EP1154799A2 - Complexes constitues d'un anticorps et de chimiokines biotinylees - Google Patents

Complexes constitues d'un anticorps et de chimiokines biotinylees

Info

Publication number
EP1154799A2
EP1154799A2 EP00911877A EP00911877A EP1154799A2 EP 1154799 A2 EP1154799 A2 EP 1154799A2 EP 00911877 A EP00911877 A EP 00911877A EP 00911877 A EP00911877 A EP 00911877A EP 1154799 A2 EP1154799 A2 EP 1154799A2
Authority
EP
European Patent Office
Prior art keywords
biotin
composition
antibody
complex
chemokine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00911877A
Other languages
German (de)
English (en)
Inventor
Walter Newman
Dominic Picarella
Dulce Soler
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Millennium Pharmaceuticals Inc
Original Assignee
Millennium Pharmaceuticals Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Millennium Pharmaceuticals Inc filed Critical Millennium Pharmaceuticals Inc
Publication of EP1154799A2 publication Critical patent/EP1154799A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6891—Pre-targeting systems involving an antibody for targeting specific cells
    • A61K47/6897—Pre-targeting systems with two or three steps using antibody conjugates; Ligand-antiligand therapies
    • A61K47/6898—Pre-targeting systems with two or three steps using antibody conjugates; Ligand-antiligand therapies using avidin- or biotin-conjugated antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • This invention relates to biotinylated compounds that, optionally, are complexed with an anti-biotin antibody or fragment thereof. More specifically, the invention relates to complexes comprising biotinylated-chemokines and an antibody or fragment thereof selectively bound to the biotin.
  • the biotinylated-chemokines and complexes containing same are useful for modulating the mechanism underlying a variety of disease states, including a chemokine-mediated cellular response, and for the selective delivery of agents to sites of disease activity.
  • Chemokines are a class of cytokine molecules that are involved in cell recruitment and activation in inflammation. These chemokines have been classified into four subgroups, depending on the nature of the spacing of two highly-conserved cysteine amino acids that are located near the amino terminus of the polypeptide. The first chemokine subgroup is referred to as "CXC”; the second subgroup is referred to as “CC”; the third chemokine subgroup is referred to as “CX3C”; and the fourth chemokine subgroup is referred to as "C”. Within these subgroups, the chemokines are further divided into related families that are based upon amino acid sequence homology. The CXC chemokine families include the IP- 10 and Mig family; the
  • the GRO ⁇ , GRO ⁇ , and GRO ⁇ family include the monocyte chemoattractant protein (MCP) family; the family including macrophage inhibitory protein- l ⁇ (MlP-l ⁇ ), macrophage inhibitory protein- l ⁇ (MlP-l ⁇ ), and regulated on activation normal T cell expressed (RANTES).
  • MCP monocyte chemoattractant protein
  • MlP-l ⁇ macrophage inhibitory protein- l ⁇
  • MlP-l ⁇ macrophage inhibitory protein- l ⁇
  • MlP-l ⁇ macrophage inhibitory protein- l ⁇
  • RANTES regulated on activation normal T cell expressed
  • the stromal cell-derived factor l ⁇ (SDF-l ⁇ ) and stromal cell-derived factor l ⁇ (SDF-l ⁇ ) represent a chemokine family that is approximately equally related by amino acid sequence homology to the CXC and CC chemokine subgroups.
  • the CX3C chemokine family includes fractalkine;
  • the C chemokine family includes lymphotactin.
  • the CXC chemokines are bound by members of the CXCR class of receptors;
  • the CC chemokines are bound by the CCR class of receptors;
  • the CX3C chemokines are bound by the CX3CR class of receptors;
  • the C chemokines are bound by the CR class of receptors.
  • Cells which express chemokine receptors include migratory cells such as lymphocytes, granulocytes, and antigen-presenting cells (APCs) that are believed to participate in immune responses or that may release other factors to mediate other cellular processes in vivo.
  • APCs antigen-presenting cells
  • chemokine gradient serves to attract migratory cells which express the chemokine receptors.
  • migratory cells can be attracted by a chemokine gradient to a particular site of inflammation, at which location they play a role in further modifying the immune response.
  • Chemokine receptors also are involved in interacting with viral proteins.
  • CXCR4(fusin), CCR5, and other chemokine receptors have been identified as co- receptors for HIV-1 and HIV-2.
  • chemokine receptors are expressed on a variety of non-motile cells such as neurons, microglia, epithelial cells and fibroblasts.
  • Chemokines are also known to affect a variety of non-migratory cell functions-such as granule release, cytokine release, angiogenesis, growth and differentiation.
  • the half-life for chemokines in vivo is relatively short. (See, e.g., D. Hechtman, et al., J. Immunol. 147(3):883-892 (1991) which reports a decline to preinjection levels of IL-8 in 30 minutes).
  • the present invention overcomes these and other obstacles by providing a biotinylated-chemokine antibody complex that can be reversibly dissociated in the presence of free biotin. Accordingly, the invention provides a method by which the concentration of biotinylated-chemokine antibody complex can be adjusted by exposing the complex to varying amounts of free biotin in vivo or in vitro.
  • the complexes of the invention are useful for a variety of applications, including modulating an immune response such as one which is mediated by the chemokine- induced recruitment of migratory cells to a site of inflammation, as well as for the targeted delivery of agents to cells which express a chemokine receptor.
  • the invention embraces biotinylated peptide ligands that bind to G-protein coupled receptors.
  • the invention permits the targeted delivery of agents
  • a composition which includes at least one type of each of the following components: (a) a biotin conjugate (also referred to as a "biotinylated agent"), including: (i) a biotin covalently coupled to (ii) a pharmacologically active agent; and (b) an anti-biotin antibody.
  • a biotin conjugate also referred to as a "biotinylated agent”
  • biotinylated agent including: (i) a biotin covalently coupled to (ii) a pharmacologically active agent; and (b) an anti-biotin antibody.
  • the anti-biotin antibody is selectively bound to the biotin to form a complex of the invention.
  • composition optionally further includes a pharmaceutically acceptable carrier and may be formulated for a variety of modes of delivery in vivo, including intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, and controlled-release delivery.
  • pharmacologically active agents are chemokines, although other peptide ligands which bind to G-protein coupled receptors also are embraced within the broader aspects of the invention.
  • the linker molecule is selected to have a structure and to provide a sufficient distance between the biotin and the pharmacologically active agent to ensure that the presence of the biotin and/or the linker molecule does not adversely affect the pharmacological activity of the agent.
  • Such linker molecules are well known in the art for coupling peptides and proteins to one another and for the attachment of other functional molecules to proteins.
  • the biotinylated pharmacologically active agents are peptides which selectively bind to G-protein coupled receptors.
  • Exemplary peptide ligands which bind to G-protein coupled receptors include Angiotensin; Bradykinin; Bombesin/Neuromedin; C3a; C5a; Calcitonin; Calcitonin Gene Related Peptide; Chemokine; Cholecystokinin; Conopressin; Corticotropin Releasing Factor (CRF); CD55 - Decay Accelerating Factor (DAF); Diuretic Hormone Receptors; Endothelin; MLP; FSH Glycoprotein Hormone; Galanin; Growth Hormone Releasing Hormone (GHRH); Growth Hormone Secretagogue (GHS); Gastric Inhibitory Peptide; Gastric Inhibitory Peptide; Glucagon-like Peptide; Glucagon; Gonadotropin Releasing Hormone
  • the pharmacologically active agents are chemokines.
  • chemokines are provided in Table 1 for use in accordance with the methods of the invention. The Tables are located immediately preceding the claims.
  • biotin is coupled to the pharmacologically active peptide via a linker molecule. More specifically, for peptides which are chemokines, biotin is coupled via a linker to the carboxyl terminus of the chemokine.
  • biotinylated-chemokines of the invention embrace biotin coupled to the complete sequence of the chemokine, as well as biotin coupled to truncated or elongated versions of such peptides (e.g., a chemokine peptide which lacks a portion of its amino terminal or carboxyl terminal sequences).
  • a chemokine peptide which lacks a portion of its amino terminal or carboxyl terminal sequences.
  • interaction of the chemokine highly basic carboxyl terminus with the negatively charged glucosaminoglycans mediates the cellular uptake of chemokines and that truncation or deletion of the highly basic carboxyl terminus of chemokines can be used to create novel chemokine agonists having improved half-life characteristics.
  • the invention also embraces biotinylated truncated or elongated chemokine peptides, in particular, at the amino terminus, for use as chemokine antagonists to inhibit the normal pharmacological activity of the chemokine.
  • biotinylated truncated or elongated chemokine peptides in particular, at the amino terminus, for use as chemokine antagonists to inhibit the normal pharmacological activity of the chemokine.
  • the complexes of the invention further include an anti-biotin antibody which selectively binds to biotin in the biotin conjugate.
  • an anti-biotin antibody which selectively binds to biotin in the biotin conjugate.
  • the term "antibody” embraces intact antibodies as well as antibody fragments, e.g., Fab' 2 fragments, CDR3 regions.
  • the anti-biotin antibody is a human, humanized, or primatized antibody and is non-antigenic in humans.
  • the anti-biotin antibodies of the invention have a biotin binding domain that selectively binds to biotin alone or coupled to a linker molecule.
  • One distinguishing feature of the anti-biotin antibodies of the invention when complexed to a biotin conjugate, is a half-life of the complex which is significantly greater than the half-life of the free chemokine (not conjugated or associated in a complex) or biotinylated chemokine.
  • the complexes of the invention have a half-life on the order of one day to one month
  • the effective half-life of the complex can be attenuated by contacting the complex with biotin (in vivo or in vitro) to shift the equilibrium in the direction of complex dissociation.
  • biotin in vivo or in vitro
  • the anti-biotin antibodies of the invention are selected which permit the dissociation of the antibody from the biotin conjugate only in the presence of supra physiological levels of free biotin (alone or coupled to the linker molecule), i.e., the complex will not become dissociated under the conditions of physiologic biotin concentrations.
  • Physiological concentrations of biotin in blood are approximately 0.5 nMol/liter or 122 ng/L (122 pg/ml).
  • the invention provides a mechanism by which the antibody can be selectively dissociated from the biotinylated chemokine, e.g., by contacting the complex with superphysiological concentrations of an exogenous source of free biotin under conditions which shift the equilibrium reaction in favor of complex dissociation.
