WO1994001131A1 - Apport transvasculaire et intracellulaire de proteines lipidisees - Google Patents

Apport transvasculaire et intracellulaire de proteines lipidisees Download PDF

Info

Publication number
WO1994001131A1
WO1994001131A1 PCT/US1993/006599 US9306599W WO9401131A1 WO 1994001131 A1 WO1994001131 A1 WO 1994001131A1 US 9306599 W US9306599 W US 9306599W WO 9401131 A1 WO9401131 A1 WO 9401131A1
Authority
WO
WIPO (PCT)
Prior art keywords
lipidized
protein
antibody
antibodies
lipoamine
Prior art date
Application number
PCT/US1993/006599
Other languages
English (en)
Inventor
Bernard Malfroy-Camine
Original Assignee
Eukarion, 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 Eukarion, Inc. filed Critical Eukarion, Inc.
Priority to JP6503580A priority Critical patent/JPH07502753A/ja
Priority to AU47727/93A priority patent/AU4772793A/en
Priority to EP93918190A priority patent/EP0607408A4/en
Publication of WO1994001131A1 publication Critical patent/WO1994001131A1/fr
Priority to FI941169A priority patent/FI941169A/fi
Priority to NO940877A priority patent/NO940877L/no

Links

Classifications

    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/395Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12Antivirals
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00Drugs for immunological or allergic disorders
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K1/00General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides

