsulfam), epoxide compound (e.g., anaxirone, mitolactoϊ, dianhydrogalactitol, teroxirone), miscellaneous alkylating agent (e.g., ipomeanol, carzelesin, methylene dimethane sulfonate, mitobronitol, bizelesin, adozelesin, piperazinedione, VNP40101M, asaley, 6- hydroxymethylacylfulvene, EO9, etoghicid, ecteinascidin 743, pipobroman), platinum compound (e.g., ZD0473, liposomal-cisplatin analogue, satraplatin, BBR 3464, spiroplatin, ormaplatin, cisplatin, oxaliplatin, carboplatin, lobaplatin, zeniplatin, iproplatin), triazene compound (e.g., imidazole mustard, CB 10-277, mitozolomide, temozolomide, procarbazine, dacarbazine), picoUne compound (e.g., penclomedine), cisplatin, dibromodulcitol, fotemustine, ifosfamide (ifosfamid), ranimustine (ranomustine), nedaplatin (latoplatin), bendamustine (bendamustine hydrochloride), eptaplatin, temozolomide (methazolastone), carboplatin, altretamine (hexamethylmelamine), prednimustine, oxaliplatin (oxalaplatinum), carmustine, thiotepa, leusulfon (busulfan), lobaplatin, cyclophosphamide, bisulfan, melphalan, and chlorambucil, or an analogue or derivative of any of the foregoing.
Intercalating agents include but are not limited to an anthraquinone compound, bleomycin antibiotic, rebeccamycin analogue, acridine, acridine carboxamide, amonafide, rebeccamycin, anthrapyrazole antibiotic, echinomycin, psoralen, LU 79553, BW A773U, crisnatol mesylate, benzo(a)pyrene-7,8-diol-9,10-epoxide, acodazole, elliptinium, pixantrone, or an analogue or derivative of any of the foregoing.
DNA adduct forming agents include but are not limited to enediyne antitumor antibiotic (e.g., dynemicin A, esperamicin Al, zinostatin, dynemicin, calicheamicin gamma II), platinum compound, carmustine, tamoxifen (e.g., 4-hydroxy-tamoxifen), psoralen, pyrazine diazohydroxide, benzo(a)pyrene-7,8-diol-9,10-epoxide, or an analogue or derivative of any of the foregoing.
Anti-metabolites include but are not limited to cytosine, arabinoside, floxuridine, 5-fluorouracil (5-FU), mercaptopurine, gemcitabine, hydroxyurea (HU), and methotrexate (MTX).
Anti-mitotic agents include but are not limited to Vinblastine, Vincristine, and Paclitaxel (Taxol).
In another embodiment, the drug portion is an amine-derived hormone, such as but not limited to catecholamine; adrenaline (or epinephrine); dopamine; noradrenaline (or norepinephrine); tryptophan derivatives, e.g., melatonin (N-acetyl-5- methoxytryptamine), serotonin (5-HT); or a tyrosine derivative, e.g., thyroxine (T4) and triiodothyronine (T3).
In another embodiment, the drug portion is a peptide hormone, including but not limited to antimullerian hormone (AMH, also mullerian inhibiting factor or hormone); adiponectin; adrenocorticotropic hormone (ACTH, also corticotropin); angiotensinogen and angiotensin; antidiuretic hormone (ADH, also vasopressin, arginine vasopressin, AVP); atrial-natriuretic peptide (ANP, also atriopeptin); calcitonin; cholecystokinin (CCK); corticotropin-releasing hormone (CRH); erythropoietin (EPO); follicle stimulating hormone (FSH); gastrin; glucagon; gonadotropin-releasing hormone (GnRH); growth hormone-releasing hormone (GHRH); human chorionic gonadotropin (hCG); growth hormone (GH or hGH); insulin; insulin-like growth factor (IGF, also somatomedin); leptin; luteinizing hormone (LH); melanocyte stimulating hormone (MSH or α-MSH); neuropeptide Y; oxytocin; parathyroid hormone (PTH); prolactin (PRL); relaxin; renin; secretin; somatostatin; thrombopoietin; thyroid-stimulating hormone (TSH); or thyrotropin-releasing hormone (TRH).
In still another embodiment, the drug portion is a steroid hormone, including but not limited to glucocorticoids, e.g., Cortisol; Mineralocorticoids, e.g., aldosterone; sex steroids, e.g., androgens (testosterone, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), androstenedione, dihydrotestosterone (DHT)), estrogen, e.g., estradiol, progestagen, e.g., progesterone, progestin.
In still another embodiment, the drug portion is a sterol hormone, including but not limited to Vitamin D derivatives, e.g., calcitriol.
In still another embodiment, the drug portion is a lipid or phospholipid hormone (eicosanoid), including but not limited to prostaglandin, leukotriene, prostacyclin, or thromboxane.
In still another embodiment, the drug portion is a cytokine such as IFN-α, IFN-β, IFN-γ, IL"2, IL-3, IL-4, IL-5, JL-6, JL-7, IL-S, IL-9, IL-IO, IL-12 and IL-15.
The drug portion can also be an agonist or antagonist to a cell surface receptor, including but not limited to G-protein-coupled receptors, acetylcholine receptors, adenosine receptors, adrenoceptors (adrenergic receptors), Type-B GABA receptors (γ- Aminobutyric acid or GABA); angiotensin receptors; cannabinoid receptors; cholecystokinin receptors; dopamine receptors; glucagon receptors; metabotropic ghitamate receptors; histamine receptors; opioid receptors; secretin receptors; serotonin receptors, somatostatin receptors; tyrosine kinase receptors, such as erythropoietin receptor; insulin receptor, growth factors and cytokines receptors; guanylyl cyclase receptors; GC-A & GC-B receptors, Le., receptors of atrial-natriuretic peptide (ANP) and other natriuretic peptides GC-C, e.g., guanylin receptor; ionotropic receptors, such as nicotinic; acetylcholine receptors; glycine receptor (GIyR); ghitamate receptors, e.g., NMDA receptor, AMPA receptor, and Kainate receptor. A RBC binding conjugate can be used in hormone replacement therapy. In one embodiment, a RBC binding conjugate comprising an estrogen or a progestagen as the drug portion can be used for contraception. In another embodiment, a RBC binding conjugate comprising a thyroxine, e.g., levothyroxine, as the drug portion can be used for treating hypothyroidism. In another embodiment, a RBC binding conjugate comprising a steroid as the drug portion can be used for treating autoimmune diseases and certain respiratory disorders. In another embodiment, a RBC binding conjugate comprising an insulin as the drug portion can be used for treating diabetics.
The drug portion can also be a diagnostic agent, e.g., a contrast agent for magnetic resonance imaging or a radioactive label for imaging of blood vessels. Examples of diagnostic agents include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bio luminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. See generally U.S. Patent No. 4,741,900 for metal ions which can be conjugated to proteins for use as diagnostics. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidiα/biotin and avidin/biotin. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. Examples of luminescent material include luminol. Examples of bioluminescent materials include luciferase, luciferin, and aequorin. Examples of suitable radioactive material include I, I, In or Tc.
In preferred embodiments of the invention, the RBC binding conjugate comprises an RBC binding portion covalently conjugated to one or more drug portions, such as but not limited to, enzymes. In preferred embodiments, the RBC binding conjugate comprises an RBC binding portion covalently-conjugated to at least 1, 2, 3, 4, 5 or 6 drug portions. Preferably, the drug portions are attached to the RBC binding portion, most preferably at different sites, in such a way that their therapeutic activity is not compromised. In preferred embodiments, the RBC binding conjugate of the invention retain at least 5%, 15%, 25%, 50%, 90% or 99% of the therapeutic activity as compared to the unconjugated drug portion. In one embodiment, the drug portion is attached at a selected residue on the RBC binding portion. Preferably, such a selected residue is selected so that the drug portion's therapeutic activity is not comprised.
If more than one drug portion is covalently-conjugated to one RBC binding portion, the drug portions can be the same or different. In embodiments in which the drug portions are different drug portions, such drug portions can have different therapeutic activities.
The RBC binding portion, e.g., an anti-CRl antibody or anti-glycophorin A antibody, and the drug portion(s) are preferably covalently conjugated by a linker. Any cross-Unking chemistry known in art for conjugating proteins can be used in conjunction with the present invention. In one embodiment of the invention, the RBC binding portion and the drug portion are produced using cross-linking agents sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (s SMCC) and N-succinimidyl-S-acetyl-thioacetate (SATA).
In a preferred embodiment, the RBC binding portion and the drug portion are covalently conjugated via a water soluble polymer linker. The polymer linker can be but is not limited to a polypeptide, a polyalkylene oxide, a polyoxyethylenated polyol, a polyacrylamide, a polyvinyl pyrrolidone, a polyvinyl alcohol, and a dextran. In one preferred embodiment of the invention, the RBC binding portion and the drug portion are conjugated via a poly-(ethylene glycol) linker (PEG). In this embodiment, the PEG moiety can have any desired length. For example, the PEG moiety can have a molecular weight in the range of 200 to 40,000 Daltons. Preferably, the PEG moiety has a molecular weight in the range of 500 to 8000 Daltons. Such a RBC binding conjugate can be produced using cross-linking agents, e.g., N-succinimidyl-S-acetyl-thioacetate (SATA) and a poly(ethylene glycol)-maleimide, e.g., monomethoxy poly(ethylene glycol)-maleimide (mPEG-MAL), NHS-poly(ethylene glycol)-maleimide (PEG-MAL), or PEG hydrazine. Such a RBC binding conjugate can also be produced using NHS- PEG-benzaldehyde (PBA) and succinimidyl C64-hydrazino-nictoinamde acetone hydrazone. Methods of producing PEG-linked polypeptides that can be used are described in WO 2004024889.
In another preferred embodiment of the invention, the RBC binding conjugate is a polypeptide comprising an RBC binding portion and a drug portion, such as but not limited to, an enzyme. Such a polypeptide can be produced using methods known in the art, e.g., using recombinant techniques (see, e.g., WO 01/80883 and WO 02/46208, each of which is incorporated herein by reference in its entirety).
In one embodiment, the RBC binding conjugate of the invention is a polypeptide molecule which consists essentially of, or alternatively comprises, a RBC binding domain, e.g., a binding domain that binds a CRl receptor, bound to the amino terminus of a polypeptide drug portion, e.g., an enzymatic portion. The binding domain can be an Fab, an Fab', an (Εab%, or an Fv fragment of an immunoglobulin molecule, or a single- chain variable region fragment (scFv), i.e., a VL fused via a polypeptide linker to a VH. The drug portion can be any one disclosed above. The RBC binding conjugate can optionally comprises a linker polypepide between the binding domain and the polypeptide drug portion.
In another embodiment, the RBC binding conjugate of the invention is a dimeric molecule consisting of a first polypeptide consisting essentially of, or comprising, a RBC binding domain, e.g., a binding domain that binds a CRl receptor, bound to the amino terminus of a first linker polypeptide, and a second polypeptide, consisting essentially of, or comprising, a second linker polypeptide with a polypeptide drug portion, e.g., an enzymatic portion, bound to the second linker polypeptide's carboxy terminus, wherein the first and second linker polypeptides are complementary to and can associate with each other. The linkers do not comprise a functional Fc domain. In a specific embodiment, the first polypeptide consists essentially of, or comprising, a variable light chain domain (VL) and constant light chain domain (CL) followed by the first linker molecule. In another specific embodiment, the first polypeptide consists essentially of, or comprising, a scFv bound to the amino terminus of the first linker molecule.
In a specific embodiment, the RBC binding conjugate comprises a RBC binding portion that binds CRl or glycophorin A covalently conjugated to a drug portion comprising a tissue-type plasminogen activator, streptokinase, staphylokinase, or urokinase via a PEG linker. In a preferred embodiment, the PEG linker is attached to the drug portion at a moiety comprising an activated carboxylic acid group or a reactive carbonyl group. In another preferred embodiment, the PEG linker is attached to the drug portion at an oxidized carbohydrate. In another preferred embodiment, the RBC binding conjugate comprises a RBC binding portion attached to a PEG linker that links with the drug portion via a hydrazide/aldehyde linkage.
π. PRODUCTION OF RBC BINDING CONJUGATES
The RBC binding conjugate of the invention can be produced in various ways. Examples of methods for production include, but are not limited to, cross-linking, recombinant technique, or protein trans-splicing. These methods as well as methods for producing the RBC binding portions, the drug portions, and methods for purification and characterization of the RBC binding conjugates are described in Sections 5.2.1-5.2.4, infra.
A. PRODUCTION OF RBC BINDING PORTIONS
The antibodies can be immunoglobulin molecules. The immunoglobulin molecules are encoded by genes which include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant regions, as well as a myriad of immunoglobulin variable regions. Light chains are classified as either kappa or lambda. Light chains comprise a variable light (VL) and a constant light (CL) domain. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD and IgE, respectively. Heavy chains comprise variable heavy (VH), constant heavy 1 (CHl), hinge, constant heavy 2 (CH2), and constant heavy 3 (CH3) domains. The IgG heavy chains are further sub-classified based on their sequence variation, and the subclasses are designated IgGl, IgG2, IgG3 and IgG4.
Antibodies can be further broken down into two pairs of a light and heavy domain. The paired VL and VH domains each comprise a series of seven subdomains: framework region 1 (FRl), complementarity determining region 1 (CDRl), framework region 2 (FR2), complementarity determining region 2 (CDR2), framework region 3 (FR3), complementarity determining region 3 (CDR3), framework region 4 (FR4) which constitute the antibody-antigen recognition domain.