  • the antibodies of the invention are selected, in part, based upon their ability to dissociate from the biotinylated pharmacologically active agent in the presence of exogenous biotin.
  • the antibodies of the invention when complexed, exhibit a half-life of the complex that is significantly shorter than the half-life of an avidin-biotin complex in the presence of a supra physiological level of free biotin.
  • the half-life of the complex in the presence of a supra physiological level of free biotin can be determined in accordance with routine procedures known to those of ordinary skill in the art.
  • the half-life for the complexes of the invention are at least one-tenth, preferably one-one hundredth, and, more preferably, one-one thousandth of the half-life of the avidin-biotin complex in the presence of a supra physiological level of free biotin.
  • the half-life for a preferred complex of the invention in the presence of a supra physiological level of free biotin, with respect to the dissociation of biotin from the anti-biotin antibody is less than about one hour, more preferably less than about 0.5 hours and, most preferably, less than about 15 minutes.
  • Exemplary anti-biotin antibodies that are publicly available and that can be tested in screening assays to determine whether the antibody exhibits an acceptable dissociation rate constant and/or affinity constant for use (or further modification for use) in accordance with present invention are identified below.
  • the antibodies of the invention further include a diagnostic or therapeutic agent for targeted delivery to a cell which expresses a receptor for the pharmacological agent (e.g., chemokine receptor).
  • a diagnostic or therapeutic agent for targeted delivery to a cell which expresses a receptor for the pharmacological agent e.g., chemokine receptor.
  • chemokine receptor e.g., chemokine receptor
  • the anti-biotin antibody exhibits a multi-specificity, preferably, a dual specificity.
  • the antibody includes a first binding domain which selectively binds to biotin and a second binding domain which selectively binds to at least one other molecule.
  • the antibody can exhibit a second specificity for a second biotin molecule, a tumor cell associated antigen, or a viral associated antigen.
  • tumor associated antigens are those from the following types of tumor cells: breast cancer cells, ovarian cancer cells, lung cancer cells, prostate cancer cells, as well as other, for example, her2-neu expressing cancer cells.
  • the tumor cell associated antigens are cell-surface antigens.
  • tumor associated antigens include carcinoembryonic antigen (tumors of epithelial origin, such as colon, lung, and breast and their metastases), EGF-R (bladder and breast cancer), prostate specific membrane antigen (prostate cancer), GD2 (neuroblastoma), membrane immunoglobulins (lymphomas), and T-cell receptors (T-cell lymphoma).
  • exemplary viral associated antigens include: gpl20 of HIV, HbsAg, immediate and early genes of hepatitis C virus, CMV, Epstein Barr virus, and respiratory syncytial virus.
  • the complex includes a biotin conjugate containing a chemokine that is involved in the recruitment of migratory cells which mediate a THl response, and the antibodies of the invention selectively bind to a tumor cell associated antigen and to the biotinylated-chemokine.
  • the complex of the invention is delivered to tumor cells which express the tumor cell antigen and, thereby, mediates recruitment of THl -type cells to the location of the tumor cells to enhance a localized THl immune response.
  • the antibodies of the invention exhibit a dual specificity to permit recruitment of pre-selected TH2-type cells to a location to treat a localized site of inflammation.
  • a biotin composition comprising a therapeutically effective amount of a biotin of the invention and a pharmaceutically acceptable carrier.
  • the biotin composition is useful for administration to a patient who has or will receive a biotinylated-pharmacologically active agent antibody complex of the invention.
  • Administration of the biotin composition facilitates dissociation of the complex in vivo and, thereby, allows one of ordinary skill in the art to further adjust the half-life of the complex in vivo.
  • the half-life of a biotinylated-chemokine of the invention is longer when the biotin conjugate is complexed with an anti-biotin antibody of the invention and is shorter when the biotin conjugate is present "free" (uncomplexed) in vivo.
  • biotin composition also can be used to adjust the concentration in vitro.
  • the biotin composition can be administered in a variety of methods and, preferably, is administered in an oral form.
  • the therapeutically effective amount of biotin is significantly greater for the oral biotin composition of the invention compared to dietary biotin supplements.
  • the oral biotin compositions of the invention are at least 10-fold, preferably 100-fold, and, more preferably, at least 1 ,000-fold greater than the concentration of biotin in dietary supplements.
  • the therapeutically effective amount of biotin that is present in the biotin composition is from about 100 ⁇ g to about 100 mg.
  • the effective amount of biotin is from about 100 ⁇ g to about 10 mg and, most preferably, the effective amount of biotin is from about 1 mg to about 10 mg.
  • a composition comprising a mixture of biotin conjugates.
  • the composition includes: (a) a first biotin conjugate, including (i) a first biotin covalently coupled to (ii) a first agent having a first pharmacological activity; and (b) a second biotin conjugate, including (i) a second biotin covalently coupled to (ii) a second agent having a second pharmacological activity.
  • the first biotin and the second biotin may be the same or different.
  • the first biotin may include a linker which differs from that included in the second biotin.
  • the biotins are coupled to the first and second agents, respectively, in a manner which does not adversely affect (diminish to a significant extent) the first and second pharmacological activities.
  • the first agent and the second agent represent different chemokines which bind to different receptors expressed on the same or different cell types.
  • the composition optionally, further includes an anti-biotin antibody that binds to the first biotin conjugate and/or the second biotin conjugate.
  • the composition also provides a means of reversibly attaching two or more chemokines by way of an anti- biotin antibody.
  • the complexes of the invention can be used to target different categories of pre-selected chemokine receptors, e.g., CCR2 and CXCR3 receptors, without requiring de novo fusion protein construction.
  • the anti-biotin antibody has a dual specificity for binding to biotin and to an antigen expressed on the surface of a pre-selected cell.
  • a first anti- biotin antibody can be used to target a receptor that is expressed on a cytotoxic T-cell (e.g., CD8)
  • a second anti-biotin antibody can be used to target a receptor that is expressed on a monocyte or a virus-infected cell.
  • the complexes of the invention containing the same or different chemokines can be delivered to the same or different cell types.
  • a biotin conjugate composition includes the above-described biotin conjugate of the invention and a pharmaceutically acceptable carrier.
  • the biotin conjugate includes a biotin covalently coupled to an agent having a pharmacological activity (e.g., a chemokine).
  • the pharmaceutically acceptable carrier is one which is tailored for in vivo use, particularly parenteral use. Exemplary pharmaceutically acceptable carriers are disclosed below.
  • a method for treating inflammation in a subject is provided. As used herein, the word treating embraces preventing, inhibiting, and ameliorating the symptoms of the particular condition which is being treated.
  • the method of treatment involves: administering to a subject in need of such treatment a therapeutically effective amount of a biotin conjugate or of a complex comprising: (a) a biotin conjugate and an anti-biotin antibody.
  • the biotin conjugate includes: (i) a biotin covalently coupled to (ii) an agent that selectively binds to a receptor expressed by a pre-selected cell associated with inflammation (e.g., a migratory cell).
  • Administration of the biotin conjugate or the complex prevents or reduces inflammation in the subject.
  • the biotin conjugate(s) and the anti-biotin antibody are simultaneously or sequentially administered to the subject.
  • the biotin conjugate is a biotinylated chemokine.
  • chemokines that can be used as the agent to which biotin is coupled in this aspect of the invention are provided in Table 1.
  • the pre-selected cell is a migratory cell, such as a T-cell or leukocyte, and the administration of a complex enhances recruitment of the migratory cell to a site of tumor cells in the subject.
  • a method for delivering a diagnostic or a therapeutic agent to a pre-selected cell is provided.
  • the pre-selected cells express on their surface a receptor which selectively binds to a biotin conjugate of the invention.
  • the method involves contacting a population of cells containing a pre-selected cell (e.g., a leukocyte) with an effective amount of a complex of the invention under conditions to deliver the complex to the pre-selected cell.
  • the complex includes: (a) a biotin conjugate including (i) a biotin covalently coupled to (ii) an agent that selectively binds to a receptor expressed by the pre-selected cell; and (b) an anti-biotin antibody selectively bound to the biotin.
  • the anti-biotin antibody further includes a diagnostic and/or therapeutic agent.
  • Exemplary therapeutic agents include cytotoxic agents such as those described elsewhere in this application.
  • Exemplary diagnostic agents include detectable labels such as those described below.
  • the diagnostic and/or therapeutic agents of the invention are attached to the anti-biotin antibodies in a manner which does not adversely affect the ability of the antibody to selectively bind to biotin.
  • a method for modulating (up regulating or down regulating/desensitizing) a pre-selected chemotactic response is provided. The method involves administering to a subject in need of such treatment a therapeutically effective amount of a biotinylated chemokine agonist or chemokine antagonist to modulate the chemotactic or other chemokine mediated proinflammatory response.
  • the method for modulating a chemokine response can be used to evaluate the role of specific chemokine receptors in various animal models of human disease.
  • the method for modulating a chemokine response also can be used to prevent recruitment and/or activation of resident inflammatory cells, e.g., by administering to a subject in need of such treatment a biotinylated chemokine antagonist of the invention, particularly a biotinylated CXC chemokine antagonist. Additionally, the method can be used to identify specific leukocyte populations which express receptors for chemokines, diagnostic imaging, reagent screening, and creating new animal models of human disease (e.g., by disrupting the normal chemotactic response and observing a change in symptoms in the animal model).
  • Fig. 2 shows that C-terminally biotinylated ITAC is equivalent to ITAC in binding to CXCR3.
  • Fig. 3 shows that biotinylated ITAC is equivalent to ITAC in chemotaxis of CXCR3 expressing RBL cells.
  • Fig. 4 shows that significant recruitment of eosinophils to the peritoneum does not begin until 8 hours post challenge in an ovalbumin model for eosinophil recruitment into the peritoneum.