Definitions

  • the invention provides methods for targeting a protein, such as an antibody, to intracellular compartments in a eu aryotic cell, methods for enhancing organ uptake of proteins, pharmaceutical compositions of modified proteins for use in human therapy, and methods for manufacturing modified proteins.
  • the modified proteins of the invention comprise an attached lipid portion, wherein one or more acyl groups are linked to the protein through a carbohydrate side-chain and various covalent linkage chemistries which are provided.
  • proteins are generally only poorly transported across vascular endothelial membranes, if at all, and usually cannot traverse cellular membranes to gain access to intracellular compartments.
  • antibodies can be raised against purified intracellular proteins, such as transcription factors, intracellular enzymes, and cytoarchitectural structural proteins, but such antibodies generally are not able to enter intact cells and bind to the intracellular antigen targets unless the cell membrane is disrupted.
  • monoclonal antibody technology in the mid 1970's heralded a new age of medicine. For the first time, researchers and clinicians had access to essentially unlimited quantities of uniform antibodies capable of binding to a predetermined antigenic site and having various immunological effector functions.
  • monoclonal antibodies were thought to hold great promise in, e.g.. the removal of harmful cells, microbial pathogens, and viruses j-n vivo. Methods allowing the development of specific monoclonal antibodies having binding specificities directed against almost any desired antigenic epitope, including antigens which are located in intracellular compartments in intact cells, promised a cornucopia of medicinal "magic bullets". Unfortunately, the development of appropriate therapeutic products based on monoclonal antibodies, as well as polyclonal antisera, has been severely hampered by a number of drawbacks inherent in the chemical nature of naturally- occurring antibodies.
  • antibodies are generally not able to efficiently gain access to intracellular locations, as immunoglobulins are not able to traverse the plasma membrane of cells, and are typically only internalized, if at all, as a consequence of inefficient endocytotic mechanisms.
  • antibodies do not generally cross vascular membranes (e.g., subendothelial basement membrane) , hampering the efficient uptake of antibodies into organs and interstitial spaces. Therefore, therapies for many important diseases could be developed if there were an efficient method to get specific, biologically active immunoglobulin molecules across capillary barriers and into intracellular locations.
  • the life cycle of a retrovirus involves intracellular replication wherein several viral-encoded polypeptides essential for production of infectious virions from an infected cell could potentially be inhibited or blocked if specific monoclonal antibodies reactive with the viral-encoded proteins could readily gain access to the intracellular locations where retroviral replication occurs.
  • Immunoliposomes have been produced as a potential targeted delivery system for delivering various molecules contained in the liposome to a targeted cell.
  • Immunoliposomes employ immunoglobulins as targeting agents, wherein an acylated immunoglobulin is anchored in the lipid bilayer of the liposome to target the liposome to particular cell types that have external antigens that are bound by the acylated immunoglobulin(s) of the immunoliposomes (Connor and Huang (1985) J. Cell Biol. 101: 582; Huang, L. (1985) Biochemistry 24: 29; Babbitt et al. (1984) Biochemistry 23 : 3920; Connor et al. (1984) Proc. Natl. Acad. Sci.
  • Immunoliposomes generally contain immunoglobulins which are attached to acyl substituents of a liposome bilayer through a crosslinking agent such as N-hydroxysuccimide and which thus become anchored in the liposome lipid bilayer.
  • the crosslinked immunoglobulin is linked to the liposome and serves to target the liposomes to specific cell types bearing a predetermined external antigen by binding to the external cellular antigen. While such methods may serve to target liposomes to particular cell types, immunoliposomes suffer from several important drawbacks that have limited their application as drug-delivery vehicles, particularly for delivering proteins to intracellular locations.
  • cationization involves carbodiimide linkage of a diamine, such as putrescine or hexanediamine, to the carboxylates of aspartate and glutamate residues in the immunoglobulin polypeptide sequence.
  • a diamine such as putrescine or hexanediamine
  • these chemical modifications of primary a ino acids likely disrupt the secondary and tertiary structure of the immunoglobulin sufficiently to account for the loss in binding affinity.
  • present methods produce some degree of cationization in glutamate and aspartate residues located in the variable domain of an immunoglobulin chain, which results in significant loss of binding affinity and/or specificity.
  • Chemical modification of small molecules has also been proposed as a method to augment transport of small bioactive compounds.
  • Feigner discloses forming lipid complexes consisting of lipid vesicles and bioactive substances contained therein.
  • Feigner et al. discloses cationic lipid compounds that are allegedly useful for enhancing transfer of small bioactive molecules in plants and animals.
  • Liposomes and polycationic nucleic acids have been suggested as methods to deliver polynucleotides into cells. Liposomes often show a narrow spectrum of cell specificities, and when DNA is coated externally on to them, the DNA is often sensitive to cellular nucleases. Newer polycationic lipospermines compounds exhibit broad cell ranges (Behr et al., (1989) Proc. Natl. Acad. Sci. USA 86 .
  • the present invention provides methods wherein lipid substituents are linked to a protein, such as an immunoglobulin, typically by covalent linkage to a carbohydrate side chain of the protein such that the lipid substituent does not substantially destroy the biological activity of the protein (e.g., antigen binding) .
  • the invention provides methods for producing lipidized proteins, generally by lipidization of a carbohydrate moiety on a glycoprotein or glycopeptide.
  • the methods of the invention are used for attaching a lipid, such as a lipoamine, to a polypeptide, typically by covalent linkage of the lipid to a carbohydrate moiety on a protein, wherein the carbohydrate moiety generally is chemically oxidized and reacted with a lipoamine to form a lipidized protein.
  • the resultant lipidized protein generally has advantageous pharmacokinetic characteristics, such as an increased capacity to cross vascular barriers and access parenchymal cells of various organs and an increased ability to access intracellular compartments.
  • lipidization of proteins such as antibodies directed against transcription factors (e.g., Fos, Jun, AP-1, OCT-1, NF-AT) , enhances intranuclear localization of the lipidized protein(s) .
  • transcription factors e.g., Fos, Jun, AP-1, OCT-1, NF-AT
  • the invention also provides methods for producing lipidized antibodies that are efficiently transported across capillary barriers and internalized into mammalian cells in vivo.
  • the methods of the invention relate to methods for chemically attaching at least one lipid substituent (e.g.. lipoamine) to a carbohydrate substituent on an immunoglobulin to produce a carbohydrate-linked lipidized immunoglobulin, wherein the lipidized immunoglobulin is capable of intracellular localization.
  • at least one lipid substituent e.g.. lipoamine
  • At least one lipid substituent e.g., lipoamine
  • a lipid substituent is covalently attached to a non-carbohydrate moiety on a protein or polypeptide (e.g., by formation of an amide linkage with a Asp or Glu residue side-chain carboxyl substituent or a thioester linkage with a Cys residue) .
  • a fatty acid can be linked to an Arg or Lys residue by the side-chain amine substituents.
  • lipid substitutents can be covalently attached to peptidomimetic compounds.
  • Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed “peptide mimetics” or “peptido imetics” (Fauchere, J. (1986) Adv. Drug Res. 15: 29; Veber and Freidinger (1985) TINS p.392; and Evans et al. (1987) J. Med. Chem 30: 1229, which are incorporated herein by reference) and are usually developed with the aid of computerized molecular modeling.
  • Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent therapeutic or prophylactic effect.
  • a particularly preferred non-peptide linkage is -CH 2 NH-.
  • Such peptide mimetics may have significant advantages over polypeptide embodiments, including, for example: more economical production, greater chemical stability, enhanced pharmacological properties (half- life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others.
  • Lipidization of peptidomimetics usually involves covalent attachment of one or more acyl chains, directly or through a spacer (e.g., an amide group) , to non-interfering position(s) on the peptidomimetic that are predicted by quantitative structure-activity data and/or molecular modeling.
  • Such non-interfering positions generally are positions that do not form direct contacts with the macromolecules(s) (e.g., receptors) to which the peptidomimetic binds to produce the therapeutic effect.
  • Lipidization of peptidomimetics should not substantially interfere with the desired biological or pharmacological activity of the peptidomimetic.
  • the invention also relates to therapeutic and diagnostic compositions of lipidized proteins, such as lipidized antibodies, that can cross vascular membranes and enter the intracellular compartment, particularly lipidized antibodies that bind to intracellular immunotherapeutic targets, such as viral-encoded gene products that are essential components of a viral life cycle (e.g., HIV-l Tat protein) , to intracellular antigens that are biologically active (e.g., an oncogene protein such as c-fos, c-src, c-myc, c-lck (p56) , c-fyn (p59) , and c-abl) , and/or to transmembrane or extracellular antigens (e.g., polypeptide hormone receptors such as an IL-2 receptor, PDGF receptor, EGF receptor, NGF receptor, GH receptor, or TNF receptor) .
  • intracellular immunotherapeutic targets such as viral-encoded gene products that are essential components of a viral life cycle (e.g.
  • lipidized antibodies include, but are not limited to, the following: c-ras p21, c-her-2 protein, c-raf, any of the various G proteins and/or G-protein activating proteins (GAPs) , transcription factors such as NF-AT, calcineurin, and cis-trans prolyl isomerases.
  • GAPs G-protein activating proteins
  • the lipidized antibodies can be used to localize a diagnostic reagent, such as a radiocontrast agent or magnetic resonance imaging component, to a specific location in the body, such as a specific organ, tissue, body compartment, cell type, neoplasm, or other anatomical structure (e.g., a pathological lesion).
  • the lipidized antibodies can also be used to localize linked therapeutic agents, such as chemotherapy drugs, radiosensitizing agents, radionuclides, antibiotics, and other agents, to specific locations in the body.
  • the lipidized antibodies of the invention can be used therapeutically for neutralizing (i.e, binding to and thereby inactivating) an intracellular target antigen, such as HIV-l Tat protein, a transmembrane or membrane-associated antigen target (e.g., 7-glutamyltranspeptidase, c-ras 11 p21, rasGAP) or an extracellular antigen target (i.e., j ⁇ -amyloid protein deposits in the brain of an Alzheimer's disease patient).
  • an intracellular target antigen such as HIV-l Tat protein, a transmembrane or membrane-associated antigen target (e.g., 7-glutamyltranspeptidase, c-ras 11 p21, rasGAP) or an extracellular antigen target (i
  • Lipidized antibodies can traverse the blood-brain barrier and react with extracellular antigen targets that are generally inaccessible to immunoglobulins which circulate in the blood or lymphatic system. Lipidized antibodies can also react with intracellular portions on transmembrane proteins, such as cytoplasmic tails of viral envelope glycoproteins or protein kinase domains of protooncogene proteins (c-src, c-abl) , and thus inhibit production of infectious enveloped virus or kinase activity, respectively.
  • transmembrane proteins such as cytoplasmic tails of viral envelope glycoproteins or protein kinase domains of protooncogene proteins (c-src, c-abl)
  • Figure 1 shows structural formulae representing various lipoamines that can be used in the invention.
  • the righthand column exemplifies branched-chain lipoamines and the lefthand column exemplifies straight-chain lipoamines.
  • Figure 2 is a schematic representation of (1) a glycosylated antibody comprising an immunoglobulin tetramer (two light chains associated with two heavy chains) , and (2) a schematic representation of carbohydrate-linked lipidized immunoglobulins of the invention.
  • a glycosylated antibody comprising an immunoglobulin tetramer (two light chains associated with two heavy chains)
  • carbohydrate-linked lipidized immunoglobulins of the invention For example but not limitation, branched-chain lipoamide substituents are shown attached to partially oxidized carbohydrate sidechains of an immunoglobulin tetramer. Such carbohydrate sidechains may be located in the C H , V H , C L , and/or V L regions.
  • Figure 3 shows the beneficial effect of a lipidized anti-Tat immunoglobulin on the jLn vitro survival of cells infected with HIV-l as compared to the lack of effect of the native (i.e., non-lipidized) anti-Tat immunoglobulin.
  • Fig. 4 shows that the lipidized anti-Tat antibody significantly inhibited CAT activity (by approximately 75%) , whereas native (unlipidized) anti-Tat antibody, lipidized anti-gpl20 antibody, or rsCD4 were far less effective in inhibiting CAT activity in HLCD4-CAT cells.