A chimeric antibody may be made by splicing the genes from a monoclonal antibody of appropriate antigen specificity together with genes from a second human antibody of appropriate biologic activity. More particularly, the chimeric antibody may be made by splicing the genes encoding the variable regions of an antibody together with the constant region genes from a second antibody molecule. This method is used in generating a humanized monoclonal antibody wherein the complementarity determining regions are mouse, and the framework regions are human thereby decreasing the likelihood of an immune response in human patients treated with the antibody (United States Patent Nos. 4,816,567, 4,816,397, 5,693,762; 5,585,089; 5,565,332 and 5,821,337, each of which is incorporated herein by reference in its entirety).
An antibody suitable for use in the present invention may be obtained from natural sources or produced by hybridoma, recombinant or chemical synthetic methods, including modification of constant region functions by genetic engineering techniques (United States Patent No. 5,624,821). The antibody of the present invention may be of any isotype, but is preferably human IgGl.
An antibody can also be a single-chain antibody (scFv) which generally comprises a fusion polypeptide consisting of a variable domain of a light chain fused via a polypeptide linker to the variable domain of a heavy chain.
An RBC binding portion that binds a human CRl receptor can be produced by known methods. In one embodiment, the RBC binding portion, e.g., an IgG, can be prepared using standard hybridoma precedure known in the art (see, for example, Kohler and Milstein, 1975, Nature 256:495-497; Hogg et al., 1984, Eur. J. Immunol. 14:236- 243; O'Shea et al., 1985, J. Immunol. 134:2580-2587; Schreiber, U.S. Patent 4,672,044). A suitable mouse is immunized with an appropriate RBC specific antigen, e.g., human CRl, which can be purified from human erythrocytes. The spleen cells obtained from the immunized mouse are fused with an immortal mouse myeloma cell line which results in a population of hybridoma cells, including a hybridoma that produces an RBC binding portion. The hybridoma which produces the antibody that can be used as a RBC binding portion is then selected, or 'cloned', from the population of hybridomas using conventional techniques such as enzyme linked immunosorbent assays (ELISA). Hybridoma cell lines expressing antibodies for use as RBC binding portions can also be obtained from various sources, for example, the murine monoclonal antibody that binds human CRl described in U.S. Patent 4,672,044 is available as hybridoma cell line ATCC HB 8592 from the American Type Culture Collection (ATCC). The obtained hybridoma cells are grown and washed using standard methods known in the art. The antibodies are then recovered from supernatants. IQ embodiments in which an anti-CRl antibody is used, the Fc domain (also referred to herein as the "effector domain") is removed or otherwise inactivated, e.g., by introducing one or more mutations into the Fc domain such that it loses the effector function.
In other embodiments, nucleic acids encoding the heavy and light chains of an RBC antibody, e.g., an IgG, that binds a RBC surface antigen, are prepared from the hybridoma cell line by standard methods known in the art. As a non-limiting example, cDNAs encoding the heavy and light chains of the IgG are prepared by priming mRNA using appropriate primers, followed by PCR amplification using appropriate forward and reverse primers. Any commercially available kits for cDNA synthesis can be used. The nucleic acids are used in the construction of expression vector(s). The expression vector(s) are transfected into a suitable host. Non-limiting examples include E. coli, yeast, insect cell, and mammalian systems, such as a Chinese hamster ovary cell line. Antibody production can be induced by standard method known in the art.
An antibody that binds a RBC antigen can be prepared by immunizing a suitable subject with an appropriate RBC-specific antigen which can be purified from human erythrocytes. The antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide. If desired, the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction.
At an appropriate time after immunization, e.g., when the specific antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256:495-497), the human B cell hybridoma technique by Kozbor et al. (1983, Immunol. Today 4:72), the EBV- hybridoma technique by Cole et al. (1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. The technology for producing hybridomas is well known (see Current Protocols in Immunology, 1994, John Wiley & Sons, Inc., New York, NY). Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide of interest, e.g., using a standard ELISA assay. Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies. For example, the monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., 1975, Nature, 256:495, or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567). The term "monoclonal antibody" as used herein also indicates that the antibody is an immunoglobulin.
In the hybridoma method of generating monoclonal antibodies, a mouse or other appropriate host animal, such as a hamster, is immunized as hereinabove described to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization (see, e.g., U.S. Patent No. 5,914,112, which is incorporated herein by reference in its entirety.)
Alternatively, lymphocytes may be immunized in vitro. Lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1986). The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the SaIk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 cells available from the American Type Culture Collection, Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, 1984, J. Immunol., 133:3001; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immuno-absorbent assay (ELISA). The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al., 1980, Anal. Biochem., 107:220.
After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI- 1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal. The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal antibody directed against an appropriate RBC-specific antigen can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the antigen. Kits for generating and screening phage display libraries are commercially available (e.g., Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene antigen SurfZAP™ Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S. Patent Nos. 5,223,409 and 5,514,548; PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO 92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs et al., 1991, Bio/Technology 9:1370-1372; Hay et al., 1992, Hum. Antibod. Hybridomas 3:81-85; Huse et al., 1989, Science 246:1275-1281; Griffiths et al., 1993, EMBO J. 12:725-734.
In addition, techniques developed for the production of "chimeric antibodies" (Morrison, et al., 1984, Proc. Natl. Acad. ScL, 81, 6851-6855; Neuberger, et al., 1984, Nature 312, 604-608; Takeda, et al., 1985, Nature, 314, 452-454) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region. (See, e.g., Cabilly et al., U.S. Patent No. 4,816,567; and Boss et al., U.S. Patent No. 4,816,397, each of which is incorporated herein by reference in its entirety)
Humanized antibodies are antibody molecules from non-human species having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule, (see e.g., U.S. Patent No. 5,585,089, which is incorporated herein by reference in its entirety.) Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT Publication No. WO 87/02671; European Patent Application 184,187; European Patent Application 171,496; European Patent Application 173,494; PCT Publication No. WO 86/01533; U.S. Patent No. 4,816,567 and 5,225,539; European Patent Application 125,023; Better et al., 1988, Science 240:1041-1043; Liu et al., 1987, Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al., 1987, Proc. Natl. Acad. Sci. USA 84:214-218; Nisbimura et al., 1987, Cane. Res. 47:999-1005; Wood et al., 1985, Nature 314:446-449; Shaw et al., 1988, J. Natl. Cancer Inst. 80:1553-1559; Morrison 1985, Science 229:1202-1207; Oi et al., 1986, Bio/Techniques 4:214; Jones et al., 1986, Nature 321:552-525; Verhoeyan et al., 1988, Science 239:1534; and Beidler et al., 1988, J. Immunol. 141:4053-4060.
Complementarity determining region (CDR) grafting is another method of humanizing antibodies. It involves reshaping murine antibodies in order to transfer full antigen specificity and binding affinity to a human framework (Winter et al. U.S. Patent No. 5,225,539). CDR-grafted antibodies have been successfully constructed against various antigens, for example, antibodies against JL-2 receptor as described in Queen et aL, 1989 (Proc. Natl. Acad. Sci. USA 86:10029); antibodies against cell surface receptors-CAMPATH as described in Riechmann et aL (1988, Nature, 332:323; antibodies against hepatitis B in Cole et al. (1991, Proc. Natl. Acad. Sci. USA 88:2869); as well as against viral antigens-respiratory syncitial virus in Tempest et al. (1991, Bio-Technology 9:267). CDR-grafted antibodies are generated in which the CDRs of the murine monoclonal antibody are grafted into a human antibody. Following grafting, most antibodies benefit from additional amino acid changes in the framework region to maintain affinity, presumably because framework residues are necessary to maintain CDR conformation, and some framework residues have been demonstrated to be part of the antigen binding site. However, in order to preserve the framework region so as not to introduce any antigenic site, the sequence is compared with established germline sequences followed by computer modeling.
Completely human antibodies are particularly desirable for administration to human patients. Such antibodies can be produced using transgenic mice which are incapable of expressing endogenous immunoglobulin heavy and light chain genes, but which can express human heavy and light chain genes. The transgenic mice are immunized in the normal fashion with an appropriate RBC-specifϊc antigen.
Monoclonal antibodies directed against the RBC-specifϊc antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see e.g., U.S. Patent 5,625,126; U.S. Patent 5,633,425; U.S. Patent 5,569,825; U.S. Patent 5,661,016; and U.S. Patent 5,545,806. In addition, companies such as Abgenix, Inc. (Freemont, CA; see, for example, U.S. Patent No. 5,985,615) and Medarex, Inc. (Princeton, NJ), can be engaged to provide human antibodies directed against the RBC-specifϊc antigen using technology similar to that described above.
Completely human antibodies which recognize and bind a selected epitope can be generated using a technique referred to as "guided selection." In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope (Jespers et al., 1994, Bio/technology 12:899-903).
A pre-existing anit-CRl antibody, including but not limited to H4, H9, H47, H48, 7G9, HB8592, 3D9, 57F, and 1B4 (see, e.g., Talyor et al., U.S. Patent No. 5,487,890, which is incorporated herein by reference in its entirety), can also be used. In a preferred embodiment, a hybridoma cell line secreting a high-affinity anti-CRl monoclonal antibody, e.g., 7G9 (murine IgG∑a, kappa), is used to generate a master cell bank (MCB). Preferably, the master cell bank is tested for mouse antibody production, mycoplasma and sterility. The RBC binding portion is then produced and purified from ascites fluid. In another preferred embodiment, the anti-CRl monoclonal antibody used for the production of the RBC binding conjugates is produced in vitro (hollow-fiber bioreactor) and purified under cGMP. Anti-CRl monoclonal antibodies without an effector domain can be generated by treating an appropriate antibody with an enzyme such as pepsin or papain.
B. PRODUCTION OF DRUG PORTIONS
The drug portion of the RBC binding conjugate of the invention can be produced by various methods known in the art.
In a preferred embodiment, the drug portion can be modified such that it can be attached to a selected residue of an RBC binding portion. Preferably, such a residue is selected so that the antigen-binding affinity is not compromised after the fragment is covalently-conjugated to the RBC binding portion. More preferably, such a residue is on the surface of the RBC binding portion. In a preferred embodiment, a cysteine residue is engineered into an appropriate location in a drug portion to allow site-specific attachment of the drug portion to an RBC binding portion (see, e.g., Lyons et al., Protein Engineering 3:703-708, which is incorporated herein in its entirety). A skilled person in the art MdIl be able to determine the location where the cysteine residue is introduced as well as the method that can be used to generate such an engineered drug portion. In a preferred embodiment, the cysteine is introduced to the C-terminus of the drug portion.
In another preferred embodiment, the drug portion is modified such that it can be attached at a selected residue to an RBC binding portion. Preferably, such a residue is selected so that the activity of the drug is not compromised after it is covalently- conjugated to the RBC binding portion. More preferably, such a residue is away from the active site of the drug portion. A skilled person in the art will be able to determine the residue to which the RBC binding portion is attached as well as the method that can be used to produce such an attachment.
In another preferred embodiment, the drug portion containing a cysteine residue is produced by a host cell in such a manner that a cysteinyl free thiol is maintained (see, e.g., Carter, U.S. Patent No. 5,648,237, which is incorporated herein in its entirety). The drug portion containing cysteinyl free thiol (also referred to as "drug-cys-SH") can then be used to produce the RBC binding conjugate of the invention directly with an appropriate RBC binding portion or an appropriately derivatized RBC binding portion which can react with the free thiol to form a covalent bond. RBC binding portion can be a maleimide derivatized anti-CRl antibody, e.g., an anti-CRl antibody derivatized with sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-l-carboxylate (sSMCC) or a poly(ethylene glycol)-maleimide, e.g., monomethoxy poly(ethylene glycol)-maleimide (mPEG-MAL) or NHS-poly(ethylene glycol)-maleimide (PEG-MAL). Alternatively, the RBC binding portion can be a thiolated RBC binding portion, e.g., an RBC binding portion derivatized with N-succinimidyl-S-acetyl- thioacetate (SATA), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). The drug-cys-SH can be covalently-conjugated with the thiolated RBC binding portion via a disulfide bond.
In still another preferred embodiment, the drug portion contains a recombinantly introduced glycosylation site. Such a drug portion can be produced use the method described in WO92/16555, which is incorporated herein by reference in its entirety.
C. PRODUCTION OF RBC BINDING CONJUGATES
The RBC binding conjugate of the present invention can be a covalent conjugate of one or more polypeptide drug portions, e.g., enzymes, with a polypeptide, e.g., an antibody, that binds the red blood cell. Such a conjugate can be produced by any standard chemical conjugating methods known in the art, such methods can employ either maleimide chemistry, biotin chemistry, or hydrazide chemistry. Preferably, a conjugating method employing a bifunctional linker is used. For example, cross-linking agents, including but not limited to, protein A, glutaraldehyde, carbodiimide, N-succinimidyl-S-acetyl-thio acetate (SATA),
N-succinirnidyl-3-(2-pyridyldithio)propionate (SPDP), sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sSMCC), succinimidyl 6- hydrazinonicotinate acetone hydrazone (SANH) or succinimidyl 4-formyl benzoate (SFB) can be used. Preferably, a conjugating method employing a bifunctional "water- soluble polymer linker, e.g., a poly(ethylene glycol) linker, is used. The conjugate of the invention can also be prepared by recombinant methods or by a method utilizing protein trans-splicing.