  • Fig. 5 shows that eosinophil recruitment to the peritoneum of ova- sensitized mice is selectively inhibited by low doses of biotinylated eotaxin complexed with murine anti-biotin antibody at 48 hours post challenge.
  • Fig. 6 shows that the complex of biotinylated eotaxin and mouse anti-biotin does not inhibit neutrophil recruitment to the peritoneum in ova-sensitized mice at 6 hours post challenge.
  • Fig. 7 shows that lymphocyte recruitment to the peritoneum of ova-sensitized and challenged mice is selectively inhibited by biotinylated ITAC but not eotaxin complexed with murine anti-biotin antibody at 72 hours post challenge.
  • Fig. 8 shows that soluble eotaxin administered 24 hours following ova challenge renders the recruited peritoneal eosinophils refractory to further chemotactic stimuli for at least 24 hours.
  • the present invention provides a biotinylated-chemokine antibody complex that can be reversibly dissociated in the presence of free biotin. Accordingly, the invention provides a method by which the concentration of a biotinylated-chemokine antibody complex can be adjusted by exposing the complex to varying amounts of free biotin in vivo or in vitro.
  • the complexes of the invention are useful for a variety of applications, including modulating an immune response such as one which is mediated by the chemokine-induced recruitment of migratory cells to a site of inflammation, as well as for the targeted delivery of agents to cells which express a chemokine receptor.
  • a pharmacologically active agent refers to a peptide that binds to a G-protein coupled receptor.
  • G protein-coupled receptor superfamily is a large group of receptor proteins which share a common structural homology.
  • Exemplary GPCR include the calcium sensing receptor (CSR) and the metabotropic glutamate receptors (mGluRs) (Tanage, Y., et al., Neuron, 1992, 8:169-179; Brown, E., et al., Nature, 1993, 366:575-580); several receptors for glycoprotein hormones (Segaloff, D., et al., Oxf. Rev. Reprod.
  • exemplary peptide ligands that bind to G-protein coupled receptors include Angiotensin; Bradykinin; Bombesin/Neuromedin; C3a; C5a; Calcitonin; Calcitonin Gene Related Peptide; Chemokine; Cholecystokinin; Conopressin; Corticotropin Releasing Factor (CRF); CD55 - Decay Accelerating Factor (DAF); Diuretic Hormone Receptors; Endothelin; MLP; FSH Glycoprotein Hormone; Galanin; Growth Hormone Releasing Hormone (GHRH); Growth Hormone Secretagogue (GHS); Gastric Inhibitory Peptide; Gastric Inhibitory Peptide; Glucagon-like Peptide; Glucagon; Gonadotropin Releasing Hormone; LH Glycoprotein Hormone; Melanocortin Receptors; Neuropeptide Y; Neurotensin; Opioid
  • the pharmacologically active agents are covalently coupled to a biotin in a manner which does not adversely affect the pharmacological activity of the pharmacologically active agent.
  • the binding of a peptide to its cognate G-protein coupled receptor is accompanied by G-protein signal transduction, an event which can be measured using conventional screening assays, such as assays that measure changes in the intracellular concentrations of calcium and/or cyclic nucleotides (see, e.g., PCT Publication No. WO 94/18959, entitled “Calcium Receptor-Active Molecules," inventors E. Nemeth et al.).
  • the chemokines are a preferred class of pharmacologically active agents. Exemplary and preferred chemokines that are useful in the compositions and methods of the invention include those which are depicted in Table 1.
  • compositions and methods that include a biotinylated-chemokine as an exemplary biotinylated pharmacologically active agent. It is to be understood that other peptides which bind to G-protein coupled receptors can be substituted for the chemokines described herein to make and use additional biotinylated pharmacologically active agents and complexes of the invention.
  • a composition which includes the following components: (a) a biotin conjugate, including: (i) a biotin covalently coupled to (ii) a pharmacologically active agent; and (b) an anti-biotin antibody.
  • the anti-biotin antibody is selectively bound to the biotin to form a complex of the invention.
  • the composition optionally further includes a pharmaceutically acceptable carrier and may be formulated for a variety of modes of delivery in vivo, including intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, and controlled-release delivery.
  • the preferred pharmacologically active agents are chemokines.
  • Biotin is a naturally occurring enzyme cofactor in its L isomeric form.
  • the invention also embraces the D isomeric form of biotin, as well as other biotin derivatives.
  • the biotin is conjugated to the pharmacologically active agent (e.g., chemokine) by way of a linker molecule to form a "biotin conjugate".
  • the linker molecule is selected to have a structure and to provide a sufficient distance between the biotin and the pharmacologically active agent to ensure that the presence of the biotin and/or the linker molecule do not adversely affect the pharmacological activity of the agent.
  • linker molecules are well known in the art for coupling peptides and proteins to one another and for the attachment of other functional molecules to proteins.
  • a biotin refers to the naturally occurring metabolic biotin which is in its L-isomeric form (Sigma Chemical Co., St. Louis, MO; Pierce Chemical Co., Rockford, IL), as well as the D-isomeric form and modifications of the foregoing biotin molecules.
  • modified biotin it is meant a biotin (either L- or D-isomer) to which a further molecule is coupled in a manner to facilitate the covalent attachment of biotin to the pharmacologically active agent.
  • modified biotins will refer to a biotin to which a linker molecule is attached.
  • biotin is coupled to the pharmacologically active peptide via a linker molecule.
  • linker molecule More specifically, for peptides which are chemokines, biotin is coupled via a linker to the carboxyl terminus of the chemokine.
  • the biotinylated- chemokines of the invention embrace biotin coupled to the complete sequence of the chemokine, as well as biotin coupled to truncated or elongated versions of such peptides (e.g., a chemokine peptide which lacks a portion of its amino terminal or carboxyl terminal sequences).
  • chemokine highly basic carboxyl terminus with the negatively charged glucosaminoglycans mediates the cellular uptake of chemokines and that truncation or deletion of the highly basic carboxyl terminus of chemokines can be used to create novel chemokine agonists having improved half-life characteristics.
  • the invention also embraces biotinylated truncated or elongated chemokine peptides, in particular, at the amino terminus, for use as chemokine antagonists to inhibit the normal pharmacological activity of the chemokine. (See, e.g., D. Hechtman, et al., J.
  • Linker molecules are used to covalently attach the biotin to the pharmacologically active agent of the invention. Such molecules are discussed in numerous books and catalogues, e.g., Pierce Catalog and Handbook, Rockford, 111. Typically, these reagents are used to assist in the determination of near-neighbor relationships in proteins, three-dimensional structures of proteins, enzyme-substrate orientation, solid-phase immobilization, hapten-carrier protein conjugation and molecular associations in cell membranes. They also are useful for preparing antibody-enzyme conjugates, immunotoxins and other labeled protein reagents. For use in accordance with the present invention, it is necessary to maintain the native structure of the pharmacologically active agent.
  • cross-linking groups are similar (homobifunctional) or different (heterobifunctional)
  • the reagent can be radio-labeled or tagged with another label.
  • Reactive groups that can be targeted using a linker molecule include primary amines, sulfhydryls, carbonyls, carbohydrates and carboxylic acids.
  • any reactive group can be coupled nonselectively using a linker molecule such as photoreactive phenyl azides.
  • Linker molecules are available with varying lengths of spacer arms or bridges. These bridges connect the two reactive ends.
  • the linker molecules for use in accordance with the instant invention must be selected to minimally inhibit the pharmacological activity of the pharmacologically active agent and the ability of biotin to bind to the anti-biotin antibody. Because steric effects dictate the distance between potential reaction sites for cross-linking, different lengths of bridges are required for the interaction. Intermolecular cross-linking is favored with a cross-linker containing a longer space arm (about three to about fifteen atoms).
  • Conjugation reagents contain at least two reactive groups.
  • Homobifunctional cross-linkers can contain at least two identical reactive groups, and heterobifunctional reagents contain two or more different reactive groups.
  • Homobifunctional and heterobifunctional cross-linkers that couple through amines, sulfhydryls or react non- specifically are available from many commercial sources.
  • Exemplary linker molecules that are available from Pierce Co., Rockford, IL are shown in Tables 2,3, 4, and 5. The tables also identify the group that the linker molecule is reactive toward, e.g., sulfhydryl, amino, etc.
  • the chemokine antagonist of the invention can be used to competitively inhibit the binding and activity of a chemokine to its respective receptor in vivo or in vitro and, thereby, inhibit intracellular signaling.
  • the chemokine agonists of the invention can be used to render cells refractory to further stimulation by circulating chemokines.
  • rendering cells refractory it is meant that the cells are no longer responsive in chemotaxis. Ca 2+ flux or degranulation assays.
  • a composition containing a biotin conjugate non-covalently coupled to an anti-biotin antibody to form a complex of the invention.
  • the biotin conjugate includes a biotin covalently coupled to a pharmacologically active agent, and the anti- biotin antibody selectively binds to the biotin to form the complex.
  • the complex compositions of the invention are useful, for example, for modulating an immune response and, in particular, are useful for treating inflammation, enhancing an anti- tumor response, and treating viral infections.
  • an anti-biotin antibody refers to an antibody or antibody fragment that selectively binds to biotin alone, coupled to a linker molecule, or coupled to a pharmacologically active agent of the invention.
  • the anti-biotin antibodies of the invention are selected to bind to biotin and, when complexed. exhibit a half-life of the complex that is significantly shorter than the half-life of an avidin- biotin complex.
  • the half-life of an avidin-biotin complex is on the order of about 200 days in vitro.
  • the half-life for the complexes of the invention are at least one-tenth, preferably one-one hundredth, and, more preferably, one-one thousandth of the half-life of the avidin-biotin complex. More particularly, the half-life for a preferred complex of the invention with respect to the dissociation of biotin from the anti-biotin antibody is less than about one hour, more preferably less than about 0.5 hours and, most preferably, less than about 15 minutes.
  • Exemplary anti-biotin antibodies that are publicly available and that can be tested in screening assays to determine whether the antibody exhibits an acceptable dissociation rate constant and/or affinity constant for use (or further modification for use) in accordance with present invention are described in H.
