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WO1991001004A1 - Molecules hybrides - Google Patents

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Publication number
WO1991001004A1
WO1991001004A1 PCT/US1990/003828 US9003828W WO9101004A1 WO 1991001004 A1 WO1991001004 A1 WO 1991001004A1 US 9003828 W US9003828 W US 9003828W WO 9101004 A1 WO9101004 A1 WO 9101004A1
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WIPO (PCT)
Prior art keywords
hybrid molecule
cells
hybrid
cytokine
molecule
Prior art date
Application number
PCT/US1990/003828
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English (en)
Inventor
Richard C. Svrluga
Cory A. Waters
Original Assignee
Seragen, 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.)
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Publication date
Application filed by Seragen, Inc. filed Critical Seragen, Inc.
Publication of WO1991001004A1 publication Critical patent/WO1991001004A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the field of the invention is cytokine hybrid molecules.
  • the hematopoietin receptor superfamily (Idzerda et al., J. Exp. Med. 171:861-873, 1990) is a family of cytokine receptors, the extracellular domains of which exhibit a significant degree of amino acid homology with each other.
  • IL-2 receptors which include the Interleukin 2 (IL-2) p75 receptor, are located on the surfaces of hematopoietic cells, including both lymphoid cells and myeloid cells, and are capable of binding and mediating the cellular proliferative effects of such cytokines as IL-2, Interleukin 3 (IL-3) , Interleukin 4 (IL-4), Interleukin 6 (IL-6) , erythropoietin (EPO) , and prolactin.
  • cytokines which bind to members of the hematopoietin receptor superfamily are herein collectively termed "hematopoietin receptor superfamily cytokines”.
  • IL-2 IL-2- receptor-bearing cells
  • IL-2 receptor-bearing cells such as allograft-activated human T-cells and certain leukemic lymphocytes
  • IL-2R IL-2 receptors
  • some of these hybrid molecules have been created by chemical conjugation of IL-2 with a toxin (Greenfield et al., Science, pp. 238, 536 (1987), more recently, recombinant DNA methodologies have been applied to the genetic assembly of hybrids such as diphtheria toxin/IL-2 hybrid (U.S. Patent No.
  • the mechanism of cell killing by recombinant toxin-IL-2 hybrids is the same, except that the toxin's own cell- binding domain is replaced with IL-2, so that the hybrid toxin binds to IL-2-receptor-bearing cells exclusively.
  • the nature of these IL-2-specific receptors has been the object of several studies (e.g., Robb et al. , J.Exp.Med. 154:1455-1464, 1981; Tsudo et al., Proc. Natl. Acad. Sci. USA 83:9694-9698, 1986; and Teshigawara et al., J.Exp.Med. 165:223-234, 1987).
  • IL-2-binds IL-2 alone or together in a heterodimeric receptor complex Yagita et al.. Cancer Res. 49, in press, 1989.
  • IL-2-binding molecule is p55 alone
  • the N-termina'l amino acid residues of IL-2, particularly Asp_ 0 are essential for binding of IL-2 to the p75 receptor molecule (Collins et al., Proc. Natl.
  • Pseudomonas exotoxin A/IL-2 recombinant hybrid has been shown to be internalized by the low and intermediate affinity receptor subunits as well as by the heterodimeric high affinity receptor (Lorberbaum-Galski et al., J. Biol. Chem. 263:18650- 18656, 1988) .
  • the Pseudomonas exotoxin is linked to IL-2 through IL-2*s carboxy- terminus.
  • the invention features a hybrid molecule containing a hematopoietin receptor superfamily cytokine (or a fragment or analog thereof capable of binding to the high-affinity receptor for such cytokine) covalently bonded, at its amino terminal end, to a chemical entity which causes the hybrid molecule to be processed via a lower affinity receptor for such cytokine to a degree less than the degree of processing of the cytokine by the lower affinity receptor, provided that the chemical entity cannot be a fragment of diphtheria toxin which includes the translocation domain of that toxin, as such domain is described in John R. Murphy U.S. Patent No. 4,675,382, hereby incorporated by reference.
  • a “lower affinity receptor” for a given cytokine is any receptor capable of processing that cytokine, other than the high-affinity receptor for that cytokine.
  • "Processing” is a term which is used to mean the binding of a cytokine by one of that cytokine*s receptors and internalization of the receptor-bound cytokine into an endo ⁇ ytic vesicle or into the cell.
  • cytokine processing means simply the binding of the cytokine by one of that cytokine•s receptors.