  • the term “corresponds to” is used herein to mean that a polynucleotide sequence is homologous (i.e., is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence.
  • the term “complementary to” is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence.
  • the nucleotide sequence "TATAC” corresponds to a reference sequence "TATAC” and is complementary to a reference sequence "GTATA".
  • substantially similarity denotes a characteristic of a polypeptide sequence or nucleic acid sequence, wherein the polypeptide sequence has at least 50 percent sequence identity compared to a reference sequence, and the nucleic acid sequence has at least 70 percent sequence identity compared to a reference sequence.
  • the percentage of sequence identity is calculated excluding small deletions or additions which total less than 25 percent of the reference sequence.
  • the reference sequence may be a subset of a larger sequence, such as a constant region domain of a constant region immunoglobulin gene; however, the reference sequence is at least 18 nucleotides long in the case of polynucleotides, and at least 6 amino residues long in the case of a polypeptide.
  • Naturally-occurring refers to the fact that an object can be found in nature.
  • a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring.
  • a lipoprotein e.g., a naturally- occuring isoprenylated or myristylated protein
  • Glycosylation sites refer to amino acid residues which are recognized by a eukaryotic cell as locations for the attachment of sugar residues.
  • the amino acids where carbohydrate, such as oligosaccharide, is attached are typically asparagine (N-linkage) , serine (O-linkage) , and threonine (O-linkage) residues.
  • the specific site of attachment is typically signaled by a sequence of amino acids, referred to herein as a "glycosylation site sequence".
  • the glycosylation site sequence for N-linked glycosylation is: -Asn-X-Ser- or -Asn-X-Thr-, where X may be any of the conventional amino acids, other than proline.
  • the predominant glycosylation site sequence for O-linked glycosylation is: -(Thr or Ser)-X-X-Pro-, where X is any conventional amino acid.
  • glycosaminoglycans a specific type of sulfated sugar
  • X is any conventional amino acid.
  • N-linked and O- linked refer to the chemical group that serves as the attachment site between the sugar molecule and the amino acid residue. N-linked sugars are attached through an amino group; O-linked sugars are attached through a hydroxyl group.
  • glycosylation site sequences in a protein are necessarily glycosylated; some proteins are secreted in both glycosylated and nonglycosylated forms, while others are fully glycosylated at one glycosylation site sequence but contain another glycosylation site sequence that is not glycosylated.
  • glycosylation site sequences that are present in a polypeptide are necessarily glycosylation sites where sugar residues are actually attached.
  • the initial N- glycosylation during biosynthesis inserts the "core carbohydrate” or “core oligosaccharide” (Proteins, Structures and Molecular Principles r (1984) Creighton (ed.), W.H. Freeman and Company, New York, which is incorporated herein by reference) .
  • glycosylating cell is a cell capable of glycosylating proteins, particularly eukaryotic cells capable of adding an N-linked "core oligosaccharide” containing at least one mannose residue and/or capable of adding an O-linked sugar, to at least one glycosylation site sequence in at least one polypeptide expressed in said cell. particularly a secreted protein.
  • a glycosylating cell contains at least one enzymatic activity that catalyzes the attachment of a sugar residue to a glycosylating site sequence in a protein or polypeptide, and the cell actually glycosylates at least one expressed polypeptide.
  • mammalian cells are typically glycosylating cells.
  • Other eukaryotic cells such as insect cells and yeast, may be glycosylating cells.
  • an antibody refers to a protein consisting of one or more polypeptides substantially encoded by genes of the immunoglobulin superfamily (e.g., see The Immunoglobulin Gene Superfamilv. A.F. Williams and A.N. Barclay, in Immunoglobulin Genes. T. Honjo, F.W. Alt, and T.H. Rabbitts, ed ⁇ ., (1989) Academic Press: San Diego, CA, pp.361- 387, which is incorporated herein by reference).
  • an antibody may comprise part or all of a heavy chain and part or all of a light chain, or may comprise only part or all of a heavy chain.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma (IgG- L , IgG 2 , IgG 3 , IgG 4 ) , delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Full-length immunoglobulin "light chains" (about 25 Kd or 214 amino acids) are encoded by a variable region gene at the NH2-terminus (about 110 amino acids) and a kappa or lambda constant region gene at the COOH - terminus.
  • Fully-length immunoglobulin "heavy chains” (about 50 Kd or 446 amino acids) are similarly encoded by a variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes, e.g.. gamma (encoding about 330 amino acids) .
  • Antibodies include, but are not limited to, the following: immunoglobulin fragments (e.g., Fab, F(ab) 2 ), Fv) , single chain immunoglobulins, chimeric immunoglobulins, humanized antibodies, primatized antibodies, and various light chain- heavy chain combinations) .
  • Antibodies can be produced in glycosylating cells (e.g., human lymphocytes, hybridoma cells, yeast, etc.), in non-glycosylating cells (e.g., in E . coli ) , or synthesized by chemical methods or produced by in vitro translation systems using a polynucleotide template to direct translation.
  • glycosylating cells e.g., human lymphocytes, hybridoma cells, yeast, etc.
  • non-glycosylating cells e.g., in E . coli
  • lipidized antibody is an antibody which has been modified by lipid derivatization (e.g., by covalent attachment of a lipoamine, such as glycyldioctadecyla ide, dilauroylphosphatidylethanolamine, or dioctadecylamidoglycylspermidine) of one or more carbohydrate moieties attached to an immunoglobulin at a glycosylation site.
  • a lipoamine such as glycyldioctadecyla ide, dilauroylphosphatidylethanolamine, or dioctadecylamidoglycylspermidine
  • the lipid substituent such as a lipoamine, is covalently attached through a naturally-occurring carbohydrate moiety at a naturally-occurring glycosylation site.
  • immunoglobulins that have altered glycosylation site sequences (typically by site- directed mutagenesis of polynucleotides encoding immunoglobulin chains) and/or altered glycosylation patterns (e.g., by expression of immunoglobulin-encoding polynucleotides in glycosylating cells other than lymphocytes or in lymphocytes of other species) .
  • Lipid substituents can be attached to one or more naturally-occurring or non- naturally ⁇ occurring carbohydrate moiety on an immunoglobulin chain.
  • the carbohydrate may be lipidized prior to attachment to the immunoglobulin
  • lipidized protein refers to a protein (including multimeric proteins, glycoproteins, and polypeptides of various sizes) that has been modified by attachment of lipid (e.g., lipoamine), generally through a carbohydrate moiety.
  • a lipidized protein is generated by derivatizing a protein such that the resultant lipidized protein is distinct from naturally-occurring lipid-linked proteins and lipoproteins.
  • proteins that are biologically active e.g., enzymes, receptors, transcription factors
  • lipidization should not substantially destroy the biological activity (e.g., at least about 15 percent of a native biological activity should be preserved in the lipidized protein) .
  • Lipidized peptidomimetics should retain at least about 25 to 95 percent of the pharmacologic activity of a corresponding non-lipidized peptidomimetic.
  • Alkyl refers to a fully saturated aliphatic group which may be cyclic, branched or straightchain. Alkyl groups include those exemplified by methyl, ethyl, cyclopropyl, cyclopropylmethyl, sec-butyl, heptyl, and dodecyl.
  • non-interfering substitutents e.g., halogen; C ⁇ - -C ⁇ alkoxy; C-L- ⁇ acyloxy; formyl; alkylenedioxy; benzyloxy; phenyl or benzyl, each optionally substituted with from 1 to 3 substituents selected from halogen, C ⁇ - -C ⁇ alkoxy or C 1 -C 4 acyloxy.
  • non-interfering characterizes the substituents as not adversely affecting any reactions to be performed in accordance with the process of this invention. If more than one alkyl group is present in a given molecule, each may be independently selected from “alkyl” unless otherwise stated.
  • Alkylene refers to a fully saturated divalent radical containing only carbon and hydrogen, and which may be a branched or straight chain radical. This term is further exemplified by radicals such as methylene, ethylene, n-propylene, t-butylene, i-pentylene, n-heptylene, and the like.
  • non-interfering sustituents e.g., halogen; C ⁇ - -C ⁇ alkoxy; C 1 -C 4 acyloxy; formyl; alkylenedioxy; benzyloxy; phenyl or benzyl, each optionally substituted with from 1 to 3 substituents selected from halogen, C 1 -C 4 alkoxy or C- -C acyloxy.
  • non- interfering characterizes the substituents as not adversely affecting any reactions to be performed in accordance with the process of this invention. If more than one alkylene group is present in a given molecule, each may be independently selected from “alkylene” unless otherwise stated.
  • Aryl denoted by Ar, includes monocyclic or condensed carbocyclic aromatic groups having from 6 to 20 carbon atoms.
  • Aryl groups include those exemplified by phenyl and naphthyl. These groups may be substituted with one or more non-interfering substituents, e.g., those selected from lower alkyl; lower alkenyl; lower alkynyl; lower alkoxy; lower alkylthio; lower alkylsulfinyl; lower alkylsulfonyl, dialkylamine; halogen; hydroxy; phenyl; phenyloxy; benzyl; benzoyl; and nitro. Each substituent may be optionally substituted with additional non-interfering substituents.
  • Amin refers to the group -NH 2 .
  • Alkylcarbonyl refers to the group -(CHR- ⁇ -CO- wherein R 2 is further designated the ⁇ -position.
  • R 2 may be hydrogen, alkyl, or an amino group.
  • Preferably R ⁇ is an amino group.
  • novel methods for chemically modifying proteins, such as antibodies, to facilitate passage across capillary barriers and into cells include the covalent attachment of at least one non-interfering lipid substituent (e.g., glycyldioctadecylamide, glycyldiheptadecylamide, glycyldihexadecylamide, dilauroylphosphatidylethanolamine, and glycyldioctadecadienoylamide) to a reactive site in the protein molecule (e.g., a periodate-oxidized carbohydrate moiety) .
  • a reactive site in the protein molecule e.g., a periodate-oxidized carbohydrate moiety
  • lipidized proteins such as lipidized antibodies of the invention.
  • lipids may be conjugated to a protein of interest to yield a lipidized protein: lipoamines, lipopolyamines, and fatty acids (e.g., stearic acid, oleic acid, and others) .
  • the lipid will be attached by a covalent linkage to a carbohydrate linked to the protein (e.g., a carbohydrate side chain of a glycoprotein) .
  • Naturally-occurring carbohydrate side-chains are preferably used for linkage to a lipoamine, although novel glycosylation sites may be engineered into a polypeptide by genetic manipulation of an encoding polynucleotide, and expression of the encoding polynucleotide in a glycosylating cell to produce a glycosylated polypeptide.
  • Glycosylated proteins can be lipidized to enhance transvascular transport, organ uptake, and intracellular localization of the lipidized protein, including intranuclear localization.
  • a glycosylated polypeptide such as an antibody
  • an oxidizing agent e.g., periodate
  • a lipoamine to form a covalent (amide or imide, respectively) bond linking the lipoamine to the protein.
  • the oxidation of the carbohydrate side- chain is a partial oxidation producing at least one reactive carboxyl or aldehyde group, although generally chemical oxidation methods will produce some molecules that are partially oxidized and others that are either unoxidized or completely oxidized.
  • the glycoprotein in order to be lipidized by reaction with a lipoamine, the glycoprotein must be oxidized to produce at least one pendant aldehyde group that can react with a lipoamine, although it may be possible to produce lipidized proteins through linkage to pendant carboxyl groups as well.
  • a pendant carboxyl or aldehyde group of an oxidized glycoprotein is a carboxyl or aldehyde group having a carbonyl carbon derived from an oxidized oligosaccharide and which is covalently attached to the protein, either directly or through a spacer (e.g., an unoxidized portion of a N- or O-linked carbohydrate side-chain) .
  • a spacer e.g., an unoxidized portion of a N- or O-linked carbohydrate side-chain
  • N-linked and O-linked carbohydrate chains are incompletely oxidized to generate a multiplicity of reactive aldehyde and carboxyl groups at each glycosylation position for subsequent reaction with lipoamines.
  • glycoproteins having one or more complex N-linked oligosaccharides such as those having a branched (manno ⁇ e) 3 (/3-N-acetylglucosamino) core, are partially oxidized by limited reaction with a suitable oxidant, generally periodate.
  • Linked oligosaccharides containing N- acetylglucosamine (NAG) , mannose, galactose, fucose (6- deoxygalactose) , N-acetylneuraminic acid (sialic acid) , glucose, N-acetylmuramic acid, N-acetylgalactosamine, xylose, or combinations of these monosaccharide units can be oxidized and reacted with lipoamines to produce lipidized proteins, more specifically carbohydrate-linked lipidized proteins.
  • Glycoproteins containing linked oligosaccharides with monosaccharide units other than those specifically listed above for exemplification, including non-naturally occurring monosaccharides can also be oxidized and covalently linked to a lipoamine to form a lipidized protein.