  • Dissociation and association rate constants can be determined using known methods. For example, such rate constants can be measured using the Biacore® systems instrument (Biacore AB, Uppsala, Sweden). According to this method, a ligand, such as a biotinylated chemokine of the invention, is immobilized onto a gold film and a binding protein, such as an anti-biotin antibody, is contacted with the gold film under conditions to allow the binding protein to bind to the ligand.
  • the Biacore® instrument employs a laser to measure the refractive index at the surface of the gold film and to provide real time measurements of the association and dissociation of the binding protein to the immobilized ligand. The instrument is used according to manufacturer's directions to determine the on-rate constant, the off-rate constant, and the affinity constant (the ratio of the on-rate constant to the off-rate constant).
  • the antibodies of the invention also can be characterized in terms of their affinity constants which can be determined according to conventional methods such as those identified above.
  • the antibodies of the invention have an affinity constant ranging from about 1.0 to about 100.0 nanomolar.
  • the anti-biotin antibodies of the invention have an affinity constant ranging from about 1 to about 50 nanomolar and, more preferably, the antibodies have an affinity constant ranging from about 1 to about 10 nanomolar.
  • the affinity constant for an avidin-biotin complex is several logs higher than those of the antibodies of the invention.
  • the antibodies disclosed herein allow for the competitive binding of soluble biotin to displace or compete with the biotin conjugates that are complexed with the antibodies of the invention in vivo or in vitro.
  • the complexes of the invention can be dissociated in vivo or in vitro by contacting the complexes with a biotin molecule (e.g., L-biotin) at a concentration that is effective to displace the equilibrium of the complex to allow dissociation of the complex.
  • a biotin molecule e.g., L-biotin
  • Antibodies with extremely high affinities for biotin are to be avoided for applications which require a reversible reaction, but are preferred for those applications where an extended half-life is desirable as in chronic inflammatory diseases and cancer.
  • An exemplary screening assay for selecting an antibody, which when complexed, has the appropriate half-life constant and the ability to permit complex dissociation is provided in the examples.
  • the invention provides for the selection of anti-biotin antibodies having particular structural characteristics which allow the antibodies to be used in accordance with the methods of the invention.
  • the invention also permits the selection of linker molecules having the appropriate spacer length to minimize interference with the pharmacological activity of the chemokine to which the biotin is coupled, as well as to select linker molecule primary structures which, when coupled to biotin, can be used to generate monoclonal antibodies that selectively bind to biotin as well as to a portion of the linker molecule.
  • the examples also provide assays for evaluating complex stability in vivo by, for example, measuring the half-life of the complex in vivo, as well as its tissue localization.
  • antibody has been used in its broadest sense to embrace full-length (“intact”) antibody molecules, as well as functionally active fragments thereof (e.g., Fab, Fab' , Fd, scFv. and antibody fragments which include a CDR3 region which binds selectively to a biotin).
  • Antibodies include polyclonal and monoclonal antibodies, prepared according to conventional methodology.
  • the antibodies for human therapeutic applications are human antibodies.
  • an antibody molecule As is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, W.R. (1986) The Experimental Foundations of Modern Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford).
  • the pFc' and Fc regions are effectors of the complement cascade but are not involved in antigen binding.
  • an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region designated an Fab fragment
  • Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd.
  • the Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.
  • CDRs complementarily determining regions
  • FRs framework regions
  • CDR1 through CDR3 complementarily determining regions
  • non-CDR regions of a mammalian antibody may be replaced with similar regions of nonspecific or heterospecific antibodies while retaining the epitopic specificity of the original antibody.
  • This is most clearly manifested in the development and use of "humanized” antibodies in which non-human CDRs are covalently joined to human FR and/or Fc/pFc * regions to produce a functional antibody.
  • PCT International Publication Number WO 92/04381 teaches the production and use of humanized murine RSV antibodies in which at least a portion of the murine FR regions have been replaced by FR regions of human origin.
  • Such antibodies, including fragments of intact antibodies with antigen-binding ability, are often referred to as "chimeric" antibodies.
  • the present invention also provides for Fab' 2 , Fab, Fv and Fd fragments; chimeric antibodies (e.g., based on the commercially available anti-biotin antibodies) in which the Fc and/or FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab') fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences: and chimeric Fd fragment antibodies in which the FR and/or CDR1 and/or CDR2 regions have been replaced by homologous human or non-human sequences.
  • chimeric antibodies e.g., based on the commercially available anti
  • the present invention also includes so-called single chain antibodies, as well as human antibodies derived from libraries such as totally synthetic V gene libraries.
  • the biotin or biotin conjugates of the invention, or a fragment thereof, or complexes of biotin can be used to screen peptide libraries, including phage display libraries, to identify and select peptide binding polypeptides that selectively bind to the biotin or biotin conjugates of the invention.
  • Such molecules can be used, as described, for screening assays, for purification protocols, for use in the complexes of the invention to treat inflammation, deliver a diagnostic or therapeutic agent, or modulate a chemotaxis response in vivo or in vitro, and for other purposes that will be apparent to those of ordinary skill in the art.
  • the antibodies of the present invention are preferably intact antibody molecules including the Fc region.
  • Such intact antibodies will have longer half-lives than smaller fragment antibodies (e.g., Fab) and are more suitable for intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or transdermal administration.
  • Fab fragments including chimeric Fab fragments, are preferred in methods in which the peptides of the invention are administered directly to a local tissue environment.
  • the Fab fragments are preferred when the peptide of the invention is administered directly to the site of the tumor or infection.
  • Fabs offer several advantages over Fab " 2 and whole immunoglobulin molecules for this therapeutic modality. First, because Fabs have only one binding site for their cognate antigen, the formation of immune complexes is precluded whereas such complexes can be generated when bivalent Fab' and whole immunoglobulin molecules encounter their target antigen. This is of some importance because immune complex deposition in tissues can produce adverse inflammatory reactions in certain instances.
  • Fabs lack an Fc region they cannot trigger adverse inflammatory reactions that are activated by Fc, such as activation of the complement cascade.
  • the tissue penetration of the small Fab molecule is likely to be much better than that of the larger whole antibody.
  • Fabs can be produced easily and inexpensively in bacteria, such as E. coli, whereas whole immunoglobulin antibody molecules require mammalian cells for their production in useful amounts. Production of Fabs in E. coli makes it possible to produce these antibody fragments in large fermenters which are less expensive than cell culture-derived products.
  • the invention involves binding polypeptides of numerous size and type that bind selectively to biotin. and conjugates (e.g., with linker molecule and/or with pharmacologically active agent) containing biotin ("biotin conjugate").
  • biotin conjugate e.g., with linker molecule and/or with pharmacologically active agent
  • binding polypeptides also may be derived from sources other than antibody technology.
  • polypeptide binding agents can be provided by degenerate peptide libraries which can be readily prepared in solution, in immobilized form, as bacterial flagella peptide display libraries or as phage display libraries.
  • Combinatorial libraries also can be synthesized of peptides containing one or more amino acids. Libraries further can be synthesized of peptides and non-peptide synthetic moieties.
  • anti-biotin antibodies of the invention can be selected to enhance clearance, e.g., for applications in which it is desirable to minimize the half-life of a complex in vivo.
  • anti-biotin antibodies of suitable isotypes can be prepared.
  • antibody fragments are administered or intact antibodies of a subclass selected from the group consisting of: IgGl, IgG2a, IgG2b, IgG3, IgA, and IgM.
  • an anti- biotin antibody which has a greater hinge region flexibility such as an IgG3 antibody subtype.
  • an anti- biotin antibody which has a greater hinge region flexibility such as an IgG3 antibody subtype.
  • antibodies which are non-antigenic in humans e.g., human monoclonal antibodies or fragments thereof.
  • other mammals e.g., domestic animals (e.g., dogs, cats) and livestock (e.g., cows, sheep, and horses)
  • the biotinylated-chemokine and antibody components of the complex should be selected to be non-antigenic in the species that is being treated.
  • the antibodies of the invention can be selected for targeted delivery to a preselected cell. This selection can be accomplished by selecting an antibody of a particular subgroup (e.g., targeted delivery of the antibody to Fc receptor-bearing cells by virtue of the nature of the Fc domain of the antibody) or by the specificity of an antibody binding domain.
  • the anti-biotin antibodies of the invention optionally include a binding domain which selectively binds to a tumor associated antigen or a viral associated antigen.
  • a tumor cell associated antigen is a term of art as used in, for example, International Application No. PCT/US98/01785, (Publication No. WO 98/33914), entitled “Chimeric Antibody Fusion Proteins for the Recruitment and Stimulation of an Anti-Tumor Immune Response,” inventors J. Rosenblatt, et al.
  • Antibodies of the invention which have a dual specificity preferably bind to a tumor cell associated antigen from tumor cells which are breast cancer cells, ovarian cancer cells, lung cancer cells, prostate cancer cells, or other cells which express on their surface a protein which is associated with cancer cell growth.
  • tumor cell associated antigens include: carcinoembryonic antigen (tumors of epithelial origin, such as colon, lung, and breast and their metastases), EGF-R (bladder and breast cancer), prostate specific membrane antigen (prostate cancer), GD2 (neuroblastoma), membrane immunoglobulins (lymphomas), and T-cell receptors (T-cell lymphoma).
  • carcinoembryonic antigen tumors of epithelial origin, such as colon, lung, and breast and their metastases