In one embodiment, SATA is used to derivatize the drug portion. A skilled person in the art will be able to determine the concentrations of the drug portion and SATA. In one embodiment, by way of example but not limitation, the following protocol is used. A solution of SATA in DMSO is prepared. The drug portion is dialyzed against PBSE buffer. The coupling reaction is initiated by combining the drug portion and SATA at a molar ratio of about 1 :6. The reactants are mixed by inversion and incubated at room temperature for a desired period of time with mixing. A hydroxylamine HCl solution is prepared by adding hydroxyamine and EDTA to MES. The Hydroxylamine HCl solution is added to the reaction mixture from the SATA coupling step at an appropriate molar ratio, e.g., a molar ratio of about 2000:1, and incubated for a desired period of time at room temperature under argon atmosphere. The reaction mixture is then desalted by chromatography, e.g., using an Amersham Hi-Prep desalting column in MES buffer. The SATA derivatized drag portion can then be used with an appropriately derivatized RBC binding portion, e.g., a maleimide derivatized RBC binding portion, to produce the RBC binding conjugate of the invention.
In another embodiment, the drug portion containing a cysteine residue is produced by a host cell in such a manner that a free thiol is maintained (see, e.g., Carter, U.S. Patent No. 5,648,237, which is incorporated herein in its entirety). Preferably, the drug portion containing a free thiol is secreted by the host cell. The drug portion containing the free thiol can then be recovered and used with an appropriately derivatized RBC binding portion, e.g., a maleimide derivatized RBC binding portion, to produce the RBC binding conjugate of the invention.
In another embodiment, the RBC binding portion is derivatized with a maleimide using any method known in the art. A skilled person in the art will be able to determine the concentrations of the RBC binding portion and maleimide to achieve a desired number of cross-Unking sites on the RBC binding portion. In a specific embodiment, the antibody is derivatized with maleimide as follows: a fresh stock solution of sSMCC Conjugation solution is prepared in PBSE buffer; the antibody is dialyzed exhaustively against PBSE buffer; the coupling reaction is initiated by combining the antibody and sSMCC at a molar ratio of about 1 :6; the reactants are mixed by inversion and incubated at room temperature for 60 min with mixing; and the sSMCC-antibody is recovered by size exclusion chromatography using FPLC with two Pharmacia 26/10 Desalting Columns in series (cat#17-5087-01). The column is preferably pre-washed with distilled water followed by PBSE buffer according to the manufacturer's instructions before loaded with the reaction mixture. The maleimide modified antibody is eluted in the void volume with PBSE buffer and should be used within 15 min. The maleimide derivatized RBC binding portion can then be allowed to react with an appropriately drug portion, e.g., a SATA derivatized drug portion, to produce the RBC binding conjugate of the invention.
In another embodiment, the RBC binding portion is thiolated, e.g., derivatized with N-succinimidyl-S-acetyl-thioacetate (SATA),
N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) . The thiolated RBC binding portion is then allowed to react with an appropriately drug portion, e.g., a SATA derivatized drug portion, to produce the RBC binding conjugate of the invention.
The derivatized RBC binding portion, e.g., antibody-maleimide, antibody-PEG- maleimide, or antibody-SH, and the drug portion containing a free thiol (drug-SH), can be combined at a desired molar ratio of derivatized RBC binding portion: drug portion. A skilled person in the art will be able to determine the molar ratio of the derivatized RBC binding portion and drug portion to achieve a desired number of drug portions to each RBC binding portion. In a preferred embodiment, the antibody-maleimide and the drug-SH are combined at a molar ratio of about 2:1 (derivatized RBC binding portion:drug portion). In another preferred embodiment, the derivatized RBC binding portion and the drug-SH are combined at a molar ratio of about 1 : 1 (derivatized RBC binding portion.drug portion). In preferred embodiments, 1, 2, 3, 4, 5 or 6 drug portions are conjugated to each RBC binding portion.
The RBC binding portion and the drug portion can also be covalently conjugated via a suitable water-soluble polymer (see, e.g., WO92/16555, which is incorporated herein by reference in its entirety). Water-soluble polymers that can be used in the present invention include but not limited to polyalkylene oxides, polyoxyethylenated polyols, polyacrylamides, polyvinyl pyrrolidone, polyvinyl alcohol, dextran, and other carbohydrate-based polymers.
The molecular weight of the polymer can be selected based upon the end use of the conjugate by an ordinary skilled person in the art. In general, the useful range of molecular weight is between about 600 and about 100,000 Daltons, and preferably between about 1,000 and about 40,000 Daltons.
The RBC binding portion and the drug portion in the RBC binding conjugates of the invention can be conjugated via a water-soluble polymer linker at desired residues using methods known in the art, including but are not limited to lysine (reactive through its e-NH2); bistidine, tryptophan, or cysteine (reactive through its sulfhydryl SH; See, e.g., Goodson et al, 1990, Biotechnology 8:343); aspartic acids (reactive through their carboxyl functionalities); arginine, serine, or threonine (reactive through its hydroxyl OH); or glutamic acid (reactive through its carboxyl functionalities). When either or both the RBC binding portion and the drug portion in the RBC binding conjugates are glycopolypeptides, they can also be attached to PEG linkers at the saccharide units (see, e.g., WO92/16555).
In one embodiment, to produce the RBC binding conjugate, a bifunctional polymer is used to derivatize the RBC binding portion or the drug portion using one of the functional groups of the polymers. The derivatized RBC binding portion or the derivatized drug portion is then conjugated with a suitably derivatized counterpart portion via the remaining functional group of the linker polymer. In another embodiment, a bifunctional polymer is reacted with both the RBC binding portion and the drug portion in one reaction. In the latter embodiment, it is preferable that the two functional groups of the polymer react to different types of residues of the RBC binding portion and the drug portion so that production of undesirable homodimers is minimized.
In a preferred embodiment, the polymer unit is attached cόvalently to the polypeptide or glycopolypeptide RBC binding portions or the polypeptide or glycopolypeptide drug portions (hereinafter referred simply as the "polypeptide" or the "glycopolypeptide") by reacting an acyl hydrazine derivative of the polymer with the polypeptide or glycopolypeptide having a reactive carbonyl group or an activated peptide carboxylic acid group. As used herein, a reactive carbonyl group refers to either a ketone or aldehyde group, excluding other carboxyl-containing groups such as amides. In a preferred embodiment, the polymers are attached to the polypeptide or glycopolypeptide by reacting with aldehyde groups, because aldehyde groups are more reactive than ketones.
The carbonyl group can be generated either on a peptide or a saccharide unit. For example, Dixon (Dixon, 1984, J. Protein Chem. 3:99) describes several methods to generate reactive carbonyl groups on the N-terminus of a polypeptide molecule, which is incorporated herein by reference in its entirety. In one embodiment, carbonyl groups can be generated on peptides by reacting a polypeptide or glycopolypeptide with a suitable heterobifunctional reagent such as a reactive ester of formyl benzoic acid, disclosed by King et al., 1986, Biochemistry 25: 5774, which is incorporated herein by reference in its entirety. In another embodiment, carbonyl groups can be generated on saccharide units of glycopolypeptides by oxidizing vicinal diols of carbohydrate moieties of glycopolypeptides with excess periodate or with an enzyme, e.g. galactose oxidase.
In one embodiment, the polymer acyl hydrazine reacts with the reactive carbonyl group on the polypeptide or glycopolypeptide to form a hydrazone linkage between the polymer and the polypeptide or glycopolypeptide (see, e.g., WO 92/16555). The hydrazone can be reduced to a more stable alkyl hydrazide by using a suitable agent, e.g., NaBHU or NaCNBHa. The reaction of polymer acyl hydrazine derivatives with carbonyl-containing polypeptides and glycopolypeptides to form a hydrazone linkage is illustrated by the exemplary reaction sequence of Scheme 1 in which R represents the water-soluble polymers, X is a molecular moiety comprising a functional terminal group on the polymer or a polypeptide linked via a functional group, Z is O, NH, S or a lower alkyl group containing up to ten carbon atoms and either or both of Rl and R2 are independently selected from oxidized carbohydrate moieties of glycopolypeptides and peptide units of polypeptides and glycopolypeptides on which, reactive carbonyl groups have been generated:
Scheme 1
X-
Figure imgf000037_0001
C-Ri Hydrazone
Figure imgf000037_0002
X-R-Z-C-NH-NH-CH-R,
Hydrazide
In another embodiment, the activated peptide carboxylic acid group is derived either from a C-terminus carboxylic acid group or a carboxylic acid group of aspartic or glutamic acid residues. Activated carboxylic acid groups are carboxylic acid groups substituted with a suitable leaving group capable of being displaced by the polymer acyl hydrazine. The polymer acyl hydrazine reacts with the activated peptide carboxylic acid group to form a diacylhydrazine linkage between the polymer and the polypeptide or glycopolypeptide. The reaction of polymer acyl hydrazine derivatives with activated peptide carboxylic acid groups of polypeptides and glycopolypeptides to form diacylhydrazides is illustrated by the reaction sequence of Scheme 2: Scheme 2
Q Activation, e.g., EDC p
R3-C-OH O=C-R4
O R3 O R3
Il ^ l Il I
X-R-Z-C-NH-NH2 + O=C-R4 X-R-Z-C-NH-NH-C-R4
Diacylhydrazide
R again represents the water-soluble polymer, X is a molecular moiety comprising a functional terminal group on the polymer or a polypeptide linked via a functional group, and Z is O, NH, S or a lower alkyl group containing up to ten carbon atoms. R3 represents a polypeptide containing aspartic acid, glutamic acid or a C-terminus carboxylic acid residues. R4 represent a leaving group substituted on the peptide carboxylic acid when the carboxylic acid group is activated. Examples of suitable leaving groups are disclosed by Bodanszky, Principles of Peptide Synthesis (Springer- Verlag, New York, 1984), which is incorporated herein by reference in its entirety. Such leaving groups include, but are not limited to, imidazolyL, triazolyl, N- hydroxysuccinimidyl, N-hydroxynorbornenedicarboximidyl and phenolic leaving groups, and are substituted onto the peptide carboxylic acid group by reacting the polypeptide or glycopolypeptide in the presence of an activating reagent with the corresponding imidazole, triazole N-hydroxysuccinimide, N-hydroxynorbornene dicarboximide and phenolic compounds.
Suitable activating reagents are also well-known and disclosed by Bodanszky, Principles of Peptide Synthesis (Springer- Verlag, New York, 1984), which is incorporated herein by reference in its entirety. Examples of such activating reagents include, but are not limited to, water-soluble carbodϋmides such as N-ethyl-N'-(3- dimethylaminopropyl) carbodϋmide (EDC) and N-cyclohexyl-N'-(2-morphoh'noethyl) carbodϋmide, p-toluene sulfonate, 5-substituted isoxazolium salts, such as Woodward's Reagent K. The acyl hydrazine polymer derivatives used in Schemes 1 and 2 are shown with the general structure (I):
O
Il
X-R-Z-C-NH-NH2 α)
X can be a hydroxyl group, in which case the polymer has two labile groups per polymer moiety capable of reacting to form a derivative that can be covalently linked with a polypeptide or glycopolypeptide. X can therefore also be a group into which the terminal hydroxyl group may be converted, including the reactive derivatives disclosed in U.S. Patent Nos. 4,179,337 and 4,847,325, both of which are incorporated herein by reference in their entireties, as well as the acyl hydrazine derivatives described in WO 92/16555, which is incorporated herein by reference in its entirety. The heterobifunctional polymers can be prepared by methods known to those skilled in the art, including the methods disclosed supra with reference to the preparation of acyl hydrazine derivatives, as well as the methods disclosed by Zalipsky et al., 1986, Polvm. Prior. 27(1):1, and Zalipsky et al., 1990, J. Bioact. Comsat. Porvm. 5:227, both of which are incorporated herein by reference in their entireties.
In another preferred embodiment, polymer hydrazides of the general formula (II) are used in Schemes 1 and 2:
X-R-Z-C-AA-NH-NH2
(H)
where AA represents an amino acid or a peptide sequence. AA can be a peptide sequence of any of the common amino acids, or at least one amino acid residue. In the case of AA being one amino acid residue, it is preferable that it is a residue that does not appear naturally in proteins. Examples of such unusual residues include, but are not limited to, α- or γ-amino butyric acid, norleucine, homoserine, β-alanine, ε-caproic acid, and the like. The selectivity of the acyl hydrazines for the reactive carbonyl or activated carboxylic acid groups over the peptide amino group prevents intermolecular cross linking between peptide amino groups and the reactive carbonyl groups and activated carboxylic acid groups, limiting occurrences of such crosslinking to instances when bifunctional polymer derivatives are employed.
In one embodiment, the acyl hydrazine derivative is prepared by reacting the terminal -OH group of methoxylated PEG (mPEGlOH) with phosgene to form mPEG- chloroformate as described in U.S. Patent No. 5,122,614, which is incorporated herein by reference in its entirety. The reaction is carried out in organic solvents in which the reactants are soluble, such as methylene chloride, and will run to completion overnight at room temperature. The solvents and excess phosgene are removed and the residue of polymeric chloro formate is then reacted with an excess of hydrazine.