  • Lipoamines are molecules having at least one acyl group and at least one free amine (i.e., a primary or second ⁇ ary amine) . It is believed that the invention can also be practiced with lipoamines that have tertiary amines which comprise at least one substituent that can be displaced by reaction with an oxidized carbohydrate. Examples of lipoamines having a primary amine are shown in Fig. 1.
  • the invention can produce lipidized proteins by reacting a glycoprotein with a straight-chain lipoamine of the formula:
  • R is: a disubstituted alkyl (alkylene) , preferably methylene (-CH 2 -) ; a 1,4-disubstituted cyclohexyl; a disubstituted aryl (arylene); preferably a 1,4-disubstituted phenyl (phenylene) ; an amido group of the formula -(CHR- j ⁇ -CO- NH- wherein R ⁇ is hydrogen or an amino group; alkylcarbonyl, preferably ⁇ -amino substituted alkylcarbonyl; or a phosphate diester, preferably of the formula -CH 2 -0-P0 2 -0-.
  • n is an integer which is typically 1 to 50, preferably about 5 to 30, more preferably about 10 to 25, and most usually about 15 to 20. In general, n is selected at the discretion of the practitioner according to the following guideline: when the molecule to be lipidized is large (i.e., a protein of more than about 10 kD) it is preferred than n is at least about 8 to 12 or more to increase the hydrophobicity of the resulting lipidized protein; when the molecule to be lipidized is small (e.g., an oligopeptide) n can typically be in the range 2 to 18, but may be larger if additional hydrophobicity of the lipidized molecule is desired.
  • n is an integer which is typically 1 to 50, preferably about 5 to 30, more preferably about 10 to 25, and most usually about 15 to 20. In general, n is selected at the discretion of the practitioner according to the following guideline: when the molecule to be lipidized is large (i.e., a protein of more than about 10 kD) it
  • branched- chain lipoamines which, for example, can include lipoamines of the formula:
  • R' is: a trisubstituted alkyl, preferably - CH 2 -CH ⁇ or 1,2,4-trisubstituted cyclohexyl; a trisubstituted aryl, preferably 1,2,4-trisubstituted phenyl; an amido group of the formula -(CHR ⁇ -CO-N ⁇ wherein R ⁇ i- s hydrogen or an amino group; an i ino group of the formula -CHR 2 -NH-CH ⁇ wherein R 2 is hydrogen or an amino group or an imino group of the formula - CH 2 -N ⁇ ; or a phosphate diester, preferably of the formula -CH 2 - CH 2 -0-P0 2 -0-CH 2 -CH(C0 2 -) 2 .
  • n is selected independently and are integers which are typically 1 to 50, preferably about 5 to 30, more preferably about 10 to 25, and most usually about 15 to 20.
  • n is selected at the discretion of the practitioner according to the following guideline: when the molecule to be lipidized is large (i.e., a protein of more than about 10 kD) it is preferred than m and/or n is at least about 8 to 12 or more to increase the hydrophobicity of the resulting lipidized protein; when the molecule to be lipidized is small (e.g., an oligopeptide) n can typically be in the range 2 to 18, but may be larger if additional hydrophobicity of the lipidized molecule is desired.
  • any glycoprotein can be lipidized according to the methods of the invention by reacting a lipoamine with an oxidized carbohydrate side-chain.
  • Fig. 2 shows schematically a glycosylated antibody and a carbohydrate-linked lipidized antibody of the invention, respectively.
  • Non-glycosylated proteins may be conjugated to a lipid by linkage through a suitable crosslinking agent (e.g. , by carbodiimide linkage chemistry) .
  • a suitable crosslinking agent e.g. , by carbodiimide linkage chemistry
  • novel lipidized antibodies capable of specifically binding to predetermined intracellular epitopes with strong affinity are provided.
  • the lipidized antibodies readily enter the intracellular compartment and have binding affinities of at least about 1 x 10 6 M “1 , preferably 1 x 10 7 M “1 to 1 x 10 8 M “1 , more preferably at least about 1 x 10 9 M “1 or stronger.
  • the lipidized antibodies typically have a lipid substituent attached to a naturally-occurring carbohydrate side chain on a donor immunoglobulin chain, which composes an antibody specifically reactive with an intracellular, transmembrane, or extracellular epitope. Since carbohydrates are located on the Fc portion of immunoglobulins, chemical modification of the carbohydrate residues by lipidization would be unlikely to produce a substantial loss of affinity of the antibodies for their antigens (Rodwell et al. (1986) Proc.
  • the lipidized antibodies generally retain substantial affinity for their antigen, and the avidity can be readily measured by any of several antibody-antigen binding assays known in the art.
  • the antibodies can be produced economically in large quantities and find use, for example in the treatment of various human disorders by a variety of techniques.
  • immunoglobulin constitutes the basic structural unit of an antibody. This form is a tetramer and consists of two identical pairs of immunoglobulin chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions are together responsible for binding to an antigen, and the constant regions are responsible for the antibody effector functions.
  • immunoglobulins may exist in a variety of other forms including, for example, Fv, Fab, and (Fab') 2 , as well as bifunctional hybrid antibodies, fusion proteins (e.g., bacteriophage display libraries), and other forms (e.g. f Lanzavecchia et al., Eur. J. Immunol. 17, 105
  • Antibodies can be produced in glycosylating cells (e.g., human lymphocytes, hybridoma cells, yeast, etc.), in non-glycosylating cells (e.g., in E. coli ) , or synthesized by chemical methods or produced by in vitro translation systems using a polynucleotide template to direct translation.
  • glycosylating cells e.g., human lymphocytes, hybridoma cells, yeast, etc.
  • non-glycosylating cells e.g., in E. coli
  • One source of hybridoma cell lines and immunoglobulin-encoding polynucleotides is American Type Culture Collection, Rockville, MD.
  • Antibodies that are produced in non-glycosylating cells can be conjugated to a lipid by use of a bifunctional crosslinking agent or preferably post-translationally glycosylated in a glycosylation system such as purified canine pancreatic microsomes (Mueckler and Lodish (1986) Cell 44: 629 and Walter, P. (1983) Meth. Enzymol. 96: 84, which are incorporated herein by reference) .
  • polynucleotides that encode antibodies may be isolated from screened prokaryotic expression libraries, such as combinatorial antibody fragment display libraries, and subsequently expressed in glycosylating cells to produce glycosylated antibodies. According to these methods, glycosylated antibodies may be obtained, having naturally- occurring and/or non-naturally-occurring glycosylation patterns. Such glycosylated antibodies can be lipidized according to the methods of the invention.
  • V domain Glycosylation of the V domain is believed to arise from fortuitous occurrences of the N-linked glycosylation signal Asn-Xaa-Ser/Thr in the V region sequence and has not been recognized in the art as playing an important role in immunoglobulin function.
  • lipidization is performed on antibodies having naturally- occurring glycosylation patterns. If glycosylation sites are engineered into an antibody, it is preferred that novel glycosylation site be introduced in a constant region or variable region framework region, which are less likely to adversely affect the antigen binding activity of the antibody. It is generally most preferred that novel glycosylation sites which are engineered into an antibody are placed in a constant region.
  • polypeptide fragments comprising only a portion of a primary antibody structure and having a carbohydrate side chain that may be derivatized with a lipid substituent (e.g., lipoamine) can be produced, which fragments possess one or more immunoglobulin activities (e.g., antigen binding activity) .
  • immunoglobulin activities e.g., antigen binding activity
  • These polypeptide fragments may be produced by proteolytic cleavage of intact antibodies by methods well known in the art, or by site-directed mutagenesis at the desired locations in expression vectors containing sequences encoding immunoglobulin proteins, such as after OE ⁇ to produce Fab fragments or after the hinge region to produce (Fab') 2 fragments.
  • Single chain antibodies may be produced by joining V L and V H with a DNA linker (see.
  • the immunoglobulin-related genes contain separate functional regions, each having one or more distinct biological activities, the genes may be fused to functional regions from other genes having novel properties.
  • Nucleic acid sequences for producing immunoglobulins for the present invention are capable of ultimately expressing the desired antibodies and can be formed from a variety of different polynucleotides (genomic or cDNA, RNA, synthetic oligonucleotides, etc.) and components (e.g., V, J, D, and C regions) , as well as by a variety of different techniques.
  • Immunoglobulins and/or DNA sequences encoding immunoglobulin chains may be obtained, for example, by hybridoma clones which can be produced according to methods known in the art (Kohler and Milstein (1976) Eur. J. Immunol. 6_: 511, incorporated herein by reference) or can be obtained from several sources ("ATCC Catalog of Cell Lines and Hybridomas", American Type Culture Collection, Rockville, MD, which is incorporated herein by reference) .
  • DNA sequences encoding immunoglobulin chains can be obtained by conventional cloning methods known in the art and described in various publications, for example, Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., (1989), Cold Spring Harbor, N.Y.
  • DNA sequences will be expressed in hosts, typically glycosylating cells, after the sequences have been operably linked to (i.e., positioned to ensure the functioning of) an expression control sequence.
  • These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA.
  • expression vectors will contain selection markers, e.g., tetracycline-resistance or G418-resistance, to permit detection of those cells transformed with the desired DNA sequences (see, e.g., U.S. Patent 4,704,362).
  • selection markers e.g., tetracycline-resistance or G418-resistance
  • E. coli is one prokaryotic host useful particularly for cloning the DNA sequences of the present invention.
  • Other icrobial hosts suitable for use include bacilli, such as Bacillus ⁇ ubtili ⁇ , and other Enterobacteriaceae, such as Salmonella , Serratia , and various P ⁇ eudomonas species.
  • bacilli such as Bacillus ⁇ ubtili ⁇
  • Enterobacteriaceae such as Salmonella , Serratia
  • various P ⁇ eudomonas species such as Salmonella , Serratia , and various P ⁇ eudomonas species.
  • expression vectors which will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication) .
  • any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a jS-galactosidase promoter system, or a promoter system from phage lambda.
  • the promoters will typically control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, for initiating and completing transcription and translation.
  • Proteins, such as antibodies, that are expressed in non-glycosylating cells can be post-translationally glycosylated in a glycosylation system (Mueckler and Lodish, op.cit.. which is incorporated herein by reference.
  • Saccharomyces is a preferred host glycosylating cell, with suitable vectors having expression control sequences, such as promoters, including 3-phosphoglycerate kinase or other glycolytic enzymes, and an origin of replication, termination sequences and the like as desired.
  • mammalian tissue cell culture may also be used to express and produce the polypeptides of the present invention (see, Winnacker, "From Genes to Clones," VCH Publishers, N.Y., N.Y. (1987)).
  • Eukaryotic cells are actually preferred, because a number of suitable host cell lines capable of secreting intact immunoglobulins have been developed in the art, and include the CHO cell lines, various COS cell lines, HeLa cells, preferably myeloma cell lines, etc, and transformed B-cells or hybridomas.
  • Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer (Queen et al. , Immunol. Rev..
  • Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, Adenovirus, cytomegalovirus, Bovine Papillo a Virus, and the like.
  • the vectors containing the DNA segments of interest can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts. (See, generally, Maniatis et al. , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, (1982) .)
  • the whole antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms of the present invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (see, generally, R. Scopes, "Protein Purification", Springer-Verlag, N.Y. (1982)).
  • Substantially pure immunoglobulins of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity most preferred, for pharmaceutical uses.
  • the polypeptides may then be used therapeutically (including extracorporeally) or in developing and performing assay procedures, immunofluorescent stainings, and the like.
  • intact immunoglobulins or their binding fragments, such as Fab are preferably used.
  • lipidized antibodies will be of the human IgM or IgG isotypes, but other mammalian constant regions may be utilized as desired.
  • Lipidized antibodies of the IgA, IgG, IgM, IgE, IgD classes may be produced.
  • the lipidized antibodies of the invention are human, murine, bovine, equine, porcine, or non-human primate antibodies, more preferably human or murine antibodies.
  • the invention can be used to produce lipidized antibodies of various types, including but not limited to: chimeric antibodies, humanized antibodies, primatized antibodies, F v fragments, toxin-antibody conjugates, isotope-antibody conjugates, and imaging agent-antibody conjugates.
  • lipidized antibodies are suitably labeled with a diagnostic label, administered to the patient, and their location determined at various times following administration.
  • diagnostic reporters e.g., with Tc", other radioligands, radiocontrast agents or radio-opaque dye