  • EGF-R bladedder and breast cancer
  • prostate specific membrane antigen prostate cancer
  • GD2 nerveroblastoma
  • membrane immunoglobulins lymphomas
  • T-cell lymphoma T-cell lymphoma
  • tumor cell associated antigens include: proteins such as Ig-idiotype of B cell lymphoma, mutant cyclin-dependent kinase 4 of melanoma, Pmel- 17 (gp 100) of melanoma, MART- 1 (Melan- A) of melanoma, p 15 protein of melanoma, tyrosinase of melanoma, MAGE 1, 2 and 3 of melanoma, thyroid medullary, small cell lung cancer, colon and/or bronchial squamous cell cancer, B AGE of bladder, melanoma, breast, and squamous cell carcinoma, gp75 of melanoma, oncofetal antigen of melanoma; carbohydrate/lipids such as mucl mucin of breast, pancreas, and ovarian cancer, GM2 and GD2 gangliosides of melanoma; oncogenes such as mutant p53 of
  • tumor antigens e.g., P1A, Connexin 37, MAGE-1, MAGE-3, MART 1/Aa, gplOO, Tyrosinase
  • additional tumor antigens e.g., P1A, Connexin 37, MAGE-1, MAGE-3, MART 1/Aa, gplOO, Tyrosinase
  • CD 19 on B cells also can be used to target certain leukemias and lymphomas.
  • Additional Melanoma tumor antigen sequences are those reported by Slingluff et al., in Curr. Opin. in Immunol. 6:733-740 (1994);
  • Additional tumor cell antigens that are peptides of the mutated APC gene product are those reported by Townsend et al., in Nature 371 :662 (1994)).
  • the anti-biotin antibodies of the invention have a dual specificity for binding to a viral associated antigen.
  • viral associated antigens include: g ⁇ l20 of HIV, HbsAg, immediate and early genes of hepatitis C virus, CMV, Epstein Ban virus, and respiratory syncytial virus.
  • the antibodies with dual specificity are useful for the targeted delivery of chemokines to a cell expressing an antigen to which the anti-biotin antibody selectively binds.
  • a biotinylated chemokine and, optionally, a further therapeutic or diagnostic agent can be selectively delivered to, for example, cells expressing tumor cell associated antigens or cells/viruses expressing virus associated antigens.
  • the antibodies with dual specificity are used to enhance a localized immune response at the site of the tumor cells or virus particles/virus infected cells.
  • the complexes of the invention can be used to enhance a localized THl response for treating a cancer by administering a complex of the invention, including a biotinylated-chemokine and an anti-biotin antibody having a binding domain that selectively binds to a tumor cell associated antigen.
  • a complex of the invention including a biotinylated-chemokine and an anti-biotin antibody having a binding domain that selectively binds to a tumor cell associated antigen.
  • Targeted delivery of the complex to the tumor cell results in the selective recruitment of THl -type cells which express receptors for the chemokine.
  • Exemplary chemokines that bind to receptors expressed on THl -type cells include: IP- 10, MIG, RANTES, and ITAC.
  • the anti-biotin antibodies that are useful in the complex for treating cancer have a binding domain that is selective for binding to biotin and at least one other binding domain that is selective for binding to a tumor cell associated antigen.
  • Administration of the complex to a subject having a cancer that is associated with the tumor cell associated antigen is performed to allow targeting of the complex to the tumor cell and, thereby, result in a localized concentration of chemokine at the site of tumor cells.
  • chemokine concentration at the site of the tumor cells results in a chemokine gradient which mediates the recruitment of THl cells to the site and enhancement of a THl immune response to inhibit or prevent further tumor cell growth and/or proliferation.
  • THl response e.g., lupus, arthritis
  • diseases or conditions which flourish, in whole or in part, due to a lack of a sufficient TH2 response e.g., leprosy, asthma, inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, diseases associated with fibrosis such as emphysema and hepatic fibrosis
  • complexes which include a anti-biotin antibody of dual specificity (to target the complex to the diseased cell) and a biotinylated-chemokine which is effective in recruiting and/or binding to receptors located on TH2-type cells.
  • chemokines that recruit and/or bind to TH2-type cells include: eotaxin, TARC, and MDC.
  • the anti-biotin antibodies of the invention optionally include a therapeutic or diagnostic agent attached to the antibody in a manner which does not adversely affect the ability of the antibody to bind to biotin or to a further antigen, if applicable.
  • therapeutic agents include any therapeutic molecule which desirably is targeted selectively to a cell expressing a tumor cell associated antigen or other infection-associated antigen.
  • Therapeutic agents include antineoplastic agents, radio iodinated compounds, toxins, other cytostatic or cytolytic drugs, and so forth.
  • Antineoplastic therapeutics include: aminoglutethimide, azathioprine, bleomycin sulfate, busulfan, carmustine, chlorambucil, cisplatin, cyclophosphamide, cyclosporine, cytarabidine, dacarbazine, dactinomycin, daunorubicin, doxorubicin, taxol, etoposide, fluorouracil, interferon- ⁇ , lomustine, mercaptopurine, methotrexate, mitotane, procarbazine HC1, thioguanine, vinblastine sulfate and vincristine sulfate.
  • Additional antineoplastic agents include those disclosed in Chapter 52, Antineoplastic Agents (Paul Calabresi and Bruce A. Chabner), and the introduction thereto, 1202-1263, of Goodman and Gilman's "The Pharmacological Basis of Therapeutics", Eighth Edition, 1990, McGraw-Hill, Inc. (Health Professions Division).
  • Toxins can be proteins such as, for example, pokeweed anti-viral protein, cholera toxin, pertussis toxin, ricin, gelonin, abrin, diphtheria exotoxin, or Pseudomonas exotoxin.
  • Toxin moieties can also be high energy-emitting radio nuclides such as cobalt-60.
  • Cytotoxic agents also include, for example, the so-called “suicide” enzymes such as thymidine kinase (TK) and its “suicide” substrate, gangcyclovir, DAB389 EGF (Pickering et. al., J. Clin. Invest. 91 :724-9 (1993)). and allylamine (Hysmith et al., Toxicology 38:141-50 (1986)).
  • TK thymidine kinase
  • DAB389 EGF Fring et. al., J. Clin. Invest. 91 :724-9 (1993)
  • allylamine Hydrochsmith et al., Toxicology 38:141-50 (1986)
  • the anti-biotin antibodies of the invention further include a diagnostic agent, such as a detectable label.
  • a diagnostic agent such as a detectable label.
  • the antibodies may be labeled using radio labels, fluorescent labels, enzyme labels, or free radical labels, using techniques known to the art.
  • the antibodies may be coupled to specific diagnostic labeling agents for imaging of cells and tissues, in vivo or in vitro, that express cancer associated antigens and/or to the above-noted therapeutic agents according to standard coupling procedures.
  • Diagnostic agents include, but are not limited to, barium sulfate, iocetamic acid, iopanoic acid, ipodate calcium, diatrizoate sodium, diatrizoate meglumine, metrizamide, tyropanoate sodium and radio diagnostics including positron emitters such as fluorine- 18 and carbon- 1 1, gamma emitters such as iodine- 123, technetium-99m. iodine-131 and indium- 11 1, nuclides for nuclear magnetic resonance such as fluorine and gadolinium.
  • the antibodies and complexes of the invention can be used in vivo or in vitro. Accordingly, the detectable labels can include more traditional in vitro labels such as those described herein.
  • Typical fluorescent labels include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, and fluorescamine.
  • Typical chemiluminescent compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, and the oxalate esters.
  • Typical bioluminescent compounds include luciferin, and luciferase.
  • Typical enzymes include alkaline phosphatase, ⁇ -galactosidase, glucose-6-phosphate dehydrogenase, maleate dehydrogenase, glucose oxidase, and peroxidase.
  • Other diagnostic agents useful in the invention will be apparent to one of ordinary skill in the art.
  • a biotin composition is provided which includes a therapeutically effective amount of a biotin and a pharmaceutically acceptable carrier. The biotin composition is useful for reducing the amount of complex in vivo or in vitro.
  • biotin compositions are useful for adjusting the concentration of the complex in vivo or in vitro and as a pharmaceutical antidote to terminate therapy, if warranted.
  • a composition comprising a therapeutically effective amount of a biotin in a pharmaceutical acceptable carrier.
  • the therapeutically effective amount of biotin is that amount needed to counteract the effect of a complex of the invention.
  • the biotin compositions disclosed herein are intended for use for adjusting the effective concentration of the complexes of the invention in vivo or in vitro.
  • the therapeutically effective amount of biotin in the biotin composition is from about 100 ⁇ g to about 100 mg. More preferably, the concentration of biotin in the biotin composition is from about 100 ⁇ g to about 10 mg and, most preferably, the concentration of biotin is from about 1 mg to about 10 mg. In general, the concentration of biotin that is administered as a dietary supplement is the U.S. recommended daily allowance of 0.3 mg. In contrast, the concentrations of biotin in the claimed biotin composition are at least 10 fold and, more preferably, 100 fold greater than those which are administered for dietary supplement applications. According to yet another aspect of the invention, a composition including a mixture of complexes of the invention is provided.
  • the composition contains: (a) a first biotin conjugate comprising (i) a first biotin covalently coupled to (ii) a first agent having a first pharmacological activity; and (b) a second biotin conjugate comprising (i) a second biotin covalently coupled to (ii) a second agent having a second pharmacological activity.
  • the first biotin and the second biotin may be the same or different from one another.
  • the first biotin may be the L isomer and the second biotin may be the D isomer.
  • the identity of the first and the second agents is dependent upon the particular use to which the mixture of complexes is to be applied.
  • the mixture of biotin conjugates can further include one or more anti-biotin antibodies to form the complexes of the invention and, optionally, to effect the targeted delivery of the complex to a particular cell population in vivo or in vitro.
  • the anti-biotin antibody can have a dual specificity for biotin and a tumor cell associated antigen.
  • the first and the second agent can be two different chemokines that mediate the recruitment of different immune system cells to the site of the tumor cell location to enhance a THl response to the tumor.
  • the mixture of complexes of the invention can be used to effect the targeted delivery of different chemokines to a virus infected cell.
  • the composition further includes an anti-biotin antibody which has a dual specificity for binding to biotin and to an antigen expressed on the surface of a pre-selected cell.
  • a first anti-biotin antibody can be used to target a receptor that is expressed on a cytotoxic T-cell (e.g., CD8)
  • a second anti-biotin antibody can be used to target a receptor that is expressed on a monocyte or a virus-infected cell.
  • complexes of the invention containing the same or different chemokines can be delivered to the same or different cell types.