The acyl hydrazine polymer derivative containing a peptide sequence can be synthesized by first preparing the polymeric chloroformate. The polymeric chloroformate is then reacted with the peptide or an amino acid derivative in a solvent in which the polymeric chloroformate is soluble, such as methylene chloride. The peptide or amino acid is preferably in the form of the ester of the C-terminus acid group, more preferably methyl or ethyl esters.
This reaction can be carried out under mild conditions and typically runs to completion at room temperature and the resulting product can be readily converted to a hydrazide by hydrazinolysis. The acyl hydrazine polymer derivative containing a peptide sequence is then recovered and purified by a method known in the art.
The acyl hydrazine polymer derivative containing a peptide sequence or an amino acid can be prepared by reacting the peptide sequence with a succinimidyl carbonate active ester of the polymer, as disclosed in U.S. Patent No. 5,122,614, or by directly reacting isocyanate derivatives of an amino acid with the terminal hydroxyl group of the polymer as disclosed by Zalipsky et al., 1987, Int. J Peptide Protein Res. 30:740, both of which are incorporated herein by reference in their entireties. Both reactions can be carried out under mild conditions, running to completion at room temperature in organic solvents in which the polymer is soluble, such as methylene chloride. The reaction of isocyanate derivatives of amino acid esters with terminal hydroxyl groups of polymers is disclosed in Zalipsky et aL, 1986, Polvm. Prior. 27(1): 1 and in Zalipsky et aL, 1987, Int. J Peptide Protein Res. 30:740. The succinimidyl carbonate derivative of the polymer is formed by the known method of reacting the above-disclosed polymeric chloroformate with N-hydroxysuccinimide, as disclosed in U.S. Patent No. 5,122,614. Either of the above polymer-polypeptide derivatives can be readily converted to a hydrazide by hydrazinolysis method to yield an acyl hydrazine.
Generally, the conjugation of a polypeptide or glycopolypeptide with a water- soluble polymer first involves either oxidizing carbohydrate moieties of the glycopolypeptide or activating carboxylic acid groups of peptide moieties of the polypeptides or glycopolypeptides. The carbohydrate moieties can be oxidized by reacting the glycopolypeptide in aqueous solution with sodium periodate or with an enzyme, e.g., galactose oxidase or a combination of neuraminidase and galactose oxidase as disclosed by Solomon et al., 1990, J. Cbromatoaraphy 510:321-9. The reaction runs rapidly to completion at room temperature. The reaction medium is preferably buffered, depending upon the requirements of the polypeptide or glycopolypeptide. The oxidized glycopolypeptide is then recovered and separated from the excess periodate by column chromatography.
Carboxylic acid groups of peptide moieties can be activated by reacting the polypeptide or glycopolypeptide with an activating reagent such as a water-soluble carbodimide, e.g., EDC. The reactants are contacted in an aqueous reaction medium at a pH between about 3.0 and 8.0, and preferably about 5.0, which medium may be buffered to maintain the pH. This reaction can be carried out under mild conditions (typically 4 to 37°C) that are tolerated well by most proteins.
Polypeptides or glycopolypeptides having peptide units on which reactive carbonyl groups have been generated may be directly reacted with the acyl hydrazine polymer derivatives in an aqueous reaction medium. This reaction medium may also be buffered, depending upon the pH requirements of the polypeptide or glycopolypeptide and the optimum pH for the reaction, which pH is generally between about 5.0 and about 7.0 and preferably about 6.0.
The optimum reaction media pH for the stability of particular polypeptides or glycopolypeptides and for reaction efficiency, and the buffer in which this can be achieved, is readily determined by those of ordinary skill in the art. For purposes of this application, mild conditions refer to conditions in which the temperatures are in the range between about 4 and about 37°C. Those of ordinary skill in the art will understand that the reactions will run somewhat faster to completion at higher temperatures, as long as the temperature of the reaction medium does not exceed the temperature at which the polypeptides or glycopolypeptides begin to denature. Furthermore, those of ordinary skill in the art will understand that certain polypeptides and glycopolypeptides will require reaction with the polymer acyl hydrazine derivatives at reduced temperatures to minimize loss of activity and/or prevent denaturing. The reduced temperature required by particular polypeptides and glycopolypeptides is preferably no lower than 4°C and more preferably no lower than 00C. Under such conditions, the reaction will still take place, although longer reaction times may be necessary.
Usually, the polypeptide or glycopolypeptide is reacted in aqueous solution with a quantity of the acyl hydrazine polymer derivative in excess of the desired degree of conjugation. This reaction also proceeds under inild conditions, typically at 4 to 37°C.
The reaction medium may be optionally buffered, depending upon the requirements of the polypeptide or the glycopolypeptide, and the optimum pH at which the reaction takes place. Following the reaction, the conjugated product is recovered and purified by diafiltration, column chromatography or the like. When the acyl hydrazine polymer derivative includes an amino acid or a peptide sequence, the degree of polymer conjugation of the polypeptide or glycopolypeptide can then be determined by amino acid analysis.
The acyl hydrazine polymer derivatives exhibit an excellent balance between reactivity and selectivity so that polymer conjugates can be formed with non-amino functional groups of polypeptides and glycopolypeptides with virtually no competition between the acyl hydrazines and the peptide amino groups for the non-amino functional groups. Thus, crosslinking is prevented and the activity of the polypeptide or glycopolypeptide is preserved.
In another preferred embodiment, a polymer/aldehyde/hydrazide system is used to link the RBC binding portion and the drug portion. Preferably, the polymer/aldehyde/hydrazide linker has a spacer of a noniπununogenic polymer, which is synthesized as an entity to the functional group, either hydrazide or aldehyde, or both. Preferably, the hydrazide and/or aldehyde has an aromatic ring attached next to it. The polymer molecule can have a size from 200 g/mole to 60,000 g/mole. The linker is a bifunctional molecule. The other end of the linker can be a functional group that covalently reacts with the protein, such as N-hydroxysuccinimide (NHS). Examples include: NHS-PEG-aryl-aldehyde for conjugating to SANH (NHS-4-hydrazino- nictoinamde acetone hydrazone); NHS-PEG-arylhydrazide for conjugating to SFB (succinimidyl foπnylbenzoate); and NHS-PEG-aryaldehyde for conjugating to NHS- PEG-arylhydrazide. In the derivatization reaction, each part of the linker is used to derivatize one of the proteins on an amine side chain. In the conjugation reaction, the two proteins are mixed so that the other ends of the linkers form a covalent bond through the hydrazine-aldehye reaction.
Preferably, the polymer/aldehyde/hydrazide is a PEG/aldehyde/hydrazide. The PEG/aldehyde/hydrazide linker (PAH) system is superior to other hydrazide systems because: (1) The PAH linkers form stable bonds and do not require further reactions. Other hydrazide linker systems form unstable hydrazone bonds that require toxic cyanoborohydride for a reduction reaction; (2) The PAH linkers have a polymeric PEG spacer that extends into the solvent and facilitates the conjugation reaction, while other hydrazide linkers have low reactivity due to their short spacers; (3) PEGylated protein evades the immune reaction; (4) PEGylated proteins have improved pharmacokinetic profiles; (5) The PEG spacer facilitates the enzymatic action of the conjugate by the RBC-bound conjugate; and (6) PEG may increase the solubility of the compounds. In another embodiment, the polymer is linked to the RBC binding portion or the drug portion via the functional group X. X can be any functional group known in the art, e.g., a maleimide or a N-Hydroxy-Succinimidyl (NHS) group. The derivatized RBC binding portion or drug portion is then reacted with a counterpart portion or a derivatized counterpart portion to form a hydrazide/aldehyde linkage. In one embodiment, X is an NHS group. The polymer is first reacted with the RBC binding portion via the NHS chemistry. The enzyme drug portion is derivatized to have an aldehyde group. The RBC binding portion and the drug portion are then conjugated via a hydrozide/aldehyde linkage. In a preferred embodiment, the RBC binding portion and the drug portion are covalently conjugated using polyethylene glycol (PEG) or a PEG copolymer (see WO . 2004/0244889). Soluble block copolymers of PEG with polypropylene glycol or polypropylene oxide can also be used in the present invention. The PEG moiety can have a molecular weight in the range of 200 to 40,000 Daltons. Preferably, the PEG moiety has a molecular weight in the range of 500 to 8000 Daltons.
In one embodiment, the RBC binding conjugates having PEG linkers are produced by a method described in US Patent No. 4,179,337, which is incorporated herein by reference in its entirety. Other methods of attaching a PEG linker to a protein can also be used (see, e.g., US Patent No. 5,122,614; Veronese et al. 1985, Applied Biochem, and Biotech,, 11: 141-152; Katre ef α/. US Patent No. 4,766,106 and 4,917,888; Roberts MJ. etal., 2002 Advanced Drug Delivery Reviews, 54: 459-476; U.S. 5,766,897; U.S. 6,433,158 Bl; U.S. 5,849,860; all of which are incorporated herein by reference in their entirety).
In still another embodiment, a polymer linker containing an aryl aldehyde functional group is used to derivatize the RBC binding portion or the drug portion. The counterpart portion is derivatized with a suitable cross-linker molecule to contain a hydrazide. The two portions are then reacted to conjugate via a aldehyde/hydrozide linkage. In a preferred embodiment, the RBC binding portion is derivatized with N- Hydroxy-Succinimidyl Polyethylene Glycol-Benzaldehyde (PBA) (see, WO 2004/0244889 and Example 6.2.). The polypeptide drug portion is derivatized with a bifunctional hydrozone, e.g., succinimidyl C64-hydrazino-nictoinamde acetone hydrazone. The derivatized RBC binding portion and the derivatized drug portion are then conjugated via a hydrozide/aldehyde linkage.
In preferred embodiments, heterofunctional PEG linkers are used to produce the RBC binding conjugates. Heterofunctional PEG linkers have the general formula X- PEG-Y, wherein X and Y represent derivatization or functional groups (e.g., activated functional groups). A "functional group", as used herein, refers to a group of covalently attached atoms, that are either electrophillically or nucleophillically activated and can derivatize another molecule through a covalent linkage. Specific examples of functional groups include but are not limited to, COOH, -COOR, where R is lower alkyl or phenyl (carboxylic ester), -COZ, wherein Z is a halide, -CHO (aldehyde), -C(O)R (ketone), - SO2Z (wherein Z is a halide or CF3), -SO2NHZ (Z is halide), -SO2NH2, -maleimide, - amino, -alkyl halide, -alkyl-Z (where Z is mesylate, triflate or tosylate), -alkyl isocyanate, -alkyl isothiocyanate, -alkyl amine, -alkyl-OH, -alkyl-SH, -alkysulfone, - alkylsulfonamide, -alkyl aldehyde, -alkyl ketone, -alkyl-COOH, -alkyl-COOR, -alkyl- COZ (Z is halide), -alkylsulfonamide, -alkylsulfone, -alkylsulfonyl halide. AU the above-mentioned functional groups may also comprise an aryl moiety rather than the alkyl moiety.
In one embodiment, the X and Y activated functional groups of the heterofunctional PEG linker are identical, and the X and Y activated functional groups are directed to modify the same amino acid type on the RBC binding portion and the drug portion of the RBC binding conjugate (e.g., an anti-CRl antibody and a tPA). In another embodiment, the X and Y activated functional groups are not the same and are directed to modifying different amino acid types of the RBC binding portion and the drug portion of the RBC binding conjugate (e.g., an anti-CRl antibody and tPA).
In a preferred embodiment, the amino acids of the RBC binding portion or the drug portion which are modified with PEG linkers on the surface of the RBC binding portion or the drug portion. In yet another embodiment, the N-terminal amino group (See e.g., Kinstler et al, Pharm. Res. 13 : 1996) or the C-terminal carboxylic acid of the RBC binding portion or the drug portion are derivatized using PEG linkers. Conditions suitable for reaction between PEG linkers and amino acid residues within the RBC binding portion or the drug portion are known to those skilled in the art. Typically these procedures involve first providing an activated PEG linker in which one or both hydroxyl groups on a PEG linker are activated, and reacting the activated PEG linker with a residue in the polypeptide selected for PEG conjugation. The general principle of PEG conjugation with proteins and common activating reagents are described in Delgado e/ al, 1992 in "The Uses and Properties of PEG-linked Proteins" from Critical Reviews in Therapeutic Drug Carrier Synthesis, 9(3,4):249-304 and the ACS Symposium Series 680 ed. Harries et al. Poly(ethylene glycol) Chemistry and Biological Applications 1997, both of which are incorporated herein by references in their entirety.
In some embodiments, the X or Y activating functional groups of the heterofunctional PEG linkers used in cross-Unking the RBC binding portion and the drug portion of the invention are electrophillically activated by methods known in the art. At least one of the hydroxyl groups on the PEG linker is activated with a functional group (X or Y) susceptible to nucleopbilic attack by the nitrogen of an amino group on a first or second recognition binding moiety. In one embodiment of the invention, electrophillically activated PEG linkers are used to modify amine residues of a first or second recognition binding moiety. The amine conjugation of PEG linkers are well known in the art, in which electrophillically activated PEG linkers target nucleophilic amine groups. Examples of functional groups of PEG linkers that can be used for the modification of amine residues of a RBC binding portion and/or a drug portion include but are not limited to, -PEG dichlorotriazene, -PEG tresylate, -PEG succinimidyl carbonate, -PEG benzotriazole carbonate, -PEG p-nitrophenyl carbonate, -PEG trichlorophenyl carbonate, -PEG carbonylimidazole, or -PEG succinimidyl succinate. In preferred embodiments, electrohilically activated PEGs used in accordance of the invention are -PEG succinimidyl succinate (-PEG-SS), succinimide of PEG propionic acid (-PEG-SPA), or succinimide of PEG Butanoate Acid (-PEG-SBA). Other Examples of PEG linkers that can be used for the modification of amine residues within a RBC binding conjugate of the invention include but are not limited to, -PEG2-H- hydroxysuccinimide (-PEG2-NHS), -PEG-Benzotriazole carbonate (-PEG-BTC), -PEG- Propionaldehyde (-PEG-ALD), -PEG-Acetaldehyde diethyl acetal (-PEG- ACET), or - PEG2-Aldehyde (-PEG2-ALD).