  • Proteins and oligopeptides i.e., polypeptides comprising from 2 to about 50 amino acid residues attached in peptidyl linkage
  • Naturally-occurring glycoproteins e.g., 7-glutamyltranspeptidase, thrombomodulin, glucose transporter proteins
  • a crosslinking agent e.g., N- hydroxysuccimide
  • At least one lipid substituent e.g., lipoamine
  • a lipid substituent is covalently attached to a non- carbohydrate moiety on a protein or polypeptide (e.g., by formation of an amide linkage with a Asp or Glu residue side- chain carboxyl substituent or a thioester linkage with a Cys residue) .
  • a fatty acid can be linked to an Arg or Lys residue by the side-chain amine substituents.
  • non-glycosylated proteins which may be lipidized for enhancing transvascular and intracellular transport include, but are not limited to, the following proteins: c-fos, c-myc, c-src, NF- AT, and HMG CoA reductase.
  • Naturally-occurring lipoproteins such as native proteins which undergo physiological farnesylation, geranylgeranylation, and palmitylation are natural products and are not defined herein as "lipidized proteins”.
  • the lipidized antibodies and pharmaceutical compositions thereof are particularly useful for parenteral administration, i.e., subcutaneously, intramuscularly or in ⁇ travenously.
  • the compositions for parenteral administration will commonly comprise a solution of the immunoglobulin or a cocktail thereof dissolved in an acceptable carrier, preferably an aqueous carrier.
  • an acceptable carrier preferably an aqueous carrier.
  • aqueous carriers can be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine and the like. These solutions are sterile and generally free of particulate matter.
  • These compositions may be sterilized by conventional, well known sterilization techniques.
  • compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, human albumin, etc.
  • concentration of antibody in these formulations can vary widely, i.e., from less than about 0.5%, usually at or at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
  • a typical pharmaceutical composition for injection could be made up to contain 1 ml sterile buffered water, and 1-10 mgs of lipidized immunoglobulin.
  • a typical composition for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution, and 150 mg of antibody.
  • Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th ed. , Mack Publishing Company, Easton, Pennsylvania (1980) , which is incorporated herein by reference.
  • the lipidized proteins and antibodies of this invention can be frozen or lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immune globulins and art-known lyophilization and reconsti- tution techniques can be employed. It will be appreciated by those skilled in the art that lyophilization and recon- stitution can lead to varying degrees of activity loss (e.g., with conventional immune globulins, IgM antibodies tend to have greater activity loss than IgG antibodies) and that use levels may have to be adjusted to compensate.
  • compositions containing the present lipidized proteins (e.g., antibodies) or a cocktail thereof can be administered for prophylactic and/or therapeutic treatments.
  • compositions are administered to a patient in an amount sufficient to cure or at least partially arrest the disease and its complications.
  • An amount adequate to accomplish this is defined as a "therapeutically effective dose.” Amounts effective for this use will depend upon the severity of the infection and the general state of the patient's own immune system, but generally range from about 1 to about 200 mg of antibody per dose, with dosages of from 5 to 25 mg being more commonly used. It must be kept in mind that the materials of this invention may generally be employed in serious disease states, that is life-threatening or potentially life-threatening situations.
  • compositions containing the present immunoglobulins or a cocktail thereof are administered to a patient not already in a disease state to enhance the patient's resistance.
  • Such an amount is defined to be a "prophylactically effective dose.”
  • the precise amounts again depend upon the patient's state of health and general level of immunity, but generally range from 0.1 to 25 mg per dose.
  • compositions can be carried out with dose levels and pattern being selected by the treating physician.
  • pharmaceutical formulations should provide a quantity of the lipidized proteins and/or lipidized antibody(ies) of this in ⁇ vention sufficient to effectively treat the patient.
  • the lipidized antibodies may either be labeled or unlabeled.
  • Unlabeled antibodies can be used in combination with other labeled antibodies (second antibodies) that are reactive with the lipidized antibody, such as antibodies specific for human immunoglobulin constant regions.
  • second antibodies labeled antibodies
  • the lipidized antibodies can be directly labeled.
  • labels may be employed, such as radionuclides, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (particularly haptens) , radiocontrast agents, metal chelates, etc. Numerous types of diagnostic imaging applications are available and are well known to those skilled in the art.
  • an antibody that binds to a tumor antigen may be lipidized and conjugated to a radiocontrast agent or magnetic imaging material, injected into a human patient, and detected so as to localize the position of a tumor or metastatic lesion.
  • the lipidized immunoglobulins of the present invention can be used for diagnosis and therapy.
  • they can be used to treat cancer, autoimmune diseases, or viral infections.
  • the antibodies will typically bind to an antigen expressed preferentially in certain cancer cells, such as c-myc gene product and others well known to those skilled in the art.
  • the lipidized antibody will bind to a mutant protein, such as a c-ras oncogene product having a pathogenic (e.g., neoplastic) sequence, such as a substitution at position 12, 13, 59, or 61 of the protein (e.g., a Ser at position 12 of p21 ras ) .
  • the antibodies will typically bind to an critical regulatory protein expressed primarily in activated T-cells, such as NF- AT, and many other intracellular proteins well known to those skilled in the art (e.g., see Fundamental Immunology. 2nd ed. , W.E. Paul, ed., Raven Press: New York, NY, which is incorporated herein by reference) .
  • the antibodies will typically bind to a protein expressed in cells infected by a particular virus such as the various viral encoded polymerases and HIV-l Tat, and many other viral proteins well known to those skilled in the art (e.g., see Virology. 2nd ed., B.N. Fields et al., eds., (1990) , Raven Press: New York, NY,' which is incorporated herein by reference) .
  • Kits can also be supplied for use with the subject lipidized antibodies in the protection against or detection of a cellular activity or for the presence of a selected cell intracellular protein or the diagnosis of disease.
  • the subject composition of the present invention may be provided, usually in a lyophilized form in a container, either alone or in conjunction with additional antibodies specific for the desired cell type.
  • the lipidized antibodies which may be conjugated to a label or toxin, or unconjugated, are included in the kits with buffers, such as Tris, phosphate, carbonate, etc., stabilizers, biocides, inert proteins, e.g., serum albumin, or the like, and a set of instructions for use. Generally, these materials will be present in less than about 5% wt.
  • a second antibody capable of binding to the lipidized antibody is employed in an assay, this will usually be present in a separate vial.
  • the second antibody is typically conjugated to a label and formulated in an analogous manner with the antibody formulations described above, as well as typically also being lipidized itself.
  • the lipidized antibodies of the present invention are also suited for use in improved diagnostic methods and protein purification methods.
  • many intracellular proteins are unstable (e.g., short half-life, susceptible to proteolysis) or prone to aggregation (e.g., /3-amyloid protein) making purification and/or diagnostic detection difficult.
  • Lipidized antibodies are able to penetrate living cells and bind to specific intracellular target antigens; such antibody- antigen binding may stabilize the target antigen and block enzymes involved in degradation of the target antigen (e.g., proteases, ubiquitin-conjugating enzymes, glycosidases) facilitating detection and/or purification of the target antigen.
  • a lipidized antibody which specifically binds to an intracellular target antigen is contacted with live cells comprising the intracellular target antigen under physiological conditions (e.g., cell culture conditions, somatic conditions) and incubated for a suitable binding period (e.g., from about 10 minutes to several hours) .
  • the lipidized antibody specifically binds to the target antigen forming an antigen- antibody complex which is less susceptible to degradation and/or aggregation that is the target antigen itself.
  • the cells are then fixed and permeabilized and the antigen-antibody complex, comprising the target antigen bound to the lipidized antibody, is detected, usually with a labeled secondary antibody that specifically binds the the lipidized antibody.
  • the secondary antibody may be lipidized and the fixation and/or permeabilization steps may be omitted and replaced with substantial washing of the cell sample to remove non-specific staining. It may also be possible to use a lipidized, labeled primary antibody directly and omit the second antibody. Labelled protein A may also be substituted for a secondary antibody for the detection of the primary (lipidized) antibody.
  • Lipidized antibodies may also be used for intracellular therapy, such as for binding to a predetermined intracellular target antigen and modifying a biochemical property of the target antigen.
  • multi-subunit proteins such as heteromultimeric proteins (e.g., transcription factors, G-proteins) or homodimeric proteins (e.g., polymerized tubulin) may possess a biochemical activity (e.g., GTPase activity) or other activity that requires inter olecular interaction(s) that may be blocked by a lipidized antibody that specifically binds to one or more subunits and prevents functional interaction of the subunits.
  • a lipidized anti-Fos antibody which binds to a portion of Fos (e.g., leucine zipper) required for binding to Jun to form a transcriptionally active AP-1 transcription factor (Fos/Jun heterodimer) may block formation of functional AP-1 and inhibit AP-1-mediated gene transcription.
  • a lipidized anti-ras antibody may bind to an epitope of ras which is required for its proper signal transduction function (e.g., a GTP/GDP-binding site, a portion of ras that binds an accessory protein such as GAP, or the like) , therebymodifying the activity of intracellular ras in living cells.
  • bovine IgG Two mg were dissolved in 400 ⁇ l of 300 mM NaHC0 3 in a 1.5 ml Eppendorf vial. Fifty ⁇ l of a freshly prepared NaI0 4 solution (42 mg/ml in H 2 0) were added and the vial was wrapped in aluminum foil and gently shaken for 90 min. at room temperature. The reaction medium was then loaded on a PD-10 column (Pharmacia) previously equilibrated with 10 mM Na 2 C0 3 (fraction 1) , and the column was eluted with 500 ⁇ l fractions. Fraction number 7 (between 3 ml and 3.5 ml) contained approximately 1.6 mg of bovine IgG as measured using the Bradford protein assay.
  • a solution of glycyldioctadecylamide in DMSO was prepared (5 mg of the lipid into 1 ml of DMSO, vigorously vortexed for several minutes) . Under those conditions the lipid was not fully dissolved. Fifty ⁇ l of this solution were taken carefully (and did not contain any undissolved lipid) and were added to 350 ⁇ l of fraction 7 obtained as described above, in an Eppendorf vial. The vial was wrapped in aluminum foil, and the mixture was gently shaken for 20 h at room temperature.
  • mice Male swiss albinos mice (20g) were used. One hundred ⁇ l of 14 C-labeled lipidized IgG or 1 C-labeled control IgG in PBS (approximately 400,000 dpm each) were administered intravenously by tail vein injection. Mice were killed after 30 min or 3 h, their blood collected in EDTA-containing tubes, and their brain (minus cerebellum and brainstem) , spleen, one kidney and one liver lobe were dissected. Organs were homogenized in 1 ml 10 mM Tris buffer, pH 7.4, and 500 ⁇ l aliquots were counted in a Beckman scintillation counter.
  • Protein concentration in these homogenates was determined by the Bradford assay (Coomassie blue) .
  • the blood was centrifuged and 20 ⁇ l fractions of the plasma were counted.
  • Table I shows the uptake of 14 C in the brain, liver, spleen and kidney, expressed as the ration of radioactivity in 1 ⁇ g protein of the organs divided by the radioactivity in 1 ⁇ l plasma (data expressed as ⁇ l/ ⁇ g protein) .
  • a monoclonal antibody which specifically binds the Tat protein of HIV-l was lipidized according to the method described in Example 1, supra. involving periodate oxidation of carbohydrate on the antibody, followed by covalent attachment of glycyldioctadecylamide to yield a carbohydrate- lipidized antibody, which was eluted from the final PD-10 column with PBS.
  • Sup Tl cells were maintained in 24-well plates (100,000 cells per ml, in 2 ml of modified RPMI 1640 culture medium) . Cells were kept in culture with either: (1) no additional treatment (two controls) , (2) in the presence of added native anti-Tat antibody (15 ⁇ g/ml) , or (3) in the presence of the lipidized anti-Tat antibody (11.7 ⁇ g/ml) during the first five days of the experiment. At the end of the first day, HIV-l IIIB was added to one well of control cells and to the cultures containing native anti-Tat antibody- treated cells or lipidized anti-Tat antibody-treated cells. Viable cells were counted daily.