  • a pharmaceutical composition containing a biotin conjugate of the invention and a pharmaceutically acceptable carrier is provided.
  • a pharmaceutically acceptable carrier is defined below in reference to the pharmaceutical compositions of the invention, preferably, with the further limitation that the pharmaceutically acceptable carriers containing the biotin conjugates exclude buffer preparations which are commonly used in in vitro assays (e.g.. phosphate buffered saline).
  • the pharmaceutically acceptable carrier for the biotin conjugates of the invention is selected to be used for an intravenous, intraperitoneal, or subcutaneous mode of administration. Accordingly, the therapeutically effective amount of the biotin conjugate also is selected to be suitable for in vivo administration.
  • the therapeutically effective amount of the biotin conjugate is at least 10 fold, more preferably 100 fold, and most preferably 1000 fold greater than the concentration of a biotin conjugate that would be used in connection with an in vitro assay, such as an ELISA assay.
  • a therapeutic method which employs the biotinylated chemokines alone or complexed with anti-biotin antibody.
  • the therapeutic method is useful for treating inflammation in a subject in need of such treatment.
  • the method involves administering to a subject a therapeutically effective amount of a complex comprising: (a) a biotin conjugate comprising (i) a biotin covalently coupled to (ii) an agent that selectively binds to a receptor expressed by a pre-selected cell (e.g., a migratory cell); and (b) an anti-biotin antibody selectively bound to the biotin to form a complex.
  • Administration of the complex prevents or reduces inflammation in the subject.
  • the pre-selected cell is a migratory cell
  • administration of the complex is believed to result in the inhibition of recruitment of the migratory cell to the site of inflammation.
  • the biotin conjugate and the anti-biotin antibody can be administered simultaneously or sequentially to the subject.
  • the method optionally involves further administering a pharmaceutically acceptable composition of biotin to modulate the effective concentration of the complex in vivo or in vitro (by shifting the equilibrium toward dissociation of the complex). Examples of chemokines, their respective receptors and the migratory cells which express these receptors are provided in Table 1.
  • a method to deliver cytotoxic agents to eliminate a specific pre-selected cell e.g., a leukocyte or virus-infected cell
  • the method can be performed in vivo or in vitro.
  • the method can be used to deliver a cytotoxic agent to a pre-selected cell population prior to infusion of the cell population into a patient.
  • the method also can be used in vitro to desensitize chemokine receptors on pre-selected cells prior to cell reinfusion, e.g., by contacting the cells with a biotin conjugate or complex of the invention at one hour, 37 C or under other conditions sufficient to desensitize the cell. In this manner, the susceptibility of the treated pre-selected cells to a chemokine gradient in vivo can be inhibited.
  • the method for delivering a therapeutic agent involves: contacting a population of cells containing a pre-selected cell (e.g., a virus infected cell) with an effective amount of a complex comprising the therapeutic agent under conditions to deliver the therapeutic agent to the pre-selected cell.
  • the complex includes: (a) a biotin conjugate comprising (i) a biotin covalently coupled to (ii) an agent that selectively binds to a receptor expressed by the pre-selected cell; and (b) an anti-biotin antibody selectively bound to the biotin to form the complex.
  • the anti-biotin antibody further comprises the therapeutic agent.
  • the method is performed under conditions whereby contacting the population of cells with the complex allows delivery of the therapeutic agent to the pre-selected cell.
  • a method for modulating (up regulating or down regulating/desensitizing) a pre-selected chemotactic response involves administering to a subject in need of such treatment a therapeutically effective amount of a biotinylated chemokine agonist or a biotinylated chemokine antagonist to modulate the chemotactic response.
  • the method is useful for evaluating the role of specific chemokine receptors in various animal models of human disease.
  • the complexes include an anti-biotin antibody that has a dual specificity to permit the targeted delivery of the biotinylated chemokine agonist or chemokine antagonist to cells expressing the chemokine receptor.
  • a method for identifying specific leukocyte populations which express receptors for chemokines is provided.
  • This method can be used to identify the cognate receptors (and cells expressing the cognate receptors) for chemokines for which receptors have not previously been identified.
  • the method involves contacting the cells suspected of expressing a cognate receptor with a labeled complex of the invention and identifying the receptor and/or cell expressing the receptor to which the complex binds.
  • the receptor can be identified either by immunoprecipitating the cognate receptor or using the responding cells to construct a cDNA library from which to expression clone the receptor from host cells transfectants expressing smaller and smaller pools of cDNAs.
  • the compositions of the invention are administered in effective amounts.
  • an effective amount is a dosage of the biotinylated pharmacologically active agent or complex of the invention sufficient to provide a medically desirable result.
  • the therapeutically effective amount of a complex of the invention is less than about 10 mg/kg body weight and, more preferably, is from about 1 mg/kg to about 5 mg/kg body weight.
  • the effective amount will vary with the particular condition being treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of the treatment, the nature of the concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner.
  • an effective amount is that amount which slows or inhibits the growth and/or proliferation of tumor cells that are associated with the cancer.
  • an effective amount for treating a viral infection would be an amount sufficient to lessen or inhibit altogether the growth and/or proliferation of infected cells so as to slow or halt the development of or the progression of the infection.
  • compositions of the invention can be used to treat the above-noted conditions prophylactically in subjects at risk of developing the foregoing conditions.
  • inhibit embraces all of the foregoing. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgment.
  • the isolated biotin conjugates and biotin complexes may be administered alone or in combination with alternative (complementary) drug therapies, by any conventional route, including injection or by gradual infusion over time.
  • the administration may, for example, be intravenous, intraperitoneal, intramuscular, intra- cavity, subcutaneous, or transdermal.
  • direct administration to the tumor or infected cell site is preferred.
  • the drug therapies are administered in amounts which are effective to achieve the physiological goals (e.g., to prevent or reduce the physiological consequences of cancer, infection, or an aberrant immune response), in combination with the biotin conjugates and biotin complexes of the invention.
  • biotin conjugates and biotin complexes may be administered alone or in combination with the above-described drug therapies as part of a pharmaceutical composition.
  • a pharmaceutical composition may include the isolated biotin conjugates and/or biotin complexes in combination with any standard physiologically and/or pharmaceutically acceptable carriers which are known in the art.
  • the compositions should be sterile and contain a therapeutically effective amount of the isolated biotin conjugates and biotin complexes in a unit of weight or volume suitable for administration to a patient.
  • pharmaceutically-acceptable carrier as used herein means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration into a human or other animal.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • the components of the pharmaceutical compositions also are capable of being co- mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.
  • Pharmaceutically acceptable further means a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism.
  • the characteristics of the carrier will depend on the route of administration.
  • Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials which are well known in the art.
  • compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the biotin conjugates and/or biotin complexes, which is preferably isotonic with the blood of the recipient.
  • This aqueous preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation also may be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butane diol.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or di-glycerides.
  • fatty acids such as oleic acid may be used in the preparation of injectables.
  • Carrier formulations suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • a variety of administration routes are available. The particular mode selected will depend, of course, upon the particular drug selected, the severity of the condition being treated, and the dosage required for therapeutic efficacy.
  • the methods of the invention may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active compounds without causing clinically unacceptable adverse effects.
  • modes of administration include rectal, topical, interdermal, or parenteral routes.
  • parenteral includes subcutaneous, intravenous, intramuscular, or infusion. Intravenous or intramuscular routes are not particularly suitable for long-term therapy and prophylaxis. They could, however, be preferred in emergency situations. Oral and nasal administration are optional, although less preferred, methods.
  • compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing the biotin conjugates and biotin complexes into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the biotin conjugates and biotin complexes into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
  • Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the biotin conjugates and biotin complexes described above, increasing convenience to the subject and the physician.
  • Many types of release delivery systems are available and known to those of ordinary skill in the art. They include the above-described polymeric systems, as well as polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Patent 5,075,109.
  • Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides; hydrogel release systems; sylastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like.
  • Specific examples include, but are not limited to: (a) erosional systems in which an agent of the invention is contained in a form within a matrix such as those described in U.S. Patent Nos. 4,452,775, 4,675,189, and 5,736,152, and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Patent Nos. 3,854,480, 5,133,974 and 5,407,686.
  • pump-based hardware delivery systems can be used, some of which are adapted for implantation.
  • Long-term sustained release means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredient for at least 30 days, and preferably 60 days.
  • Long-term sustained release implants are well-known to those of ordinary skill in the art and include some of the release systems described above. The invention will be more fully understood by reference to the following examples. These examples, however, are merely intended to illustrate the embodiments of the invention and are not to be construed to limit the scope of the invention.