In another embodiment, the X or Y activating groups of the heterofunctional PEG linkers used in producing the RBC binding conjugates are Lysine-active PEGs. The most preferred PEG derivative for lysine modification are N-hydroxylsuccinimide ("NHS") active esters such as PEG succinimidyl succinate (-PEG-SS) and succinimidyl propionate (-PEG-SPA). In one embodiment, by way of example and not limitation, the following protocol is used. Equal masses of lysine-active PEG (MW, 5000) and a first or second recognition binding moiety of the invention (i.e., anti-CRl antibody) to be derivatized are mixed at pH 8-9.5, at room temperature for 30 minutes, or a time sufficient for derivatization to take place. In some embodiments, if the protein amino acid composition is known, a molar ratio of PEG (MW 5000) to protein amino groups of 1-5 to 1 is used. In another embodiment, the X or Y activating functional groups of the heterofunctional PEG linkers used in producing the RBC binding portion and/or the drug portion are used for modification of cysteine residues in a polypeptide. Examples of functional groups in bifunctional PEG linkers that can be used for the modification of cysteine residues in a RBC binding conjugate of the invention include but are not limited to, -PEG2-forked maleimide, -PEG-forked maleimide, -PEG-maleimide, or -PEG2 maleimide. Methods for attaching PEG linkers to cysteine residues are disclosed in US Patent No. 5,766,897 which is incorporated herein by reference in its entirety. In one embodiment, site-specific derivitization of a cysteine residue using a PEG linker can be achieved using the methods and compositions of the invention by engineering specific cysteine mutants by site-directed mutagenesis methods known in the art (Kunkel et al, 1988, Nucleic Acids and Molecular Biology, Eckstein, F. Lilley, eds., Springer- Verlag, Berling and Heidelberg, vol. 2 p.124). In yet another preferred embodiment, the RBC binding conjugates of the invention are conjugated using Sulfhydryl-selective PEGs. The most preferred PEG linkers for sulfhydryl modification are vinylsulfone, iodoacetamide, and maleimide.
Examples of other hetereofunctional PEG linkers that can be used in the invention include but are not limited to NHS-vinylsulfone and NHS-Maleimide (NHS- PEG-VS and NHS-PEG-Maleimide, respectively), bis-hydrazide-PEG, bis-hydrazine- PEG, and aldehyde-PEG-NHS.
In another embodiment, the heterofunctional PEG linker is a compound of Formula (III) as follows (see WO 2004/0244889):
Figure imgf000047_0001
or a pharmaceutically acceptable salt therof, wherein R is phenyl, naphthyl, or aromatic heterocycle, any of which is substituted with at least one -C(O)H or -NH-NH2 group.
"Aromatic heterocycle" refers to a 5- to 10-membered monocyclic or bicyclic aromatic carbocycle in which 1-4 of the ring carbon atoms have been independently replaced "with a N, O or S atom. Representative examples of an aromatic heterocycle group include, but are not limited to, pyrrolyl, imidazolyl, benzimidazolyl, tetrazolyL, indolyl, isoquinolinyl, quinolinyl, quinazolinyl, purinyl, isoxazolyl, benzisoxazolyl, furanyl, furazanyl, pyridyl, oxazolyl, benzoxazolyl, thiazolyl, benzthiazolyl and thiophenyl.
In one embodiment, R is phenyl.
In another embodiment, R is pyridyl.
In a preferredembodiment, R is
Figure imgf000048_0001
In another preferred embodiment, R is
Figure imgf000048_0002
In a specific embodiment, the RBC binding portion that binds a C3b-]ike receptor (i.e., an anti-CRl antibody, e.g., an anti-CRl monoclonal antibody without a functional Fc domain) is derivatized with NHS-PEG-maleimide. By way of example, and not limitation, the protocol for NHS-PEG-maleimide can be as follows: The anti- CRl antibody is derivatized with NHS-PEG-maleimide at a molar ratio of 6: 1; 6X NHS- PEG-maleimide: IX anti-CR antibody, such that the reaction proceeds at room temperature for two hours at gentle inversion every 15-30 minutes, wherein the anti- CRl antibody is derivatized at one or more sites with NHS-PEG-maleimide. The resulting product from the derivitization is then desalted by chromatography using standard procedures known in the art (e.g., using an Amersham Hi-Prep 26/10 desalting column in MES buffer).
In yet another specific embodiment, the RBC binding portion that binds a C3b- like receptor (i.e., an anti-CRl antibody, e.g., an anti-CRl monoclonal antibody without a functional Fc domain) is derivatized with NHS-PEG-benzaldehyde. Modification using NHS-PEG-benzaldehyde may have several advantages relative to other modification procedures such as those involving maleimide chemistry. Although not intending to be bound by a particular mechanism of action, molecules, e.g., antibodies, modified with NHS-PEG-benzaldehyde tend to be stable over an extended period of time, e.g., at least one month, because the hydrazone or aldehyde moiety is stable under the pH range where the antibody is typically stored. Therefore, the antibody derivatization reaction can be carried out well in advance of the conjugation reaction. Modification using NHS-PEG-benzaldehyde may thus be preferred for commercial production, because the production schedule can be more flexible and the unconjugated monomeric fraction can be recycled. Another benefit of modifying antibodies with NHS-PEG-benzaldehyde is that the hydrazine or aldehyde chemistry will not lead to bond formation with other functional groups in the antibody; any weak bond that could form between the amino group and the aldehyde is hydrolyzed in the aqueous buffer under physiological conditions. When modifying antibodies using maleimide chemistry, however, the derivatized antibodies might react with the free sulfhydryl group on the antibody, leading to an undesired modification. Yet another particular benefit of the NHS-PEG-benzaldehyde linker of the invention is that it requires no reducing agent for a stable bond formation over the pH range where antibodies are typically maintained in the stable form. While sulfhydryl modified proteins may form homodimers, there is no homodimer formation of the antibody using the hydrazone linker. Yet another benefit of using the hydrazine chemistry is that the reaction kinetics of hydrazine/carbonyl linkage is fast and can be carried out in a condition where the antibody can be maintained in the active form.
The RBC binding portion or the drug portion can be derivatized with PEG linkers using any protocol known to those skilled in the art. It will be apparent to one skilled in the art that the molar ratio of the PEG linker used in derivatizing the RBC binding portion or the drug portion depends on the molecular weight of the PEG linker used and the molecular weight of the molecule being derivatized. One skilled in the art can determine the molar ratio of the PEG linker to be used in the derivitization of the RBC binding portion or the drug portion using routine experimentation. In a specific embodiment, for derivitazation of NHS-PEG-maleimide to the RBC binding portion or the drug portion of the invention the molar ratio of the NHS-PEG-maleimide to the RBC binding portion orthe drug portion is 3:1, 4:1, 5:1, 6:1, or 8:1.
Linear PEG linkers are the preferred linking reagents for use in the invention. In some embodiments, other types of linking reagents may also be used. Examples of additional linking reagents include but are not limited to, modified PEG linkers, branched PEG linkers (e.g., PEG2), linear forked PEG linkers, branched forked PEG linkers, or cross-linked PEG linkers.
Techniques for activating or derivatizing the RBC binding portion and/or the drug portion are well known in the art and any method known in the art can be used in accordance with the invention. For example, the RBC binding portion and/or the drug portion can be thiolated using reagents and methods known in the art, in order to react with PEG derivatives directed at sulfhydryl groups. For examples, amines of the RBC binding portion and/or the drug portion can be indirectly thiolated by reaction with succinimidyl 3-(2-pyridyldithio)propionate ("SPDP"), followed by reduction with DTT or tris-(2-carboxyethyl) phospohine ("TCEP"). Amines can also be thiolated by reaction with succinimidyl acetylthioacetate ("SATA") followed by removal of the acetyl group with 5OmM hydroxylamine or hydrazine at or near neutral pH. Additionally, thiols can be incorporated at carboxylic acid groups by an EDAC mediated reaction with cystamine followed by reduction of the disulfide with DTT or TCEP. Other techniques for thiolation of the RBC binding portion or the drug portion are well known in the art and can be used in the methods of the invention.
The RBC binding portion and/or the drug portion can be modified using hydrazine or aldehyde amine modification reagents for example with, "SANH"; succinimidyl 6-hydrazinonicotinate acetone hydrazone or "SFB"; succinimydyl 4- formylbenzoate.
In some embodiments, covalent conjugation of the RBC binding portion and the drug portion of the RBC binding conjugate of the invention are carried out in a site- directed manner. For example, PEG linker can be conjugated site-specifically to oxidized carbohydrate residues in the RBC binding portion or the drug portion. Methods to oxidize carbohydrates are well known in the art, and include but are not limited to enzymatic oxidation (e.g. glucose oxidase) or chemical oxidation (e.g., periodate). Oxidation of carbohydrate residues generates multiple reactive aldehyde groups which can be conjugated with PEG linkers that have for example, an amine or a hydrazide functional group.
In one embodiment, a glycosylation site is first introduced into the RBC binding portion and/or the drug portion using a recombinant method (see, e.g., WO 92/16555, which is incorporated herein by reference in its entirety).
PEG linker can also be conjugated site-specifically to a residue having a free thiol. In another embodiment, the RBC binding portion and/or the drug portion containing a cysteine residue is produced by a host cell in such a manner that a free thiol is maintained (see, e.g., Carter, U.S. Patent No. 5,648,237, which is incorporated herein in its entirety). Preferably, the polypeptide containing a free thiol is secreted by the host cell. The RBC binding portion and/or the drug portion containing the free thiol can then be recovered and used with an appropriately derivatized PEG, e.g., a maleimide derivatized PEG, to produce the RBC binding portion and/or the drug portion.
The RBC binding conjugate of the present invention can also be produced by a method utilizing protein trans-splicing, see e.g., WO02/46208, which is incorporated herein by reference in its entirety. The method can be used to directly or via a linker conjugate a RBC binding portion, e.g., an anti-CRl mAB, with a drug portion, e.g., tPA, to form the RBC binding conjugate.
In the method using protein trans-splicing, the RBC binding portion is conjugated to the N-terminus of an N-intein of a suitable split intein to produce an N- intein RBC binding portion fragment, whereas the drug portion is conjugated to the C- terminus of the C-intein of the split intein to produce a C-intein drug portion fragment. The N-intein RBC binding portion fragment and the C-intein drug portion fragment are then brought together such that they reconstitute and undergo trans-splicing to produce the RBC binding conjugate. The RBC binding conjugate produce by protein trans-splicing can contain a single drug portion conjugated to the RBC binding portion. Alternatively, the RBC binding conjugate of the invention can also contain two or more drug portions conjugated to different regions of the RBC binding portion. For example, the RBC binding conjugate can contain two drug portions conjugated to each of the heavy chains of a first antigen recognition monoclonal antibody. When two or more drug portions are contained in the RBC binding conjugate, such drug portions can be the same or different. For example, the two drug portions can be different enzymes.
Various split inteins can be used for the production of the RBC binding conjugates of the present invention. In one embodiment, naturally occurring split inteins are used for the production of the RBC binding conjugates. In another embodiment, engineered split intein based on naturally occurring non-split inteins are used for the production of the RBC binding conjugates. In various embodiments of the invention, a split intein can be modified by adding, deleting, and/or mutating one or more amino acid residues to the N-intein and/or the C-intein such that the modification improves or enhances the intein's proficiency in trans-splicing and/or permits control of trans- splicing processes. In one preferred embodiment, a Cys residue can be included at the carboxy terminus of a C-intein so that the requirement that the molecular moiety conjugated to the C-intein must start with a Cys is alleviated. In other preferred embodiments, one or more native proximal extein residues are added to the N- and/or C- intein to facilitate trans-splicing in a foreign extein content.
In a preferred embodiment, the trans-splicing system of the split intein encoded in the DnaE gene of Synechocystis sp. PCC6803 is used for the production of the RBC binding conjugates of the present invention. In another embodiment of the invention, an engineered split intein system based on the Mycobacterium tuberculosis RecA intein is used. The production of the RBC binding conjugates can be carried out in vitro wherein the intein antigen recognition portion fragments are expressed in separate hosts. The production of the RBC binding conjugates can also be carried out in vivo. In one embodiment, nucleic acids encoding the intein antigen recognition portion fragments are inserted into separate vectors which are then co-transfected into a host for in vivo production of the RBC binding conjugate. In another embodiment, nucleic acids encoding the intein fragments are inserted into the same vector which is then transfected into a host for in vivo production of the RBC binding conjugate.