  • a cytokine molecule is said to be "processed via a lower affinity receptor for such cytokine" when the cytokine molecule has bound to a low or intermediate-affinity receptor for that cytokine and has also been internalized by the cell (for example, in an endocytic vesicle) as a ligand/ receptor complex.
  • the degree of such processing of a hybrid molecule of the invention can be quantitatively compared to the degree of processing of a naturally-occurring cytokine molecule by, for example, measuring the amount of each of such molecule which is internalized by cells bearing one type of receptor.
  • the ability of a hybrid molecule to distinguish between high and lower affinity receptors is determined by comparing the amount of the molecule internalized by cells bearing only high-affinity receptors to the amount internalized by cells bearing only the lower affinity receptors: those hybrids which are capable of distinguishing between receptor types to a significant degree are useful for the purposes cited herein.
  • a "fragment" of a cytokine molecule is a polypeptide having an a ino acid sequence exactly corresponding to some portion of the naturally-occurring molecule that is less than all of that naturally- occurring molecule, and can be generated, for example, by proteolytic digestion of the naturally-occurring molecule, by chemical synthesis, or by recombinant DNA techniques.
  • an "analog" of a cytokine molecule is a polypeptide which differs from the naturally-occurring cytokine in that it has substitutions, additions, and/or deletions of one or more amino acids at one or more locations: for example, an analog of a given cytokine would include those polypeptides which contain a contiguous segment at least half as long as the naturally-occurring cytokine and which has at least 80% sequence homology with some portion of the naturally- occurring molecule.
  • the amino acid substitutions may be either conservative or non-conservative, and may be designed, for example, to remove proteolytically sensitive sites from the polypeptide.
  • substituted amino acyl residue is chemically similar (e.g., acidic, basic, hydrophobic, aromatic) to the residue for which it is substituted: for example, substitution of a valine for a leucine.
  • Such analogs may be conveniently obtained by recombinant DNA techniques, or by any other method known to those in the field. Once generated, any such fragments or analogs can be tested for their ability to be processed via the high-affinity and the lower affinity receptors of the cytokine from which they are derived. Those which behave comparably to the naturally-occurring cytokine can be incorporated into the hybrid molecules of the invention.
  • the cytokine is selected from the group consisting of IL-2, IL-3, IL-4, IL-6, EPO, and prolactin;
  • the chemical entity interferes with the binding by the cytokine portion of the hybrid molecule to the lower- affinity receptor;
  • the chemical entity is an X-ray-opaque moiety, a fluorescent moiety, a radioactive moiety, a polypeptide (herein defined as two or more amino acids linked by peptide bonds) such as an antibody molecule, ricin, streptavidin, gelanin, or ferritin, or an enzymatically- active fragment or analog of such a polypeptide;
  • the covalent bond is a peptide bond;
  • the hybrid molecule is capable of affecting the rate of proliferation of cells bearing high-affinity receptors.
  • the hybrid molecule of the invention may be used in a method for selectively binding a moiety to a cell having a high-affinity receptor for the cytokine of interest, by exposing the cells to hybrid molecule which contains the moiety covalently bonded to the amino terminus of cytokine (or a fragment or analog thereof capable of binding to the high-affinity receptor) .
  • the moiety includes a label and the method is used for imaging high-affinity receptors or cells bearing high-affinity receptors; or the moiety is a molecule complexed with iron and the method is used for separating cells having a relatively high number of the high-affinity receptors from other cells having a lower number of such high-affinity receptors; or the hybrid molecule is capable of decreasing the rate of proliferation of cells to which it binds, and the method is used for therapeutic treatment of a condition characterized by overproduction of cells having high- affinity receptors.
  • the hybrid molecule of the invention can also be used in a method for selectively isolating cells having high-affinity receptors for the cytokine of interest, out of a population of cells some of which lack such high- affinity receptors, which method involves (1) immobilizing the hybrid molecule, (2) causing the population of cells to contact the immobilized hybrid molecules under conditions permitting binding of high- affinity receptors to the immobilized hybrid molecules, and (3) separating unbound cells from bound cells.
  • This method may be used to remove, from bodily fluids or tissues, cells having such high-affinity receptors; alternatively, the method includes the additional step of eluting the bound cells from the immobilized hybrid molecules, and is used for isolating, from bodily fluids or tissues, cells bearing high-affinity receptors.
  • the hybrid molecule in which the chemical entity is a polypeptide may be made, for example, by providing a recombinant DNA molecule containing a DNA sequence encoding that polypeptide fused to a DNA sequence encoding the cytokine of interest (or a high-affinity- receptor-binding fragment or analog thereof) , introducing the recombinant DNA molecule into an appropriate expression system, and expressing the recombinant DNA molecule.