  • biotinylated pharmacologically active agents of the invention are prepared in accordance with routine procedures known to those of ordinary skill in the art or are commercially available.
  • biotinylated chemokines include: biotinylated human MCP-1, human MIP-1 alpha, and human MIP-1 beta (R&D Systems, Minneapolis, MN).
  • biotin conjugates embraced within the scope of the invention can be tested in high throughput screening assays, e.g., ELISA and other colorimetric assays, to select biotin conjugates having the optimum properties (e.g., retention of chemotactic binding activity, optimum binding affinities with anti-biotin antibodies) for use in accordance with the methods of the invention,
  • a) Preparation of Biotinylated Chemokines Biotinylated chemokines and chemokines in general were synthesized by automated solid phase f-moc chemistry (t-boc chemistry can also be used) using a 433 automated peptide synthesizer (PE Biosystems: Foster City, CA).
  • Biotinylated chemokines were synthesized by incorporating during synthesis an extra C-terminal Lysine derivatized at the ⁇ -NH2 group with aminocaproic acid- biotin (f-moc-Lys (aminocaproil-biotin) is commercially available from Calbiochem- Novabiochem, UK).
  • the chemokines described herein were made with the biotin- aminocaproyl-Lys amino acid being the second one starting from the C-terminus. However, this modification can be introduced into any other lysine or lysines in the sequence.
  • any other spacer groups having a carboxyl and an amino functionalities at each end can be used.
  • this synthesis reaction involves a 2-step coupling reaction, in which the spacer group is added to the lysine residue first and then the biotin is coupled to the spacer.
  • synthesizing chemokines containing an extra C-terminal cysteine modifications of this cysteine can be made with any biotin- crosslinker reagents containing sulfhydryl reactive groups (see tables 2-5) for examples of crosslinking reagents). This latter modification can be performed after synthesis, cleavage and folding and purification of the protein.
  • Example 2 Preparation of an Anti-biotin Antibody
  • the anti-biotin antibodies of the invention are prepared in accordance with routine procedures known to those of ordinary skill in the art or are commercially available.
  • An exemplary protocol for preparing a monoclonal antibody that selectively binds to biotin with a relatively high affinity is provided in H. Bagci, et al., FEBS 322(l):47-50 (1993). See also, F. Kohen, et al., Meth. in Enzymol. 279:451-463 (1997); Vincent, P., and Samuel, D., J. Immunol. Meth. 165:177-182 (1993); K. Dakshinamurti, et al, Meth. in Enzymol.
  • mice transgenic for human Vh and VI genes are commercially available from Medarex. Annandale, NJ, or from phage display libraries of human Vh and VI genes, prepared according to the procedure described in M.D. Sheets, et al., PNAS (USA) 95(11):6157-62 (1998), entitled, "Efficient Construction of a Large Nonimmune Phage Antibody Library: the production of high-affinity human single-chain antibodies to protein antigens".
  • Protocols are provided below as exemplary methods for preparing a representative anti-biotin antibody; however, it is to be understood that alternative antigenic compositions (containing biotin alone or coupled to linker molecules of various structures and lengths) can be substituted for the particular antigenic compositions described in the Examples to make anti-biotin antibodies having a range of pre-selected kinetic characteristics (e.g., half-life and affinity constants) using no more than routine experimentation.
  • kinetic characteristics e.g., half-life and affinity constants
  • the immunogen consists of a soluble chemokine conjugated via an aminocaproic acid linker covalently bound to the ⁇ -amino group of a lysine residue added at the carboxyl terminus.
  • mice are boosted with an intravenous injection of about 10-100 ⁇ g of soluble biotinylated chemokine.
  • mice Four days later the mice are euthanized and the spleen cells fused with an appropriate murine fusion partner in a ratio of about 5-10:1 (splee myeloma).
  • the entire fusion is plated in 10 96-well plates and incubated for 8-10 days until the macroscopic appearance of clones.
  • the supematants are then analyzed according to the following protocol, (b) Primary Screen
  • the purpose of the primary screen is to identify all wells containing antibodies that bind conjugated biotin.
  • each supernatant is assayed in the presence and absence of 0.5 nM biotin to approximate the concentration of biotin in peripheral blood.
  • Supematants where binding activity is not affected by the presence of soluble biotin are reassayed in the presence of increasing concentrations of a biotinylated chemokine, preferably other than the immunogen, (and/or a lysine-aminocaproic acid biotin) vs. free biotin.
  • the assay is developed as in the primary assay.
  • the relative binding ratio is calculated from the concentration of free biotin that results in a 50% reduction in antibody binding/concentration of conjugated biotin that results in a 50% reduction in binding.
  • Clones are grouped according to ratio and representatives from each group are subcloned by limiting dilution. The subclones are re-tested, production is scaled up, and the antibodies purified. The purified antibodies are then assessed for their relative ability to bind conjugated biotin in the presence of free biotin in an ELISA with an immobilized biotinylated chemokine (1 biotin moiety/molecule protein) using the same mass amount of each antibody and increasing concentrations of free biotin.
  • the selection criteria for selecting antibodies for use in accordance with the methods of the invention are as follows.
  • Antibodies useful for therapeutic intervention preferably have an affinity for conjugated biotin 1-4 orders of magnitude greater than the affinity for free biotin.
  • the antibody also would be dissociable from the conjugated biotin in the presence of free biotin at higher levels than found in blood.
  • an increased dietary intake of biotin can be employed as an antidote to dissociate the biotinylated chemokine-MAb complexes when medically necessary.
  • Example 3 Screening Assay for Binding to a Chemokine Receptor
  • binding reaction was performed with 500,000 receptor cell transfectants and 0.1 nM radiolabeled chemokine in binding buffer (50mM HEPES ⁇ pH 7.5, 1 mM CaC ⁇ , 5 mM MgC ⁇ , 0.5% BSA and 0.05% azide) for 1 hour at room temperature in the presence or absence of cold chemokine in a range of 8 different concentrations from 0.0008-20 nM.
  • Non-specific binding is determined by residual binding obtained in the presence of 250 nM cold ligand.
  • Cells are then washed with binding buffer containing 0.5 M NaCl and cell associated radioactivity counted with a top count radioactivity counter. Specific binding was calculated by subtracting non-specific binding from the total binding observed at each concentration and IC50 values were calculated by KaleidGraph software.
  • Example 4 Screening Assay for Chemotaxis Activity in vitro
  • a chemotaxis assay was performed in a 24 well transwell plate using LI .2 transfectants expressing the murine or human form of the cognate chemokine receptor.
  • increasing concentrations of the unmodified chemokine or the biotinylated form of the chemokine were added to the bottom chamber of appropriate wells in the transwell plate while 1 ,000,000 chemokine-receptor transfectants were placed in the top chamber. The cells were allowed to migrate for 3-5 hours at 37°C.
  • mice of the BALB/c or C57B1/6 strain were sensitized by subcutaneous administration on days 0 and 7 with 100 ⁇ g of ovalbumin mixed with an adjuvant consisting of 2 mg of aluminum hydroxide.
  • the mice were challenged by intraperitoneal administration of 10 ug of soluble ovalbumin.
  • leukocytes infiltrating into the peritoneum were harvested by peritoneal lavage and the number of each type of leukocyte was determined by differential cell count.
  • Complexes of biotinylated chemokine with the biotin-specific monoclonal antibody are formed ex vivo by mixing 2 ug or 0.2 ug of chemokine with 75 ug of antibody in a volume of 200 ⁇ l of saline, PBS or HBSS for 1 hour at 37°C. This represents a molar ratio of antibody to chemokine of 2 : 1 and 20 : 1 respectively.
  • the complexes are then administered subcutaneously to ovalbumin sensitized mice 15 minutes prior to i.p. challenge.
  • the effect of biotinylated chemokine/MAb complexes on the recruitment of eosinophils to the peritoneum was assessed at 48 hours post challenge.
  • the biotinylated eotaxin alone had no significant effect on eosinophil recruitment when administered at the high dose (2 ug) or the low dose (0.2 ug).
  • the complex of biotinylated eotaxin with the biotin-specific monoclonal antibody significantly inhibited eosinophil recruitment. This was true for both the high dose (2 ug) as well as the low dose (0.2 ug) of eotaxin.
  • complexes of biotinylated ITAC with antibody, produced and administered under identical conditions had no significant effect on eosinophil recruitment.
  • Example 4d Lymphocyte Recruitment to the Peritoneum Is Inhibited by Complexes with Biotinylated ITAC but not Eotaxin
  • the specificity and broad applicability of this invention is demonstrated in the ability to dissect the role of different chemokine receptors in modulating the recruitment of specific leukocyte populations.
  • complexes of biotinylated eotaxin with Mab had no significant effect on lymphocyte recruitment to the peritoneum at 72 hours.
  • complexes of biotinylated ITAC with Mab significantly inhibited lymphocyte recruitment to the peritoneum at 72 hours.
  • the stability of the complex in animal models can be assessed by forming the biotinylated chemokine-MAb complexes with radiolabeled chemokine.
  • the immunoglobulin fraction of the blood is separated from small molecular weight species such as biotin by precipitation with ammonium sulfate (or by binding to protein G-sepharose beads).
  • the ratio of labeled material in the supernatant relative to the immunoglobulin-containing precipitate (or protein G-sepharose bead associated) is a function of the dissociation rate of the chemokine from the antibody.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nanotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Medical Informatics (AREA)
  • Communicable Diseases (AREA)
  • Epidemiology (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Oncology (AREA)
  • Molecular Biology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Virology (AREA)
  • Medicinal Preparation (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne des agents actifs pharmacologiquement biotinylés, ainsi que des complexes renfermant lesdits agents. L'invention concerne, en particulier, des chimiokines biotinylées. Les complexes précités comprennent également un anticorps anti-biotine liant sélectivement la biotine. Ces complexes, qui peuvent se dissocier au contact de biotine libre, sont particulièrement utiles pour accroître la réponse immunitaire aux cellules tumorales et aux cellules infectées par virus, in vivo ou in vitro.
EP00911877A 1999-02-22 2000-02-18 Complexes constitues d'un anticorps et de chimiokines biotinylees Withdrawn EP1154799A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US12100699P 1999-02-22 1999-02-22
US121006P 1999-02-22
PCT/US2000/004312 WO2000050088A2 (fr) 1999-02-22 2000-02-18 Complexes constitues d'un anticorps et de chimiokines biotinylees