The N-intein the RBC binding portion fragment is preferably produced by fusing an appropriate antigen recognition moiety that binds a C3b-lilce receptor to the N- terminus of the N-intein of a suitable split intein. In a preferred embodiment, the C- terminus of the heavy chain of an anti-CRl mAb is fused to the N-terminus of the N- intein of a split intein. The C-intein the drug portion fragment is preferably produced by fusing the drug portion to the C-terminus of the C-intein of a suitable split intein. The amino acid residue immediately at the C-terminal side of the splice junction of the C- intein is a cysteine, serine, or threonine.
The RBC binding conjugate is produced by mixing the N-intein RBC binding portion fragment and the C-intein drug portion fragment in vitro so that the fragments reconstitute and undergo trans-splicing.
The RBC binding conjugates used in the present invention can also be produced recombinantly, where nucleotide sequences which encode antibody variable domains with binding specificity to an RBC surface receptor or antigen are fused to nucleotide sequences which encode a polypeptide drug molecule, see e.g., WO 01/80883, which is incorporated herein by reference in its entirety. In one embodiment, the nucleic acid encoding a polypeptide drug portion is fused to the nucleic acid encoding an antibody variable domain with binding specificity to an RBC surface receptor or antigen to obtain a fusion nucleic acid encoding a single polypeptide RBC binding conjugate. The nucleic acid is then expressed in a suitable host to produce the RBC binding conjugate.
D. PURIFICATION AND CHARACTERIZATION OF RBC BINDING CONJUGATES
The RBC binding conjugates produced by a method such as described supra are then preferably purified. RBC binding conjugates can be purified by any method known to one skilled in the art using molecular size or specific binding affinity or a combination thereof. In one embodiment, the RBC binding conjugates can be purified by ion exchange chromatography using columns suitable for isolation of the RBC binding conjugates of the invention including DEAE, Hydroxylapatite, Calcium Phosphate (see generally Current Protocols in Immunology, 1994, John Wiley & Sons, Inc., New York, NY).
In another embodiment, RBC binding conjugates are purified by three-step successive affinity chromatography (Corvalan and Smith, 1987, Cancer Immunol. Immunother., 24: 127-132): the first column is made of protein A bound to a solid matrix, wherein the Fc portion of the antibody binds protein A, and wherein the antibodies bind the column; followed by a second column that utilizes C3b-like receptor bound to a solid matrix which assays for C3b-like receptor binding via the RBC binding portion portion of the RBC binding conjugate; and followed by a third column that utilizes specific binding of an antigenic molecule of interest which binds the antigen recognition portion of the RBC binding conjugate.
The RBC binding conjugates can also be purified by a combination of size exclusion HPLC and affinity chromatography. In one embodiment, the appropriate fraction eluted from size exclusion HPLC is further purified using a column containing an antigenic molecule specific to the antigen recognition portion of the RBC binding conjugate.
The RBC binding conjugates can be characterized by various methods known in the art. The yield of RBC binding conjugate can be characterized based on the protein concentration. In one embodiment, the protein concentration is determined using a Lowry assay. Preferably, the RBC binding conjugate produced by the method of the present invention has a protein concentration of at least 0.100 mg/ml, more preferably at least 2.0 mg/ml, still more preferably at least 5.0 mg/ml, most preferably at least 10.0 mg/ml. In another embodiment, the concentration of the RBC binding conjugates is determined by measuring UV absorbance. The concentration is determined as the absorbance at 280nm. Preferably, the RBC binding conjugate produced by the method of the present invention has an absorbance at 280nm of at least 0.14.
The RBC binding conjugate of the invention can also be characterized using any other standard method known in the art. In one embodiment, high-performance size exclusion chromatography (HPLC-SEC) assay is used to determined the content of contamination by free IgG proteins. In preferred embodiments, the RBC binding conjugate composition produced by the method of the present invention has a contaminated IgG concentration of less than 6.0 mg/ml, more preferably less than 2.0 mg/ml, still more preferably less than 0.5 mg/ml, most preferably less than 0.03 mg/ml. In one embodiment, the RBC binding conjugates can be characterized by using SDS- PAGE to determine the molecular weight of the RBC binding conjugate.
The RBC binding conjugate can also be characterized based on the functional activity of the RBC binding conjugates. In one embodiment, the anti-CRl binding activity is determined using ELISA with immobilized CRl receptor molecules (attached to a solid phase, e.g., a microtiter plate) (see Porter et al., U.S. provisional application No. 60/380,211, which is incorporated herein by reference in its entirety). The assay is also referred to as a CRl/Antibody assay or CAA, and can be used generally to measure any RBC binding portion, or HP or AHP containing an RBC binding portion. In a preferred embodiment, ELISA/CR1 plates are prepared by incubating ELISA plates, e.g., high binding flat bottom ELISA plates (Costar EIA/RIA strip plate 2592) with a suitable amount of a bicarbonate solution of CRl receptors. Preferably, the concentration of the bicarbonate solution of CRl receptors is 0.2 ug/ml prepared from 5 mg/ml sCRl receptors stock (Avant Technology Inc.) and a carbonate-bicarbonate buffer (pH 9.6, Sigma C-3041). In a preferred embodiment, 100 ul CRl-bicarbonate solution is dispensed into each well of the ELISA plates and the plates are incubated at 4°C overnight. The plates are then preferably washed using, e.g., a wash buffer (PBS, 0.1% Tween-20, 0.05% 2-Chloroacetamide). In another preferred embodiment, a SuperBlock Blocking Buffer in PBS (Pierce) is added to the plates for about 30-60 min at room temperature after the wash. The plates can then be dried and stored at 4°C . The titration of anti-CRl Abs or RBC binding conjugates can be carried out using a CRl binding protein, e.g., human anti-CRl IgG, as the calibrator. In a preferred embodiment, the calibrator a human anti-CRl IgG having a concentration of 0.2 or 0.6 mg/ml. In one embodiment, the titration of the purified composition of RBC binding conjugates of the invention is carried out using PBS, 0.25% BSA, 0.1% Tween-20 as the diluent buffer, PBS, 0.1% Tween-20, 0.05% 2-Chloroacetamide as the wash buffer, TMB-Liquid Substrate System for ELISA (3,3',5,5'-Tetramethyl-Benzidine) and 2N H2SO4 as the stop solution. Preferably, the RBC binding conjugate composition produced by the method of the present invention has an CAA titer of at least 0.10 mg/ml, more preferably at least 0.20 mg/ml, still more preferably at least 0.30 mg/ml, and most preferably at least 0.50 mg/ml. In some embodiments, a specific anti-CRl activity is determined. The specific anti-CRl activity is a ratio of CAA and Lowry.
In a specific embodiment, where the RBC binding conjugate comprises a protein, the specific amino acids that have been modified with a linker can be determined. The method can measure the loss of the specific amino acids in unmodified form due to the modification. In one specific embodiment, where a lysine residue in a RBC binding portion and/or a drug portion has been derivatized with a linker, unmodified lysine groups can be determined using the "Habeeb Method" where unmodified lysine groups react with trinitrobenzenesulfonic acid followed by UV measurement (Habeeb, 1966 Anal Biochem. 14:328; Karr et al, 1986, J. Chrom. 354:269; Abuchowski et al, 1977 J. Biol. Chem. 252:3578). Another method for determining the unmodified lysine groups is the fluorescamine method of Stocks in which fluorescamine is reacted with unmodified lysine groups yielding a fluorescent derivative (Karr etal. 1994, Methods in Enzymo logy/ 228: 377).
In another embodiment, where a cysteine residue in a RBC binding portion and/or a drug portion has been derivatized with a PEG linker, available cysteine groups can be determined by a spectrophotometric assay based on reaction with 2,2'-dipyridyl disulfide which forms 2-thiopyridone, which absorbs at 343nm with e=7060 at pH 7.2. Another approach is reaction with Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acids) (See Grassetti et al, 1967 Biochem. Biophys. 119:41; Riddles et al, 1979, Anal. Bioch 94:75).
πi. USES OF RBC BINDING CONJUGATES
The RBC binding conjugates of the present invention can be used in treating or preventing a disease or disorder associated with the presence of a pathogenic substance in the blood. The pathogenic substance can be any substance that is present in the circulation and that is injurious to or undesirable in the subject to be treated, including but not limited to physiologically produced substances, e.g., clots, and foreign pathogenic substances, e.g., toxins and microorganisms. The RBC binding conjugates of the present invention convert or degrade such pathogenic substances into nonpathogenic substances, thereby treating or preventing the disease or disorder. The RBC binding conjugates of the present invention can react with such pathogenic substances directly. The RBC binding conjugates of the present invention can also catalyze reactions of such pathogenic substances with other substances. The RBC binding conjugates of the present invention can also be used in a combination therapy in which one or more other therapeutic agents are also administered. In a preferred embodiment, the RBC binding conjugates of the present invention are used as a means for delivery therapeutic enzymes.
The RBC binding conjugates of the present invention can be used as a means for delivery of anticoagulants or thrombolytics. Such RBC binding conjugates can be used to treat or prevent disease conditions associated with the formation of clots in the blood of a subject. In one embodiment, the drug portion of the RBC binding conjugate comprises a streptokinase, staphylokinase, tissue-type plasminogen activator or tPA, or urokinase, which act to dissolve intravascular clots by activating the protease plasmin to digest fibrin.
The RBC binding conjugates of the present invention can be used in treating or preventing a disease or disorder associated with the deficiency of a substance in a subject. For example, the RBC binding conjugates of the present invention can be used as a means for delivery of enzymes in an enzyme replacement therapy. Such RBC binding conjugates can be used to treat or prevent disease conditions associated with enzyme deficiency in a subject, e.g., various metabolic diseases. In an exemplary embodiment, the invention provides a conjugate comprising β-ghicocerebrosidase or an analogue covalently-conjugated to an anti-CRl or anti-glycophorin A antibody for treating Gaucher disease. In another exemplary embodiment, the invention provides a conjugate comprising an α-galactosidase A or an analogue covalently-conjugated to an anti-CRl or anti-glycophorin A antibody for treating Fabry disease.
The RBC binding conjugates of the present invention can also be used as a means for delivery of an anti-cancer agent. Such RBC binding conjugates can be used to treat or prevent cancers in a subject. For example, the anti-cancer agent can be an agent that degrades small molecules for which cancer cells need. The anti-cancer agent can also degrade macromolecules such as membrane polysaccharides, structural and functional protein, or nucleic acids. In an exemplary embodiment, the invention provides a conjugate comprising L-asparaginase or an analogue covalently-conjugated to an anti-CRl or anti-glycophorin A antibody for preventing and treating various types of cancers, including but not limited to, acute lymphocytic leukaemia. The cancers cells of these types of cancers are deficient in their ability to synthesize the nonessential amino acid L-asparagiαe and must extract the amino acid from the body. On the other hand, most normal cells can produce their own L-asparagine. Thus, the RBC binding conjugate can be used to degrade L-asparagine in the blood stream, and thereby prevent the growth of cancer cells. Other enzymes that can be used in treating or preventing cancers include but are not limited to L-glutaminase-L-asparaginase, L-methioninase, L- phenylalanine ammonialyase, L-arginase, L-tyrosinase, L-serine dehydratase, L- threonine deaminase, indolyl-3-alkane hydroxylase, neuraminidase, ribonuclease, various proteases, pepsin, various carboxypeptidases that are capable of hydrolyzing the L-glutamyl moiety of folic acid, or any analogue thereof. Such a therapy can also be used in conjunction with other therapies that target a specific cancer.
The RBC binding conjugates of the present invention can also be used as a means for delivery of an anti-infectious agent. Such RBC binding conjugates can be used to treat or prevent infectious diseases in a subject, e.g., various bacterial or viral infections. In an exemplary embodiment, the invention provides a conjugate comprising a lysozyme, e.g., lysostaphin, or an analogue covalently-conjugated to an anti-CRl or anti-glycophorin A antibody for treating bacterial infections, e.g., infections caused by Staphylococcus aureus. Lysostaphin kills bacteria by cleaving the glycoprotein of the bacterial wall and resulting in lyses of the bacterial cells. Lysostaphin is capable of destroying bacteria whether they are active or resting and is thus capable of killing large numbers of microorganisms. It is particularly useful in instances where an initial and rapid reduction in bacterial count is necessary. Such an RBC binding conjugate can be administered in combination with standard antibiotics.
The RBC binding conjugates of the present invention can also be used as a means for delivery of an antidote. Such RBC binding conjugates can be used to treat an overdose of certain substance in a subject, e.g., an overdose of methotrexate. In an exemplary embodiment, the invention provides a conjugate comprising a carboxypeptidase Gl or an analogue covalently-conjugated to an anti-CRl or anti- glycophorin A antibody for treating methotrexate overdose.
The preferred subject for administration of a RBC binding conjugate of the invention, for therapeutic or prophylactic purposes, is a mammal including but is not limited to non-human animals (e.g., horses, cows, pigs, dogs, cats, sheep, goats, mice, rats, etc.), and in a preferred embodiment, is a human or non-human primate.
Various purified RBC binding conjugates can be combined into a "cocktail" of RBC binding conjugates. Such cocktail of RBC binding conjugates can include RBC binding conjugates each having an RBC binding portion conjugated to any one of several desired drug portions. For example, the RBC binding conjugate cocktail can comprise a plurality of different RBC binding conjugates, wherein each different RBC binding conjugate in the plurality contains a different drug portion that targets a different pathogenic substance. Such RBC binding conjugate cocktails are useful as personalized medicine tailored according to the need of individual patients. Alternatively, a cocktail of RBC binding conjugates can include RBC binding conjugates each having a different RBC binding portion which binds a different blood cell antigen conjugated to a desired drug portion. Such RBC binding conjugate cocktails can be used to increase the drug load on each red blood cell.