  • the streptavidin-containing hybrid molecule of the invention may be used in a method for imaging high- affinity receptors specific for the cytokine of interest, or, in a population of cells, a cell bearing such high- affinity receptors, by (a) exposing the high-affinity receptors to an amount of the hybrid molecule sufficient substantially to maximize the difference between (1) the amount of the hybrid molecule which binds to the high- affinity receptors and (2) the amount which binds to all lower-affinity receptors on the population of cells; (b) exposing the receptor-bound hybrid molecules to a labelled probe covalently linked to biotin; and (c) detecting the labelled probe which binds to the receptor- bound hybrid molecules.
  • the invention takes advantage of the ability of each of the hematopoietin receptor superfamily cytokines, when linked at its N-terminus to a chemical entity, to be processed via its lower-affinity receptors to a lesser degree than is the cytokine itself, resulting in enhanced discrimination between high- and lower-affinity receptors.
  • This enhanced discrimination between receptor types by the hybrid molecule of the invention permits selective labelling, isolation, removal, or killing of cells bearing the high-affinity receptor, to the virtual exclusion of cells lacking this receptor, and thus presents an important tool for the study, diagnosis, and therapeutic treatment of various conditions in which myeloid or lymphoid cells are implicated as critical elements.
  • a hybrid molecule of the invention having IL-2 as the cytokine portion would target the high-affinity receptors on activated T cells, and so would be useful for applications involving transplant rejection, autoimmune disease, and certain lymphatic cancers.
  • Fig. 1 is a representation of the DNA coding sequence, and the corresponding amino acid sequence, for the IL-2 gene portion of plasmid pDW15, following Sphl digestion of the plasmid.
  • Fig. 2 is a diagram illustrating the stepwise construction of plasmid PSI130 bearing the CRM197/IL-2 recombinant gene.
  • Fig. 3(a) is an illustration of a recombinant DNA plasmid intermediate in the construction of a ricin A/IL- 2 hybrid gene.
  • Fig. 3(b) is an illustration of a recombinant DNA vector bearing the ricin A/IL-2 hybrid gene and suitable for expressaj n in E.coli.
  • Fig. 4(a) is an illustration of a recombinant DNA plasmid intermediate in the construction of a streptavidin/IL-2 hybrid gene.
  • Fig. 4(b) is an illustration of a recombinant DNA vector bearing the streptavidin/IL-2 hybrid gene and suitable for expression in E.coli. Discrimination Among IL-2 Receptors By IL-2 Hybrid And Native IL-2.
  • the invention is based upon the observation that attachment of a chemical entity to the N-terminal region of IL-2 interferes with processing of the IL-2 portion of the hybrid via intermediate-affinity (p75) receptors on T-cells, possibly due to structural constraints imposed on the chemical entity portion of the hybrid by the p75 receptor.
  • p75 intermediate-affinity
  • Cells displaying the high-affinity (p75+p55) receptor required approximately a 100-fold higher concentration of IL-2 hybrid than of IL-2 to effect 50% displacement.
  • the affinity of hybrid-IL-2 for the p55 receptor is within an order of magnitude of the affinity of IL-2 for that receptor
  • the affinity of hybrid-IL-2 for the p75 receptor is about 1% of the affinity of IL-2 for that receptor
  • the affinity of hybrid-IL-2 for the high-affinity (p75+p55) receptor is about 1% of the affinity of IL-2 for that receptor.
  • IL-2 gene used for these fusions was obtained from plasmid pDW15 (Fig. 2) (Williams et al., Nucleic Acids Res. 16:10453-10467, 1988) containing a synthetic form of the IL-2 gene which, when cloned into E.coli JM101, expresses IL-2 protein at a rate about 16 times that of the native cDNA sequence cloned into the same strain of E.coli. . Standard DNA cloning techniques were employed. Plasmids were introduced into E.coli by CaCl, transformation, isolated by the alkaline lysis procedure, and purified by CsCl density gradient centrifugation (Maniatis et al.. Molecular Cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring
  • the genetic fusion was made at the Sphl site of pDW15 so that the IL-2 domain of the fused gene would encode 133 amino acids of IL-2, plus one additional amino acid on its amino terminus encoded by the Sph site (Fig. 1) .