  • the untreated, HIV-infected cells grew up to a density of approximately 500,000 cells per ml, and their number began to decrease after approximately eight days due to the cytotoxic effect of the virus. Uninfected cells grew up to a density of approximately 1,000,000 cells per ml.
  • Treatment of infected cells with the native anti-Tat antibody did not protect the cells from the cytotoxic effect of the virus.
  • the lipidized anti-Tat antibody led to an almost complete protection of the cells from the cytopathic effects of the HIV-l virus. This protection continued for at least about 5 days after the treatment with the lipidized antibody was interrupted. The results are presented in Fig. 3.
  • Sup Tl cells were maintained in culture as described in the previous example and kept in culture without any treatment and without any infection, infected with HIV-l IIIB with no treatment, treated with the native anti-Tat antibody (1 ⁇ g/ml) and infected, or treated with the lipidized anti-Tat antibody (1 ⁇ g/ml) and infected.
  • the virus was added at the end of the first day in culture.
  • the native or lipidized antibody was present from day 1 until day 7.
  • HIV-1-infected SupTl cells were treated daily with anti-Tat antibody in native or lipidized form or with rsCD4 (all proteins used at 1 ⁇ g/ml) starting from Day 1 before addition of HIV-l virus containing supernatants until 10 days post infection.
  • Cell numbers and reverse transcriptase activity (RT) in the culture medium were determined every day starting from Day 2 post-infection.
  • RT reverse transcriptase activity
  • the native anti-Tat still had no significant effect on either cell counts or RT activity, whereas the lipidized anti-Tat antibody increased cell viability as compared to untreated, infected cells by approximately 70% and decreased RT activity by approximately the same extent. Cultures were continued for 3 days without further addition of antibodies.
  • a HeLa cell line stably transfected with a polynucleotide expressing CD4, the membrane receptor mediating HIV-l infection, and also containing a reporter construct comprising an HIV-l long terminal repeat (LTR) in operable linkage to and driving transcription of a linked reporter gene (chloramphenicol acetyltransferase; CAT) .
  • LTR HIV-l long terminal repeat
  • CAT chloramphenicol acetyltransferase
  • lipidized anti-Tat antibody significantly inhibited CAT activity (by approximately 75%)
  • native (unlipidized) anti-Tat antibody, lipidized anti-gpl20 antibody, or rsCD4 were far less effective in inhibiting CAT activity.
  • the data showing passage of the lipidized anti-Tat antibody into HeLa cells indicates that the transport mechanism does not likely require endosome formation, since
  • HeLa cells are reported to undergo little if any phagocytosis.
  • Glycyldioctadecylamide is obtained by linking a glycine residue to dioctadecylamine according to the method described by Behr et al. (1989) Proc. Natl. Acad. Sci. (U.S.A.) 86: 6982, which is incorporated herein by reference. Benzyloxycarbonyl-glycyl-p-nitrophenol at 1 equivalent and triethylamine at 1.1 equivalents in CH 2 Cl 2 are reacted for 5 hours, followed by addition of H 2 , 10% Pd/C in CH 2 Cl 2 /EtOH and reaction for 1 hour.
  • Glycosylated murine immunoglobulins that bind specifically to human c-myc protein are prepared by separately culturing the hybridoma cell lines MYC CT9-B7.3 (ATCC CRL 1725), MYC CT 14-G4.3 (ATCC CRL 1727), and MYC 1-9E10.2 (ATCC CRL 1729) in RPMI 1640 with 10 percent fetal bovine serum under specified conditions (Evan et al. (1985) Mol. Cell. Biol. 5_: 3610, incorporated herein by reference) and the monoclonal antibodies secreted are collected and purified by conventional methods known in the art.
  • each purified monoclonal antibody is dissolved in 400 ⁇ l of 300 mM NaHC0 3 in a 1.5 ml Eppendorf vial. Fifty ⁇ l of a freshly prepared NaI0 4 solution (42 mg/ml in H20) is added and the vial is wrapped in aluminum foil and gently shaken for 90 min. at rooir temperature. The reaction medium is then loaded on a PD-lc column (Pharmacia) previously equilibrated with 10 mM Na 2 C0 3 (fraction 1) , and the column is eluted with 500 ⁇ l fractions. The fraction(s) containing at least approximately 500 ⁇ g of IgG as measured using the Bradford protein assay are collected.
  • a solution of glycyldioctadecylamide in DMSO is prepared (5 mg of the lipid into 1 ml of DMSO, vigorously vortexed for several minutes) . Under those conditions the lipid is not fully dissolved. Fifty ⁇ l of this solution is taken carefully and added to 350 ⁇ l of the purified IgG fractions obtained as described above, in an Eppendorf vial. The vial is wrapped in aluminum foil, and the mixture is gently shaken for 20 h at room temperature. One hundred ⁇ l of a solution of NaBH 4 (10 mg/ml in
  • Anti-HMG CoA Reductase Ig Glycosylated murine immunoglobulins that bind specifically to the intracellular enzyme HMG CoA reductase are prepared by separately culturing the hybridoma cell line A9 (ATCC CRL 1811) in DMEM with 4.5 g/1 glucose, 5% horse serum and 2.5% fetal bovine serum as described (Goldstein et al. (1983) J. Biol. Chem. 258: 8450, incorporated herein by reference) and the monoclonal antibodies secreted are collected and purified by conventional methods known in the art.
  • each purified monoclonal antibody is dissolved in 400 ⁇ l of 300 mM NaHC0 3 in a 1.5 ml Eppendorf vial. Fifty ⁇ l of a freshly prepared NaI0 4 solution (42 mg/ml in H20) is added and the vial is wrapped in aluminum foil and gently shaken for 90 min. at room temperature. The reaction medium is then loaded on a PD-10 column (Pharmacia) previously equilibrated with 10 mM Na 2 C0 3 (fraction 1) , and the column is eluted with 500 ⁇ l fractions. The fraction(s) containing at least approximately 500 ⁇ g of IgG as measured using the Bradford protein assay are collected.
  • a solution of glycyldioctadecylamide in DMSO is prepared (5 mg of the lipid into 1 ml of DMSO, vigorously vortexed for several minutes) . Under those conditions the lipid is not fully dissolved. Fifty ⁇ l of this solution is taken carefully and added to 350 ⁇ l of the purified IgG fractions obtained as described above, in an Eppendorf vial. The vial is wrapped in aluminum foil, and the mixture is gently shaken for 20 h at room temperature.
  • Glycyldioctadecylamide is obtained by linking a glycine residue to dioctadecylamine according to the method described by Behr et al. (1989) Proc. Natl. Acad. Sci. (U.S.A.)
  • Anti-Ras Ig Glycosylated murine immunoglobulins that bind specifically to ras oncogene protein are prepared by separately culturing the hybridoma cell line 142-24E5 (ATCC HB 8679; U.S. Pats.
  • each purified monoclonal antibody is dissolved in 400 ⁇ l of 300 mM NaHC0 3 in a 1.5 ml Eppendorf vial. Fifty ⁇ l of a freshly prepared NaI0 4 solution (42 mg/ml in H20) is added and the vial is wrapped in aluminum foil and gently shaken for 90 min. at room temperature. The reaction medium is then loaded on a PD-10 column (Pharmacia) previously equilibrated with 10 mM Na 2 C0 3 (fraction 1) , and the column is eluted with 500 ⁇ l fractions. The fraction(s) containing at least approximately 500 ⁇ g of IgG as measured using the Bradford protein assay are collected.
  • a solution of glycyldioctadecylamide in DMSO is prepared (5 mg of the lipid into 1 ml of DMSO, vigorously vortexed for several minutes) . Under those conditions the lipid is not fully dissolved. Fifty ⁇ l of this solution is taken carefully and added to 350 ⁇ l of the purified IgG fractions obtained as described above, in an Eppendorf vial. The vial is wrapped in aluminum foil, and the mixture is gently shaken for 20 h at room temperature.
  • Hybridoma cell lines referred to in the above examples may be obtained from American Type Culture Collection, Rockville, MD (ATCC Cell Lines and Hybridomas (1992) 7th Ed, which is incorporated herein by reference) .
  • Example 6 Lipidization of a Transmembrane Enzyme
  • the enzyme gamma-glutamyltranspeptidase (GGT: EC 2.3.2.2) is a widely distributed enzyme that catalyzes the degradation of glutathione and other 7-glutamyl compounds by hydrolysis of the 7-glutamyl moiety or by its transfer to a suitable acceptor.
  • GGT is a heterodimeric glycoprotein, which is synthesized as a precursor protein that is glycosylated and cleaved into the two subunits of the mature enzyme. GGT is anchored to the cell membrane through the N-terminal portion of its heavy subunit. The active site of the enzyme lies on the extracellular portion of the molecule, which is heavily glycosylated.
  • GGT is separately purified from rat kidney and a cultured human hepatoma cell line according to procedures described previously in the art (Barouki et al. (1984) J. Biol. Chem. 259: 7970; Curthoys and Hughey (1979) Enzyme 24: 383; Matsuda et al. (1983) J. Biochem. 93 : 1427; Taniguchi et al. (1985) J. Natl. Cancer Inst. 75: 841; Tate and Meister (1985) Methods Enzymol. 113: 400; and Toya et al. (1983) Ann. N.Y. Acad. Sci. 417: 86, which are incorporated herein by reference) .
  • the fraction(s) containing at least approximately 100 ⁇ g of GGT as measured using the Bradford protein assay are collected.
  • a solution of glycyldioctadecylamide in DMSO is prepared (5 mg of the lipid into 1 ml of DMSO, vigorously vortexed for several minutes) . Under those conditions the lipid is not fully dissolved. Fifty ⁇ l of this solution is taken carefully and added to 350 ⁇ l of the purified GGT fractions obtained as described above, in an Eppendorf vial. The vial is wrapped in aluminum foil, and the mixture is gently shaken for 20 h at room temperature.
  • the lipidized human and rat GGT is radiolabeled by iodination with 125 I using chloramine T and approximately 50 ⁇ g of the radioiodinated lipidized GGT is administered to rats by intraperitoneal injection. After 24 hours, the rats are sacrificed and tissue samples removed for autoradiography to determine the pattern of localization of the lipidized GGT in the various organs.
  • an anti-actin antibody was lipidized and evaluated for its ability to penetrate cultured Swiss 3T3 fibroblasts and bind to the cytoskeletal protein actin. Native anti-actin antibody (unlipidized) was used as a control.
  • Protein A-purified rabbit anti-actin polyclonal antibodies were lipidized according to the following procedure.
  • a lipoamine, glycyldioctadecylamide was covalently linked to the carbohydrate moieties of the anti-actin antibodies by periodate oxidation-sodium borohydride reduction.
  • Antibodies were dissolved in 0.8 ml of 300 mM NaHC0 3 at a concentration of approximately 0.2 to 1.0 mg/ml.
  • Fifty ⁇ l of a freshly prepared aqueous solution of NaI0 4 (42 mg/ml) were added and the incubation vials were wrapped in aluminum foil and gently shaken for 90 minutes at room temperature.
  • reaction mixture was then purified on a PD-10 column (Pharmacia, Piscataway, NJ) equilibrated in and eluted with 10 mM Na 2 C0 3 .
  • Fifty ⁇ l of a 10 mg/ml solution of glycyldioctadecylamide in benzene are added to the fraction containing the antibodies (e.g., as determined by A 280 monitoring, Bradford assay) and the resulting reaction was incubated for 20 hours at room temperature with gentle shaking.
  • Lipidized anti-actin and lipidized anti-Tat were evaluated for their binding affinity for specific antigen relative to native (unlipidized) anti-actin or anti-Tat antibody by ELISA assay. Lipidization of either the anti-actin antibody or the anti-Tat antibody did not produce a measurable loss of affinity of the antibodies for their respective antigens as compared to their native (unlipidized) antibody.
  • lipidized anti-actin antibodies are able to bind intracellular actin in live cells
  • lipidized anti-actin antibody or native anti-actin antibody were contacted with cultured Swiss 3T3 cells for 1 hour, followed by extensive washing to remove residual anti-actin antibodies.
  • the cells were subsequently fixed and permeabilized and the anti-actin antibodies were detected with a fluorescent-labeled secondary antibody. While no specific staining could be detected in cells preincubated with the native (unlipidized) anti-actin antibody, specific actin staining (e.g., stained actin cables) was clearly evident in cells preincubated with the lipidized anti-actin antibodies.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Virology (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Veterinary Medicine (AREA)
  • General Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Communicable Diseases (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des procédes pour cibler une protéine telle qu'un anticorps sur des compartiments intracellulaires dans une cellule eucaryotique, des procédés pour stimuler la fixation des protéines par les organes, des compositions pharmaceutiques de protéines modifiées s'utilisant dans des traitements chez l'homme, et des procédés pour fabriquer ces protéines modifiées. Ces dernières comportent une partie lipidique accolée, dans laquelle un ou plusieurs groupes acyle sont liés à la protéine par l'intermédiaire d'une chaîne latérale glucidique et de diverses propriétés chimiques de covalence qui sont obtenues. Les anticorps lipidisés de la présente invention peuvent s'utiliser pour des applications diagnostiques et thérapeutiques.
PCT/US1993/006599 1992-07-13 1993-07-13 Apport transvasculaire et intracellulaire de proteines lipidisees WO1994001131A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
JP6503580A JPH07502753A (ja) 1992-07-13 1993-07-13 脂質化タンパク質類の血管通過輸送および細胞内輸送
AU47727/93A AU4772793A (en) 1992-07-13 1993-07-13 Transvascular and intracellular delivery of lipidized proteins
EP93918190A EP0607408A4 (en) 1992-07-13 1993-07-13 Transvascular and intracellular delivery of lipidized proteins.
FI941169A FI941169A (fi) 1992-07-13 1994-03-11 Lipidisoitujen proteiinien transvaskulaarinen ja intracellulaarinen antaminen
NO940877A NO940877L (no) 1992-07-13 1994-03-11 Transvasculær og intracellulær avgivelse av lipidiserte proteiner