The RBC binding conjugates of the present invention can also be used as a means for delivery of a diagnostic agent. Such RBC binding conjugates can be used to image internal tissues or organs and blood vessels in a subject. Examples of diagnostic agents include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. See generally U.S. Patent No. 4,741,900 for metal ions which can be conjugated to proteins for use as diagnostics. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. Examples of luminescent material include luminol. Examples of bioluminescent materials include luciferase, luciferin, and aequorin.
Examples of suitable radioactive material include I, ϊ, In or Tc. IV. DOSEAGE OF RBC BINDING CONJUGATES
The dose can be determined by a physician upon conducting routine tests. Prior to administration to humans, the efficacy is preferably shown in animal models. Any animal model for a blood borne disease known in the art can be used.
For example, the dose of the RBC binding conjugate can be determined based on the red blood cell concentration and the number of target surface antigen, e.g., C3b-like receptor epitope sites, bound by the RBC binding portion per red blood cell. A therapeutically effective amount of RBC binding conjugate (i.e., an effective dosage) may range from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 0.1 to 10 mg/kg body weight.
The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but is not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a RBC binding conjugate can include a single treatment or, preferably, can include a series of treatments. In a preferred example, a subject is treated with a RBC binding conjugate in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. It will also be appreciated that the effective dosage of a RBC binding conjugate, used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein.
It is understood that appropriate doses of RBC binding conjugate agents depends upon a number of factors within the ken of the ordinarily skilled physician, veterinarian, or researcher. The dose(s) of the RBC binding conjugate will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the RBC binding conjugate to have upon a pathogenic antigenic molecule or autoantibody. It is also understood that appropriate doses of RBC binding conjugates depend upon the potency of the RBC binding conjugate with respect to the biological activity it is to carry out. Such appropriate doses may be determined using a suitable assay. When one or more of these RBC binding conjugates is to be administered to an animal (e.g., a human) in order to cause the degradation of a pathogenic substance, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the RBC binding conjugate employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the concentration of pathogenic substance to be cleared.
V. PHARMACEUTICAL FORMULATION AND ADMINISTRATION
The RBC binding conjugates of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise RBC binding conjugate and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the RBC binding conjugate, use thereof in the compositions is contemplated. Supplementary RBC binding conjugates can also be incorporated into the compositions.
A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. The preferred route of administration is intravenous. Other examples of routes of administration include parenteral, intradermal, subcutaneous, transdermal (topical), and transmucosal. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF; Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that the viscosity is low and the RBC binding conjugate is injectable. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for example, water, ethanoL, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the RBC binding conjugate (e.g., one or more RBC binding conjugates) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the RBC binding conjugate into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
In one embodiment, the RBC binding conjugates are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811 which is incorporated herein by reference in its entirety.
It is advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of RBC binding conjugate calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the RBC binding conjugate and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such a RBC binding conjugate for the treatment of individuals.
The pharmaceutical compositions can be included in a kit, in a container, pack, or dispenser together with instructions for administration.
VI. EX VTVO PREPARATION OF THE RBC BINDING CONJUGATE
In alternative embodiments, the RBC binding conjugate is prebound to red blood cells of the subject ex vivo, prior to administration. For example, red blood cells are collected from the individual to be treated (or alternatively red blood cells from a non- autologous donor of the compatible blood type are collected) and incubated with an appropriate dose of the therapeutic RBC binding conjugate for a sufficient time so as to allow the conjugate to bind the antigen on the surface of the red blood cells. The red blood cell/RBC binding conjugate mixture is then administered to the subject to be treated in an appropriate dose (see, for example, Taylor et al., U.S. Patent No. 5,487,890).
Accordingly, in a specific embodiment, the invention provides a method of treating a mammal having an undesirable condition associated with the presence of a pathogenic substance, comprising the step of administering a red blood cell/RBC binding conjugate complex to the subject in a therapeutically effective amount, the complex consisting essentially of a red blood cell bound to one or more RBC binding conjugates. The method alternatively comprises a method of treating a mammal having an undesirable condition associated with the presence of a pathogenic substance comprising the steps of (a) contacting a RBC binding conjugate with red blood cells to form a red blood cell/RBC binding conjugate complex; and (b) administering the red blood cell/RBC binding conjugate complex to the mammal in a therapeutically effective amount.
The invention also provides a method of making a red blood cell/RBC binding conjugate complex comprising contacting a RBC binding conjugate with red blood cells under conditions conducive to binding, such that a complex consisting essentially of a red blood cell bound to one or more RBC binding conjugates forms.
In yet another embodiment, the RBC binding conjugate, such as a RBC binding conjugate, is prebound to red blood cells in vitro as described above, using a blend of at least two different RBC binding conjugates that bind different surface antigens on the red blood cells, e.g., different and non-overlapping recognition sites on the C3b-like receptor. By using at least two different RBC binding conjugates, the number of RBC binding conjugates that can bind to a single red blood cell is increased.
VII. KITS
The invention also provides kits comprising in one or more containers the RBC binding conjugates of the invention. Kits containing the pharmaceutical compositions of the invention are also provided. EXAMPLES
The following examples describe the production of an RBC binding conjugate comprising anti-CRl mAb 7G9 and a tissue-type plasminogen activator (t-PA). Example 6.1 describes the production of the conjugate. Example 6.2 describes the production of the bifunctional polymeric NHS-PEG-benzaldehyde.
EXAMPLE 1 : PRODUCTION OF CONJUGATE tPA-7G9
The following example describes the production of a tPA - RBC binding portion conjugate. The RBC binding portion was an anti-CRl monoclonal antibody 7G9. The antibody was derivatized with the bifunctional polymeric NHS-PEG-benzaldehyde (PBA). Tissue plasminogen activator (tPA) was derivatized with the bifunctional compound succinimidyl C64-hydrazino-nictoinamde acetone hydrazone (Hz) (Solulink). Thirty nmoles of Hz was used to modify 5 nmole of 7G9 in sample-buffer: 0.15M NaCl, 50 mM potassium phosphate, pH 7.4. After 1 hour of stirring at 25°C, small molecules were removed from the reaction mixture using a 10 ml PDlO column (Amersham) which had been equilibrated with the conjugation buffer: 0.1M citrate, pH 5. In a separate reaction, 60 nmoles of PBA was used to modify 5 nmoles of 7G9 in the sample buffer. After 1 hour of stirring at 25 0C, the small molecules were removed using a PDlO column.
The conjugation reaction was initiated by mixing the 0.6 mg of tP A-Hz with 0.3 mg of 7G9-PBA (weight ratio of tPA:7G9 was at 2: 1, molar ratio at 4.6: 1) at a total protein concentration of -0.61 mg/ml. The reaction was allowed to proceed for 16 hours at room temperature. The conjugate sample was then purified on a Suprose 6 column (Amersham) which had been equilibrated with 2XPBS: 20 mM phosphate, 0.3M NaCl, pH7.4. The conjugates had molecular weights greater than 220,000 g/mol. The fractions were analyzed by SDS-PAGE (Figure 1) and the isolated conjugates contained 1-2 tPA molecules for each 7G9 molecule. The conjugates were concentrated to yield a sample that has tPA enzyme activity equivalent to 989 ug of tPA per ml. The CRl- binding activity was equivalent to 36.2 ug of anti-CRl per 100 ug of the conjugate. EXAMPLE 2: SYNTHESIS OF N-HYDROXY-SUCCINIMIDYL POLYETHYLENE
GLYCOL-BENZALDEHYDE fPBA>
In a 25-mL round bottomed flask, 500 mg of carboxyl-polyethylene glycol- amine (0.147 mmole) (Shearwater) was diluted with 25 ml of 10 mM phosphate buffer, pH 7.5. To the resulting solution was added 49.42 mg of N-hydroxysuccinimidyl- formylbenzoate (Solulink) which had been dissolved in dimethyl sulfoxide. The resulting reaction was stirred at room temperature under argon in the dark. After 4 hours, the aqueous phase was extracted with dichloromethane (DCM). The DCM phase was dried over MgSO4 and concentrated under reduced pressure to provide a residual liquid which was extracted with ether (3 x 50 mL). Carboxy-PEG-benzaldehyde (CPB) was precipitated by adding cold isopropyl alcohol (IPA) to the combined ethereals. The precipitate was then washed with cold IPA then dissolved in 8 ml of DCM. To the resulting solution was added 0.8 ml of 10% of sodium phosphate buffer at pH 5.0, followed by 150 mg of (l-ethyl-3-(3-dimethylamino propyl)carbodiimide (EDC), and 102 mg of N-hydroxysucciiiimide (NHS). The resulting reaction was stirred under argon for 2 hours, the DCM phase was collected, dried over MgSO4 and concentrated in vacuo to provide an oily residue which was washed using IPA and dried in vacuo to provide the compound (yield = 238 mg). The molecular weight of PEG is 3400 Da.
The residual carboxyl group in the intermediate product was completely converted to the final product by another reaction with EDC and NHS. For instance, 50 mg of the intermediate product was dissolved in 2.5 ml of ethyl acetate. 16.11 mg of NHS and 28.475 mg of EDC were added. The reaction mixture was stirred for 1.5 hours under Argon. The reaction mixture was concentrated down to a colorless gumlike material. Two ml of ether was added to allow a precipitate to form. Ether was decanted and the residue was washed with ether for two more times. A solid material (23 mg) was collected as the final product. The compound was analyzed on a thin layer chromatography plate and was obeserved as a distinct spot. The ultraviolet spectrum of the final product was identical to NHS-benzaldehyde. This compound (N-hydroxy- succinimidyl-polyethylene glycol-Benzaldehyde) is referred to as PBA and has Formula HL supra. REFERENCES CITED
All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.
Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is to be Limited only by the terms of the appended claims along with the full scope of equivalents to which such claims are entitled.

Claims

WHAT IS CLAIMED IS:
1. A red blood cell (RBC) binding conjugate comprising a monoclonal antibody or a fragment thereof covalently conjugated to a mammalian serum protein or to an enzyme, wherein said monoclonal antibody or fragment thereof (i) binds a C3b-like receptor on a red blood cell, and (ii) lacks a functional effector domain.
2. The RBC binding conjugate of claim 1, wherein said monoclonal antibody or fragment binds a CRl receptor on a red blood cell.
3. The RBC binding conjugate of claim 2, wherein said monoclonal antibody or fragment thereof is covalently conjugated to a mammalian serum protein that is a human serum protein.
4. The RBC binding conjugate of claim 1, wherein said monoclonal antibody or fragment thereof is covalently conjugated to a mammalian serum enzyme or a functional fragment thereof.
5. The RBC binding conjugate of claim 1, wherein said mammalian serum protein or said enzyme is covalently conjugated at a selected residue to said monoclonal antibody or fragment thereof.
6. The RBC binding conjugate of claim 5, wherein said selected residue is selected from the group consisting of a cysteine residue, a residue comprising a reactive carbonyl or carboxylic acid group when oxidized, and a lysine residue.
7. The RBC binding conjugate of claim 6, wherein said monoclonal antibody or fragment thereof is selected from the group consisting of an Fab, an Fab', an (FabΗ and an Fv fragment of an immunoglobulin molecule that binds said C3b-like receptor.
8. The RBC binding conjugate of claim 6, wherein said monoclonal antibody or fragment thereof comprises a monoclonal antibody, and wherein the effector domain of said monoclonal antibody is inactivated.
9. The RBC binding conjugate of claim 8, wherein the effector domain of said monoclonal antibody comprises one or more mutations such that said effector domain loses its effector function.
10. The RBC binding conjugate of claim 9, wherein said monoclonal antibody is selected from the group consisting of a murine monoclonal antibody, a humanized monoclonal antibody, and a human monoclonal antibody.
11. The RBC binding conjugate of claim 9, wherein said monoclonal antibody is selected from the group consisting of anti-CRl antibodies H4, H9, H47, H48, 7G9, HB8592, 3D9, 57F3 and 1B4.
12. The RBC binding conjugate of claim 1, wherein said RBC binding conjugate preserves at least 5%, 15%, 25%, 50%, 90%, or 99% of the biological activity of said mammalian serum protein or said enzyme when unconjugated.
13. The RBC binding conjugate of any one of claims 1-12, further comprising a polymer linker, wherein said mammalian serum protein or enzyme is covalently conjugated to said monoclonal antibody or fragment thereof via said polymer linker.
14. The RBC binding conjugate of claim 13, wherein said polymer linker is selected from the group consisting of polypeptide, polyalkylene oxide, polyoxyethylenated polyol, polyacrylamide, polyvinyl pyrrolidone, polyvinyl alcohol, and dextran.
15. The RBC binding conjugate of claim 14, wherein said polymer linker is a polyethylene glycol (PEG) linker.
16. The RBC binding conjugate of claim 15, wherein said polymer linker is a PEG hydrazide/aldehyde linker.
17. The RBC binding conjugate of claim 16, wherein said monoclonal antibody or fragment thereof is covalently conjugated to a mammalian serum protein selected from the group consisting of a tissue-type plasminogen activator, a receptor of a tissue- type plasminogen activator, a streptokinase, a staphylokinase, a urokinase, and Factor VHI.
18. The RBC binding conjugate of claim 16, wherein said monoclonal antibody or fragment thereof is covalently conjugated to mammalian serum protein that is β- glucocerebrosidase.