  • Plasmid pABM6508 containing a gene coding for the N-terminal 485 amino acids of diphtheria toxin joined to ⁇ -melanocyte-stimulating hormone (Bishai et al., J. Bacteriol. 169:5140-5151, 1987), was digested completely with Hindlll and partially with Sphl a 6kB Sphl - Hindlll vector fragment was gel-purified and ligated to the 0.5 kB Sphl - Hindlll IL-2-gene-containing fragment from pDW15, resulting in a plasmid designated pABI6508.
  • pB197 carries the gene for CRM197, which is a full-length (535 amino acids) Gly 5 ⁇ Glu 52 missense mutant form of diphtheria toxin that is devoid of ADP-ribosyl transferase activity and thus is nontoxic (Uchida et al. , J. Biol. Chem. 248:3838-3844, 1973).
  • the missense mutation occurs within the 0.8kB Accl - Xmnl fragment of pB197, and so*,appears in pSI130 as well.
  • Example 2 Biological assay for IL-2 receptor discrimina ion.
  • rIL-2 recombinant human IL
  • FCS Fetal calf serum
  • Hyclone Laboratories Hyclone Laboratories, Logan, UT
  • FCS Fetal calf serum
  • the radiolabeled IL-2 binding assay was performed essentially as described by Smith and co-workers (Robb et al.; Teshigawara et al.). Cells were harvested and washed three times with RPMI 1640 medium containing 10%
  • FCS Total volume 150 l
  • rIL-2 or PT/IL-2 total volume 150 l
  • increasing concentrations of unlabeled rIL-2 or PT/IL-2 0.5- to 2000-fold molar excess
  • the binding reaction was terminated by microcentrifuging
  • Ricin cDNA is prepared as described by Hailing et al. (Nucleic Acids Res. 13:8019-8033, 1985) or Lamb et al. (Eur. J. Biochem. 148:265-270, 1985). Following digestion with BaroHI and Haelll, an 856-bp BamHI - Haelll 5 fragment of ricin cDNA (encoding the 5' end of the ricin gene, the signal sequence and ricin amino acids 1 through 272) is isolated by agarose gel electrophoresis.
  • This plasmid is then digested with Ncol and Hindlll, yielding a 1.3 kb DNA fragment coding for all of the following: mature ricin A, part of the ricin A linker, the synthetic protease-sensitive site, and IL-2.
  • Ncol - Hindlll fragment is isolated and cloned onto Ncol - Hindlll digested pKK233-2 vector (Fig. 3(b)) (Pharmacia; Piscataway, NJ; Armann et al.. Gene 40:183-190, 1985) for expression in E.coli.
  • Expression of the ricin A/IL-2 protein is induced by isopropyl- ⁇ -D-thiogalactoside (IPTG) .
  • IPTG isopropyl- ⁇ -D-thiogalactoside
  • the protein may be purified by immunoaffinity chromatography followed by HPLC size exclusion chromatography.
  • streptavidin gene is isolated from Streptomvces avidinii as described by Argarana et al., Nucleic Acids Res. 14:1871-1882, 1986. Following Nael digestion of the gene, a -433 bp fragment is isolated and further digested with Tagl.
  • a -414 bp Nael - Taql fragment is thereupon isolated and its 5' Nael blunt end is ligated with the following synthetic DNA oligomer (synthesized on a Milligen 7500 DNA synthesizer according to manufacturer instructions) : 5 « CC ATG GAC CCC TCC AAG GAC TCG AAG GCC CAG GIG ⁇ S' GTACGG TAC CTG GGG AGG TTC CTG AGC TTC CGG GTC CAG- encoding: fMet Asp Pro Ser Lys Asp Ser Lys Ala Gin Val- (cont.): -TCG GCC GCC GAG GCC 3'
  • Frozen or paraffin sections of a human lymphoma biopsy are prepared on microscope slides by standard techniques. Prior to staining, frozen sections are first fixed with acetone or another suitable fixative, then washed with a buffer such as .lm Tris, pH 7.2. Unlabeled biotin and/or free fluorochrome is then added to saturate all available nonspecific binding sites. After removal of unbound biotin by rinsing in buffer, the slides are treated with streptavidin/IL-2 hybrid protein and then rinsed in buffer to remove unbound streptavidin/IL-2. The slides are then treated with a solution containing a fluorochrome-biotin conjugate.
  • a buffer such as .lm Tris, pH 7.2. Unlabeled biotin and/or free fluorochrome is then added to saturate all available nonspecific binding sites. After removal of unbound biotin by rinsing in buffer, the slides are treated with streptavidin/IL-2 hybrid protein and then rinsed in
  • the slides are again rinsed in buffer to remove unbound labeled biotin, and observed under a fluorescence microscope illuminated at an appropriate wavelength of light.