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US91245392A 1992-07-13 1992-07-13
US07/912,453 1992-07-13

Publications (1)

Publication Number Publication Date
WO1994001131A1 true WO1994001131A1 (fr) 1994-01-20

Family

ID=25431949

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1993/006599 WO1994001131A1 (fr) 1992-07-13 1993-07-13 Apport transvasculaire et intracellulaire de proteines lipidisees

Country Status (10)

Country Link
EP (1) EP0607408A4 (fr)
JP (1) JPH07502753A (fr)
KR (2) KR20030097604A (fr)
AU (1) AU4772793A (fr)
CA (1) CA2118586A1 (fr)
FI (1) FI941169A (fr)
NZ (1) NZ255043A (fr)
OA (1) OA09893A (fr)
RU (1) RU2157239C2 (fr)
WO (1) WO1994001131A1 (fr)

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0869937A1 (fr) * 1995-07-21 1998-10-14 Genta Incorporated Nouveaux lipides cationiques a base d'amides
US5827819A (en) * 1990-11-01 1998-10-27 Oregon Health Sciences University Covalent polar lipid conjugates with neurologically active compounds for targeting
US6063759A (en) * 1990-11-01 2000-05-16 Oregon Health Sciences University Conjugate of biologically active compound and polar lipid conjugated to a microparticle for biological targeting
WO2003080115A1 (fr) * 2002-03-22 2003-10-02 Bipha Corporation Complexes peptide hydrophile - immunoglobuline
US7045543B2 (en) 2001-11-05 2006-05-16 Enzrel Inc. Covalent conjugates of biologically-active compounds with amino acids and amino acid derivatives for targeting to physiologically-protected sites
US7122656B2 (en) 2002-01-10 2006-10-17 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Splice variant of MyD88 and uses thereof
EP2096121A1 (fr) * 2008-02-29 2009-09-02 Institut Pasteur Of Shanghai Peptides antiviraux comprenant des signaux de fixation de lipides et leurs procédés d'utilisation
WO2010035261A2 (fr) 2008-09-29 2010-04-01 Ben Gurion University Of The Negev Research And Development Authority Beta-peptides amyloides et procédés d'utilisation associés
US7858593B2 (en) 2006-01-17 2010-12-28 Vib Vzw Inhibitors of prolyl-hydroxylase-1 for the treatment of skeletal muscle degeneration
WO2012001178A1 (fr) 2010-07-02 2012-01-05 Vib Vzw Rôle du gène responsable du syndrome du x fragile et de la protéine associée dans les métastases cancéreuses
WO2012013821A1 (fr) 2010-07-30 2012-02-02 Vib Vzw Inhibition de la fonction dicer pour le traitement du cancer
US8173423B2 (en) 2006-11-07 2012-05-08 Vib Vzw Diagnosis and treatment of T-cell acute lymphoblastic leukemia
WO2013038158A1 (fr) 2011-09-14 2013-03-21 Abeterno Limited Sélection de cellules intracellulaires
WO2013121042A1 (fr) 2012-02-16 2013-08-22 Vib Vzw Sous-unités de pp2a dans la réparation de l'adn, la sous-unité b55α de pp2a en tant que nouvelle protéine d'interaction avec phd2, et implications pour le cancer
US8835654B2 (en) 2004-12-22 2014-09-16 Bhi Limited Partnership Method and compositions for treating amyloid-related diseases
WO2014167282A1 (fr) 2013-04-11 2014-10-16 Abeterno Limited Imagerie de cellule in vivo
US9499480B2 (en) 2006-10-12 2016-11-22 Bhi Limited Partnership Methods, compounds, compositions and vehicles for delivering 3-amino-1-propanesulfonic acid
WO2018226992A1 (fr) 2017-06-07 2018-12-13 Adrx, Inc. Inhibiteur d'agrégation de tau
WO2019036725A2 (fr) 2017-08-18 2019-02-21 Adrx, Inc. Inhibiteurs peptidiques d'agrégation de tau
US10967070B2 (en) 2013-08-29 2021-04-06 City Of Hope Cell penetrating conjugates and methods of use thereof

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7790847B2 (en) * 2006-03-16 2010-09-07 The Scripps Research Institute Genetically programmed expression of proteins containing the unnatural amino acid phenylselenocysteine
AR083495A1 (es) * 2010-10-22 2013-02-27 Esbatech Alcon Biomed Res Unit Anticuerpos estables y solubles
KR101470793B1 (ko) * 2014-06-30 2014-12-08 순천향대학교 산학협력단 흡수촉진제로서의 펩타이드와 이를 포함하는 조성물

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990010448A2 (fr) * 1989-03-07 1990-09-20 Genentech, Inc. Conjugue covalent de lipides et d'oligonucleotides