19. The RBC binding conjugate of claim 16, wherein said monoclonal antibody or fragment thereof is covalently conjugated to a mammalian serum protein that is o> galactosidase A.
20. The RBC binding conjugate of claim 16, wherein said monoclonal antibody or fragment thereof is covalently conjugated to an enzyme selected from the group consisting of L-asparagine, L-glutaminase-L-asparaginase, L-methioninase, L- phenylalanine ammonialyase, L-arginase, L-tyrosinase, L-serine dehydratase, L- threonine deaminase, indolyl-3-alkane hydroxylase, neuraminidase, ribonuclease, a protease, pepsin, and a carboxypeptidase.
21. The RBC binding conjugate of claim 16, wherein said monoclonal antibody or fragment thereof is covalently conjugated to a mammalian serum protein that is a cytokine.
22. The RBC binding conjugate of claim 21, wherein said cytokine is selected from the group consisting of IFN-α, IFN-β, EFN-γ, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, EL- 8, 3X-9, IL-10, IL-12 and IL-15.
23. The RBC binding conjugate of claim 16, wherein said monoclonal antibody or fragment thereof is covalently conjugated to a mammalian serum protein that is a peptide hormone.
24. The RBC binding conjugate of claim 23, wherein said peptide hormone is selected from the group consisting of antimullerian hormone (AMH), adiponectin, adrenocorticotropic hormone (ACTH), angiotensinogen and angiotensin, antidiuretic hormone (ADH), atrial-natriuretic peptide (ANP), calcitonin, cholecystokinin (CCK), corticotropin-releasing hormone (CRH), erythropoietin (EPO), follicle stimulating hormone (FSH), gastrin, glucagon, gonadotropin-releasing hormone (GnRH), growth hormone-releasing hormone (GHRH), human chorionic gonadotropin (hCG), growth hormone (GH), insulin, insulin-like growth factor (IGF), leptin, luteinizing hormone (LH), melanocyte stimulating hormone (MSH or α-MSH), neuropeptide Y, oxytocin, parathyroid hormone (PTH), prolactin (PRL), relaxin, renin, secretin, somatostatin, thrombopoietin, thyroid-stimulating hormone (TSH), and thyrotropin-releasing hormone (TRH).
25. The RBC binding conjugate of claim 16, wherein said monoclonal antibody or fragment thereof is covalently conjugated to an enzyme that is lysostaphin.
26. The RBC binding conjugate of any one of claims 7-12, wherein said RBC binding conjugate is a single polypeptide comprising said monoclonal antibody or fragment thereof fused to said mammalian serum protein or said enzyme.
27. The RBC binding conjugate of claim 26, wherein said monoclonal antibody or fragment thereof is covalently conjugated to a mammalian serum protein selected from the group consisting of a tissue-type plasminogen activator, a receptor of a tissue- type plasminogen activator, a streptokinase, a staphylokinase, a urokinase, and Factor VIII.
28. The RBC binding conjugate of claim 26, wherein said monoclonal antibody or fragment thereof is covalently conjugated to a mammalian serum protein that is β- glucocerebrosidase.
29. The RBC binding conjugate of claim 26, wherein said monoclonal antibody or fragment thereof is covalently conjugated to mammalian serum protein that is α- galactosidase A.
30. The RBC binding conjugate of claim 26, wherein said monoclonal antibody or fragment thereof is covalently conjugated to an enzyme selected from the group consisting of L-asparagine, L-glutaminase-L-asparaginase, L-methioninase, L- phenylalanine ammonialyase, L-arginase, L-tyrosinase, L-serine dehydratase, L- threonine deaminase, indolyl-3-alkane hydroxylase, neuraminidase, ribonuclease, a protease, pepsin, an interferon, lysostaphin, and a carboxypeptidase.
31. The RBC binding conjugate of claim 26, wherein said monoclonal antibody or fragment thereof is covalently conjugated to a mammalian serum protein that is a cytokine.
32. The RBC binding conjugate of claim 31, wherein said cytokine is selected from the group consisting of IFN-α, IFN-β, IFN-γ, EL-2, IL-3, IL-4, IL-5, IL-6, EL-7, DL- 8, IL-9, IL-IO, IL-12 and IL-15.
33. The RBC binding conjugate of claim 26, wherein said monoclonal antibody or fragment thereof is covalently conjugated to a mammalian serum protein that is a peptide hormone.
34. The RBC binding conjugate of claim 33, wherein said peptide hormone is selected from the group consisting of antimullerian hormone (AMH), adiponectin, adrenocorticotropic hormone (ACTH), angiotensinogen and angiotensin, antidiuretic hormone (ADH), atrial-natriuretic peptide (ANP), calcitonin, cholecystokinin (CCK), corticotropin-releasing hormone (CRH), erythropoietin (EPO), follicle stimulating hormone (FSH), gastrin, glucagon, gonadotropin-releasing hormone (GnRH), growth hormone-releasing hormone (GHRH), human chorionic gonadotropin (hCG), growth hormone (GH), insulin, insulin-like growth factor (IGF), leptin, luteinizing hormone (LH), melanocyte stimulating hormone (MSH or α-MSH), neuropeptide Y, oxytocin, parathyroid hormone (PTH), prolactin (PRL), relaxin, renin, secretin, somatostatin, thrombopoietin, thyroid-stimulating hormone (TSH), and thyrotropin-releasing hormone (TRH).
35. The RBC binding conjugate of claim 26, wherein said monoclonal antibody or fragment thereof is covalently conjugated to an enzyme that is lysostaphin.
36. A method of treating a mammal having an undesirable condition associated with the formation of clots in its circulation, comprising the step of administering to the mammal a therapeutically effective amount of the RBC binding conjugate of claim 17.
37. A method of treating a patient having Gaucher disease, comprising the step of administering to the patient a therapeutically effective amount of the RBC binding conjugate of claim 18.
38. A method of treating a mammal having Fabry disease, comprising the step of administering to the patient a therapeutically effective amount of the RBC binding conjugate of claim 19.
39. A method of treating a mammal having a cancer, comprising the step of administering to the mammal a therapeutically effective amount of the RBC binding conjugate of claim 20.
40. A method of treating a mammal having cancer or a bacterial or viral infection, comprising the step of administering to the mammal a therapeutically effective amount of the RBC binding conjugate of claim 21 or 22.
41. A method for hormone replacement therapy in a mammal, comprising the step of administering to the mammal a therapeutically effective amount of the RBC binding conjugate of claim 23 or 24.
42. A method of treating a mammal having a bacterial infection, comprising the step of administering to the mammal a therapeutically effective amount of the RBC binding conjugate of claim 25.
43. A method of treating a mammal having an undesirable condition, associated with the formation of clots in its circulation, comprising the step of administering to the mammal a therapeutically effective amount of the RBC binding conjugate of claim 27.
44. A method of treating a patient having Gaucher disease, comprising the step of administering to the patient a therapeutically effective amount of the RBC binding conjugate of claim 28.
45. A method of treating a mammal having Fabry disease, comprising the step of administering to the patient a therapeutically effective amount of the RBC binding conjugate of claim 29.
46. A method of treating a mammal having a cancer, comprising the step of administering to the mammal a therapeutically effective amount of the RBC binding conjugate of claim 30.
47. A method of treating a mammal having cancer or a bacterial or viral infection, comprising the step of administering to the mammal a therapeutically effective amount of the RBC binding conjugate of claim 31 or 32.
48. A method for hormone replacement therapy in a mammal, comprising the step of administering to the mammal a therapeutically effective amount of the RBC binding conjugate of claim 33 or 34.
49. A method of treating a mammal having a bacterial infection, comprising the step of administering to the mammal a therapeutically effective amount of the RBC binding conjugate of claim 35.
50. A pharmaceutical composition comprising a therapeutically effective amount of the RBC binding conjugate of any one of claims 1-35 and a pharmaceutically acceptable carrier.
51. The pharmaceutical composition of claim 50, wherein said RBC binding conjugate is purified.
52. A method of producing a red blood cell (RBC) binding conjugate, said method comprising contacting a monoclonal antibody or fragment thereof with a mammalian serum protein or with an enzyme, wherein said monoclonal antibody or fragment binds a C3b-like receptor on a red blood cell, wherein said monoclonal antibody or fragment or said mammalian serum protein or enzyme is derivatized with a bifunctional polymer linker such that said monoclonal antibody or fragment thereof or said mammalian serum protein or enzyme comprises a reactive hydrazide group, and wherein (i) said mammalian serum protein or enzyme, if said monoclonal antibody or • fragment is derivatized, or (ϋ) said monoclonal antibody or fragment thereof, if said mammalian serum protein or enzyme is derivatized, comprises a reactive carbonyl or carboxylic acid group, under conditions conducive for reaction between said reactive hydrazide group and said reactive carbonyl or carboxylic acid group, thereby producing said RBC binding conjugate.
53. The method of claim 52, wherein at least one of said reactive hydrazide group and said reactive carbonyl or carboxylic acid group is attached to an aromatic ring.
54. The method of claim 53, wherein said polymer is polyethylene glycol (PEG).
55. A method of producing a red blood cell (RBC) binding conjugate, said method comprising contacting a monoclonal antibody or fragment thereof with a mammalian serum protein or with an enzyme, wherein said monoclonal antibody or fragment binds a C3b-like receptor on a red blood cell, wherein said monoclonal antibody or fragment thereof or said mammalian serum protein or enzyme is derivatized with a bifunctional polymer linker such that said monoclonal antibody or fragment thereof or said mammalian serum protein or enzyme comprises a reactive carbonyl or carboxylic acid group, and wherein (i) said mammalian serum protein or enzyme, if said monoclonal antibody or fragment thereof is derivatized, or (ii) said monoclonal antibody or fragment thereof, if said mammalian serum protein or enzyme is derivatized, comprises a reactive hydrazide group, under conditions conducive for reaction between said hydrazide group and said reactive carbonyl or carboxylic acid group, thereby producing said RBC binding conjugate.
56. The method of claim 55, wherein at least one of said reactive hydrazide group and said reactive carbonyl or carboxylic acid group is attached to an aromatic ring.
57. The method of claim 56, wherein said polymer is polyethylene glycol (PEG).
58. The method of claim 57, wherein said bifunctional polymer linker is N- hydroxy-succinimidyl polyethylene glycol-benzaldehyde.
59. The method of claim 58, said reactive hydrozide group is introduced to said monoclonal antibody or fragment thereof or said mammalian serum protein or enzyme by derivatizing said monoclonal antibody or fragment thereof or said mammalian serum protein with succinimidyl C64-hydrazino-nictoinamde acetone hydrazone.
60. The method of any one of claims 52-59, further comprising producing said derivatized monoclonal antibody or fragment thereof or said derivatized mammalian serum protein or enzyme.
61. A red blood cell (RBC)ZRBC binding conjugate that consists essentially of a red blood cell bound to one or more RBC binding conjugate, wherein each of said RBC binding conjugate is as claimed in any one of claims 1-35.
62. A red blood cell (RBC) binding conjugate comprising a monoclonal antibody or a fragment thereof covalently conjugated to a hormone selected from the group consisting of an amine-derived hormone, a steroid hormone, and sterol hormone, wherein said monoclonal antibody or fragment thereof (i) binds a C3b-like receptor on a red blood cell, and (ii) lacks a functional effector domain.
63. The RBC binding conjugate of claim 62, wherein said monoclonal antibody or fragment thereof is covalently conjugated to an amine-derived hormone selected from the group consisting of catecholamine, epinephrine, dopamine, norepinephrine, melatonin, serotonin, thyroxine and triiodothyronine.
64. The RBC binding conjugate of claim 62, wherein said monoclonal antibody or fragment thereof is covalently conjugated to a steroid hormone selected from the group consisting of glucocorticoid, mineralocorticoid, androgen, estrogen, and progestagen.
65. The RBC binding conjugate of claim 64, wherein said androgen is selected from the group consisting of testosterone, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), androstenedione, and dihydrotestosterone (DHT).
66. The RBC binding conjugate of claim 62, wherein said monoclonal antibody or fragment thereof is covalently conjugated to a sterol hormone that is a Vitamin D derivative.
67. A red blood cell (RBC) binding conjugate comprising a monoclonal antibody or a fragment thereof covalently conjugated to a molecule selected from the group consisting of a DNA damaging agent, an anti-metabolite, and an anti-mitotic agent, wherein said monoclonal antibody or fragment thereof (i) binds a C3b-like receptor on a red blood cell, and (ii) lacks a functional effector domain.
68. The RBC binding conjugate of claim 67, wherein said monoclonal antibody or fragment thereof is covalently conjugated to a DNA damaging agent selected from the group consisting of camptothecin, topotecan, doxorubicin, etoposide phosphate, teniposide, sobuzoxane, anthracycline antibiotic, mitomycin antibiotic, cisplatin, busulfan, cyclophosphamide, bleomycin, and tamoxifen
69. The RBC binding conjugate of claim 67, wherein said monoclonal antibody or fragment thereof is covalently conjugated to an anti-metabolite selected from the group consisting of cytosine, arabinoside, floxuridine, 5-fluorouracil (5-FU), mercaptopurine, gemcitabine, hydroxyurea (HU), and methotrexate (MTX).
70. The RBC binding conjugate of claim 67, wherein said monoclonal antibody or fragment thereof is covalently conjugated to an anti-mitotic agent selected from the group consisting of vinblastine, vincristine, and paclitaxel (Taxol).
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