Publications (1)

Publication Number Publication Date
EP1154799A2 true EP1154799A2 (fr) 2001-11-21

Family

ID=22393879

Family Applications (1)

Application Number Title Priority Date Filing Date
EP00911877A Withdrawn EP1154799A2 (fr) 1999-02-22 2000-02-18 Complexes constitues d'un anticorps et de chimiokines biotinylees

Country Status (6)

Country Link
EP (1) EP1154799A2 (fr)
JP (1) JP2002537362A (fr)
AU (1) AU3370000A (fr)
CA (1) CA2371849A1 (fr)
MX (1) MXPA01008465A (fr)
WO (1) WO2000050088A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110088129A (zh) * 2016-12-27 2019-08-02 豪夫迈·罗氏有限公司 新的生物素特异性单克隆抗体及其用途

Families Citing this family (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010029317A2 (fr) 2008-09-10 2010-03-18 Ibd Column Therapies International Ab Traitement d’états inflammatoires
KR20130113493A (ko) * 2011-01-03 2013-10-15 에프. 호프만-라 로슈 아게 항―ｄｉｇ 항체 및 펩티드에 접합되어 있는 디그옥시게닌의 복합체의 약학적 조성물
WO2012172343A2 (fr) 2011-06-13 2012-12-20 Ith Immune Therapy Holdings Traitement de l'angiocholite sclérosante primitive
EP2718311B1 (fr) 2011-06-13 2018-01-17 TLA Targeted Immunotherapies AB Traitement de la sclérose en plaques
ES2688081T3 (es) 2011-06-13 2018-10-30 Tla Targeted Immunotherapies Ab Tratamiento de dolencias asociadas a septicemia
EP2718310B1 (fr) 2011-06-13 2017-08-23 TLA Targeted Immunotherapies AB Traitement de troubles mentaux
EP3693041A1 (fr) 2011-06-13 2020-08-12 TLA Targeted Immunotherapies AB Traitement du cancer
EP2717940B1 (fr) 2011-06-13 2020-04-22 TLA Targeted Immunotherapies AB Traitement d'états associés à un syndrome métabolique
WO2012172345A2 (fr) 2011-06-13 2012-12-20 Ith Immune Therapy Holdings Traitement d'états associés à l'allergie
ES2760300T3 (es) 2011-06-13 2020-05-13 Tla Targeted Immunotherapies Ab Diagnóstico y tratamiento de la enfermedad inflamatoria del intestino y del síndrome del intestino irritable
WO2012172337A2 (fr) 2011-06-13 2012-12-20 Ith Immune Therapy Holdings Traitement de maladie cardiovasculaire
EP3228631B1 (fr) 2011-06-13 2020-06-24 TLA Targeted Immunotherapies AB Traitement de troubles respiratoires
WO2012172339A2 (fr) 2011-06-13 2012-12-20 Ith Immune Therapy Holdings Traitement de l'arthrite inflammatoire
WO2012172336A2 (fr) 2011-06-13 2012-12-20 Ith Immune Therapy Holdings Traitement de maladies inflammatoires de la peau
US9726666B2 (en) 2011-06-13 2017-08-08 Tla Targeted Immunotherapies Ab Diagnosing and treating inflammatory diseases
JP6324376B2 (ja) 2012-07-04 2018-05-16 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ 抗テオフィリン抗体および使用方法
JP6247287B2 (ja) * 2012-07-04 2017-12-13 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ 抗ビオチン抗体および使用方法
AU2013285422B2 (en) 2012-07-04 2017-04-27 F. Hoffmann-La Roche Ag Covalently linked antigen-antibody conjugates
KR102051433B1 (ko) * 2013-08-27 2020-01-08 (주)네오팜 근육 분화 촉진 및 근육 강화용 조성물 및 외용제
RU2694981C2 (ru) 2014-01-03 2019-07-18 Ф. Хоффманн-Ля Рош Аг Ковалентно связанные конъюгаты хеликар-антитело против хеликара и их применения
BR112016013849A2 (pt) 2014-01-03 2017-10-10 Hoffmann La Roche conjugados de anticorpos biespecíficos antihapteno/antirreceptores da barreira hematoencefálica, seus usos, e formulação farmacêutica
EP3089759B1 (fr) 2014-01-03 2018-12-05 F. Hoffmann-La Roche AG Conjugués covalents entre un anticorps et une toxine polypeptidique
MX2016015280A (es) 2014-06-26 2017-03-03 Hoffmann La Roche Anticuerpos anti-bromodesoxiuridina(brdu) y metodos de uso.
KR20210126135A (ko) 2016-12-27 2021-10-19 에프. 호프만-라 로슈 아게 신규한 비오틴-특이적 단일클론 항체 및 그 용도
KR102448439B1 (ko) * 2016-12-27 2022-09-27 에프. 호프만-라 로슈 아게 신규한 비오틴-특이적 단일클론 항체 및 그 용도
CN111171154B (zh) * 2020-02-25 2021-05-11 杭州百凌生物科技有限公司 一种抗生物素抗体及其应用

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IT1248685B (it) * 1990-06-04 1995-01-26 Tecnogen Scpa Procedimento per la identificazione di sequenze polinucleotidiche e dispositivo relativo
US6579696B1 (en) * 1992-12-21 2003-06-17 Promega Corporation Polymyxin B conjugates
EP1012275A1 (fr) * 1997-01-31 2000-06-28 University Of Rochester Proteines de fusion d'anticorps chimeriques utilisees pour induire et stimuler une reponse immunitaire antitumorale
WO2000012554A1 (fr) * 1998-08-28 2000-03-09 Commissariat A L'energie Atomique Procede de synthese d'une chimiokine marquee, chimiokine marquee et trousse d'analyse
EP2386565A3 (fr) * 1999-01-12 2013-11-20 Cambridge Enterprise Ltd. Composés et procédés pour inhiber ou augmenter une réponse inflammatoire

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0050088A2 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110088129A (zh) * 2016-12-27 2019-08-02 豪夫迈·罗氏有限公司 新的生物素特异性单克隆抗体及其用途

Also Published As

Publication number Publication date
AU3370000A (en) 2000-09-14
JP2002537362A (ja) 2002-11-05
WO2000050088A3 (fr) 2000-12-21
MXPA01008465A (es) 2003-06-06
CA2371849A1 (fr) 2000-08-31
WO2000050088A2 (fr) 2000-08-31

Similar Documents

Publication Publication Date Title
WO2000050088A2 (fr) Complexes constitues d'un anticorps et de chimiokines biotinylees
JP6901493B2 (ja) 抗ｂｃｍａポリペプチド及びタンパク質
CA2172630C (fr) Proteine serique et cellulaire d'ancrage et conjugues
CN104039320B (zh) 血液恶性肿瘤用抗-cxcr4抗体的治疗
EP0602290B1 (fr) Antigène de surface du virus de l'hépatite B conjugué à des anticorps et utilisation d'un tel conjugué
US11584799B2 (en) Anti-CD30 antibodies and methods for treating CD30+ cancer
CN104619722A (zh) 新型vista-ig 构建体和vista-ig 用于治疗自身免疫性、过敏性和炎性疾病的用途
JP2001515918A (ja) ケモカインペプチド類、その変異体、誘導体および類似体ならびに炎症応答を阻害または増大する方法へのそれらの使用
JPH08510909A (ja) Ｃｄ１４媒介細胞活性化を抑制するための方法および組成物
CA2370004A1 (fr) Ciblage pharmaceutique intracellulaire
JP2010155852A (ja) リンホトキシンβ経路の遮断によるウイルス誘導性全身性ショックおよび呼吸窮迫の逆転
WO2017012591A1 (fr) Complexe comprenant une nanoparticule métallique, des séquences de liaison et des anticorps
AU758576C (en) Chemokines with amino-terminal modifications
NZ578635A (en) Pharmaceutical composition, comprising an anti-cd6 monoclonal antibody used in the diagnosis and treatment of rheumatoid arthritis
US20180271997A1 (en) Methods and Reagents to Treat Tumor and Cancer
WO2018231661A1 (fr) Méthodes et réactifs pour traiter une tumeur et le cancer
CN110520151A (zh) 胰高血糖素受体结合蛋白和其使用方法
US6852508B1 (en) Chemokine with amino-terminal modifications
AU665763B2 (en) Interleukin receptor targeted molecules for treatment of inflammatory arthritis
CZ20013270A3 (cs) Protilátkové a chemokinové konstrukty a jejich pouľití při léčbě autoimunitních chorob
US6869606B1 (en) Biotinylated-chemokine antibody complexes
US20220280615A1 (en) Immune tolerant elastin-like recombinant peptides and methods of use
Lorenz Biological agents: a novel approach to the therapy of rheumatoid arthritis
CN117715941A (zh) 抗cd154抗体及其用途
CN115819581A (zh) 一种靶向人cd47的抗体、制备方法和应用

Legal Events

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

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20010704

AK Designated contracting states

Kind code of ref document: A2

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

AX Request for extension of the european patent

Free format text: AL;LT;LV;MK;RO;SI

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

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20030902