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4429008B1 (en) * 1981-12-10 1995-05-16 Univ California Thiol reactive liposomes
AU626797B2 (en) * 1987-09-08 1992-08-13 Albany Medical College Immunogenic composites capable of selectively inducing antibody production, pharmaceutical compositions employing the same and method of selectively inducing antibody production
ATE136211T1 (de) * 1989-06-23 1996-04-15 Liposome Co Inc Zielliposomen und verfahren zur kupplung von liposomen-proteinen
US5264618A (en) * 1990-04-19 1993-11-23 Vical, Inc. Cationic lipids for intracellular delivery of biologically active molecules
ATE148713T1 (de) * 1990-07-24 1997-02-15 Seikagaku Kogyo Co Ltd Glykosaminoglykan gemischt mit phospholipid oder lipid, seine herstellung und krebszellenmetastaseninhibitor

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990010448A2 (fr) * 1989-03-07 1990-09-20 Genentech, Inc. Conjugue covalent de lipides et d'oligonucleotides

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ANNUAL REVIEW OF BIOCHEMISTRY, Volume 60, issued April 1991, Y.N. VAISHNAU et al., "The Biochemistry of AIDS", pages 577-630. *
GENE ANALYSIS TECHNIQUES, Volume 5, issued May 1988, W.S. THOMPSON et al., "Antibodies Introduced Into Living Cells with Liposomes Localize Specifically and Inhibit Specific Intracellular Processes", pages 73-79. *
PROC. NATL. ACAD. SCI. U.S.A., Volume 83, issued April 1986, J.D. RODWELL et al., "Site-Specific Covalent Modification of Monoclonal Antibodies: In Vitro and In Vivo Evaluations", pages 2632-2636. *
See also references of EP0607408A4 *

Cited By (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5827819A (en) * 1990-11-01 1998-10-27 Oregon Health Sciences University Covalent polar lipid conjugates with neurologically active compounds for targeting
US6024977A (en) * 1990-11-01 2000-02-15 Oregon Health Sciences University Covalent polar lipid conjugates with neurologically active compounds for targeting
US6063759A (en) * 1990-11-01 2000-05-16 Oregon Health Sciences University Conjugate of biologically active compound and polar lipid conjugated to a microparticle for biological targeting
US6339060B1 (en) 1990-11-01 2002-01-15 Oregon Health & Science University Conjugate of biologically active compound and polar lipid conjugated to a microparticle for biological targeting
US6436437B1 (en) 1990-11-01 2002-08-20 Oregon Health And Science University Covalent polar lipid conjugates with neurologically active compounds for targeting
US6858582B2 (en) 1990-11-01 2005-02-22 Oregon Health And Sciences University Composition containing porous microparticle impregnated with biologically-active compound for treatment of infection
US7423010B2 (en) 1994-05-19 2008-09-09 Oregon Health & Science University Nonporous microparticle-prodrug conjugates for treatment of infection
EP0869937A1 (fr) * 1995-07-21 1998-10-14 Genta Incorporated Nouveaux lipides cationiques a base d'amides
EP0869937A4 (fr) * 1995-07-21 2004-07-21 Promega Biosciences Inc Nouveaux lipides cationiques a base d'amides
US7045543B2 (en) 2001-11-05 2006-05-16 Enzrel Inc. Covalent conjugates of biologically-active compounds with amino acids and amino acid derivatives for targeting to physiologically-protected sites
US7122656B2 (en) 2002-01-10 2006-10-17 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Splice variant of MyD88 and uses thereof
WO2003080115A1 (fr) * 2002-03-22 2003-10-02 Bipha Corporation Complexes peptide hydrophile - immunoglobuline
US8835654B2 (en) 2004-12-22 2014-09-16 Bhi Limited Partnership Method and compositions for treating amyloid-related diseases
US7858593B2 (en) 2006-01-17 2010-12-28 Vib Vzw Inhibitors of prolyl-hydroxylase-1 for the treatment of skeletal muscle degeneration
US9499480B2 (en) 2006-10-12 2016-11-22 Bhi Limited Partnership Methods, compounds, compositions and vehicles for delivering 3-amino-1-propanesulfonic acid
US11020360B2 (en) 2006-10-12 2021-06-01 Bellus Health Inc. Methods, compounds, compositions and vehicles for delivering 3-amino-1-propanesulfonic acid
US10857109B2 (en) 2006-10-12 2020-12-08 Bellus Health, Inc. Methods, compounds, compositions and vehicles for delivering 3-amino-1-propanesulfonic acid
US10238611B2 (en) 2006-10-12 2019-03-26 Bellus Health Inc. Methods, compounds, compositions and vehicles for delivering 3-amino-1-propanesulfonic acid
US8173423B2 (en) 2006-11-07 2012-05-08 Vib Vzw Diagnosis and treatment of T-cell acute lymphoblastic leukemia
EP2096121A1 (fr) * 2008-02-29 2009-09-02 Institut Pasteur Of Shanghai Peptides antiviraux comprenant des signaux de fixation de lipides et leurs procédés d'utilisation
WO2010035261A2 (fr) 2008-09-29 2010-04-01 Ben Gurion University Of The Negev Research And Development Authority Beta-peptides amyloides et procédés d'utilisation associés
WO2012001178A1 (fr) 2010-07-02 2012-01-05 Vib Vzw Rôle du gène responsable du syndrome du x fragile et de la protéine associée dans les métastases cancéreuses
WO2012013821A1 (fr) 2010-07-30 2012-02-02 Vib Vzw Inhibition de la fonction dicer pour le traitement du cancer
WO2013038158A1 (fr) 2011-09-14 2013-03-21 Abeterno Limited Sélection de cellules intracellulaires
WO2013121042A1 (fr) 2012-02-16 2013-08-22 Vib Vzw Sous-unités de pp2a dans la réparation de l'adn, la sous-unité b55α de pp2a en tant que nouvelle protéine d'interaction avec phd2, et implications pour le cancer
WO2014167282A1 (fr) 2013-04-11 2014-10-16 Abeterno Limited Imagerie de cellule in vivo
US10967070B2 (en) 2013-08-29 2021-04-06 City Of Hope Cell penetrating conjugates and methods of use thereof
WO2018226992A1 (fr) 2017-06-07 2018-12-13 Adrx, Inc. Inhibiteur d'agrégation de tau
WO2019036725A2 (fr) 2017-08-18 2019-02-21 Adrx, Inc. Inhibiteurs peptidiques d'agrégation de tau

Also Published As

Publication number Publication date
OA09893A (en) 1994-09-15
FI941169A0 (fi) 1994-03-11
NZ255043A (en) 1997-03-24
RU2157239C2 (ru) 2000-10-10
KR20030097604A (ko) 2003-12-31
EP0607408A4 (en) 1997-12-10
RU94024566A (ru) 1996-09-27
FI941169A (fi) 1994-05-04
CA2118586A1 (fr) 1994-01-20
EP0607408A1 (fr) 1994-07-27
KR100372119B1 (ko) 2003-06-28
JPH07502753A (ja) 1995-03-23
AU4772793A (en) 1994-01-31

Similar Documents

Publication Publication Date Title
WO1994001131A1 (fr) Apport transvasculaire et intracellulaire de proteines lipidisees
RU2198895C2 (ru) Конъюгат, обладающий способностью активировать иммунную систему, и фармацевтическая композиция, включающая указанный конъюгат
EP0490998B1 (fr) Conjugues anticorps-agent neuropharmaceutique specifiques au recepteur de transferrine
US7150872B2 (en) Polyalkylene oxide-modified single chain polypeptides
JP4114951B2 (ja) 改変/キメラスーパー抗原およびその使用
US5270199A (en) Human mannose-binding protein
CZ418691A3 (en) CHIMERIC IMMUNOGLOBULINS SPECIFIC FOR RECEPTOR IL-2 PROTEIN p 55 TAC
EP0602290B1 (fr) Antigène de surface du virus de l'hépatite B conjugué à des anticorps et utilisation d'un tel conjugué
US20050181987A1 (en) Compositions and methods for treatment of cancer
JP2005539067A (ja) ポリエチレングリコールリンカーを用いる二重特異性分子の産生
US20110142756A1 (en) Method And Composition For The Treatment Of Cancer By The Enzymatic Conversion Of Soluble Radioactive Toxic Precipitates In The Cancer
JPS61122224A (ja) 部位選択性プラスミノ−ゲン活性化因子及びその製造方法
RU2153885C2 (ru) Способ осуществления селективного метионинового голодания клеток в организме млекопитающего, полинуклеотид, каталитически активный полипептид рекомбинантной метазы
US5474766A (en) Methods and compositions for inhibition of hepatic clearance of tissue-type plasminogen activator
WO1996040248A1 (fr) Liberation transvasculaire et intracellulaire de proteines lipidisees
WO1994014471A9 (fr) Procedes et compositions d'inhibition de la clairance hepatique de l'activateur plasminogene de type tissulaire
US6825319B1 (en) Synthetic peptides and pharmaceutical compositions comprising them for diagnosis and treatment of anti-phospholipid syndrome
AU714868B2 (en) Transvascular and intracellular delivery of lipidized proteins
EP0365087B1 (fr) Immunotoxines pour le traitement et la prophylaxie de maladies auto-immunitaires
US6440733B1 (en) Monoclonal antibodies recognizing antigens on the surface of endothelial cells of tumor vessel
PT100568A (pt) Anticorpos monoclonais e antigeneos para o melanoma humano
WO2000027420A9 (fr) Compositions et methodes de traitement du cancer
WO2000027420A1 (fr) Compositions et methodes de traitement du cancer

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AT AU BB BG BR BY CA CH CZ DE DK ES FI GB HU JP KP KR KZ LK LU MG MN MW NL NO NZ PL PT RO RU SD SE SK UA US VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

WWE Wipo information: entry into national phase

Ref document number: 2118586

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 255043

Country of ref document: NZ

WWE Wipo information: entry into national phase

Ref document number: 941169

Country of ref document: FI

WWE Wipo information: entry into national phase

Ref document number: 1993918190

Country of ref document: EP

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWP Wipo information: published in national office

Ref document number: 1993918190

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWW Wipo information: withdrawn in national office

Ref document number: 1993918190

Country of ref document: EP