  • the presence of a high proportion of fluorochrome-labeled cells in a biopsy of a lymphoma would be an indicator of that tumor's potential susceptibility to chemotherapeutic treatment with diphtheria toxin/IL-2 hybrid or another IL-2 N-terminus-linked toxic hybrid.
  • the cytokine portion of the hybrid molecule could be contributed by any of the hematopoietin receptor superfamily cytokines, or fragments or analogs thereof (prepared by standard techniques) .
  • the chemical entity could be ferritin, for use in separating cells bearing high affinity cytokine receptors from those which do not; an inert moiety, the function of which is simply to enhance the cytokine's discrimination between receptor types; or an antigen, the function of which is to induce a host- mediated immunological attack on the host's own high- affinity-receptor-bearing cells.
  • the chemical entity could be an enzyme, a carbohydrate, a lipid, a synthetic polymer, a viral particle, or an inorganic molecule.
  • the linkage between the cytokine and the chemical entity can be a peptide bond or any other type of covalent bond.
  • the hybrid protein could be synthesized by utilizing recombinant DNA techniques, beginning with either a synthetic DNA sequence or a cloned natural gene, or could be assembled by chemically conjugating a chemical entity with the cytokine. After synthesis, the hybrid may be modified enzymatically or chemically (as by the addition of fluorescent or radioactive label) .
  • the polypeptide portion of any hybrid could be synthesized in a system incorporating one or more radioactive amino acids, so that the resulting hybrid protein is intrinsically labelled.
  • An IL-2-containing hybrid could be used as a drug delivery device to deliver a lethal dosage of, for example, a radioactive isotope or a toxin such as ricin or gelanin specifically to high-affinity-receptor-bearing T-cells, which are implicated in certain cancers of the lymphatic system, and which play a crucial role in cases of transplant rejection.
  • the hybrid could be used to image high-affinity receptors, either in vivo or .in vitro. by selecting an appropriate chemical entity to attach to the cytokine portion of the hybrid.
  • Examples would include the treatment of a frozen section of lymphoma tissue with an IL-2 hybrid containing a radioactive isotope such as 125Iodine, coupled with autoradiographic analysis of the labeled tissue, and administration of an IL-2 hybrid containing an X-ray-opaque entity to a transplant patient, coupled with a CAT-scan of the transplanted organ to monitor transplant rejection.
  • Attaching a hybrid of the invention to an appropriate solid support material would create an efficient and reusable means of separating high-affinity-receptor- bearing T-cells from cells lacking such receptors, permitting the collection of either fraction of cells for therapeutic or other use. What is claimed is:

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Abstract

Molécule hybride contenant une cytokine de la super famille des récepteurs d'hématopoïétine (ou un fragment ou analogue de celle-ci pouvant se lier au récepteur de grande affinité pour cette cytokine) lié par covalence, à son extrémité terminale amino, à une entité chimique qui provoque le traitement de la molécule hybride via un récepteur d'affinité plus faible pour cette cytokine à un degré moindre que le degré de traitement de la cytokine par le récepteur d'affinité plus faible, à condition que l'entité chimique ne soit pas un fragment de la toxine de la diphtérie qui comprend le domaine de transposition de cette toxine.
PCT/US1990/003828 1989-07-06 1990-07-06 Molecules hybrides WO1991001004A1 (fr)

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EP0584251A1 (fr) * 1991-05-17 1994-03-02 Seragen, Inc. Molecules ciblees sur un recepteur de cytokine par le traitement de la croissance de cellules neoplasiques
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US6210661B1 (en) * 1992-03-13 2001-04-03 Aventis Pharma Deutschland Gmbh IL-4 receptor for the therapy, prophylaxis and diagnosis of allergic, viral, and bacterial diseases and of fungal infections
US6482927B1 (en) * 1995-11-27 2002-11-19 Millennium Pharmaceuticals, Inc. Chimeric proteins comprising the extracellular domain of murine Ob receptor
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US6620413B1 (en) 1995-12-27 2003-09-16 Genentech, Inc. OB protein-polymer chimeras
US7074397B1 (en) 1996-01-08 2006-07-11 Genentech, Inc. Method for enhancing proliferation or differentiation of a cell using ob protein
US7524937B2 (en) 1996-01-08 2009-04-28 Genentech, Inc. WSX receptor agonist antibodies
US8105608B2 (en) 2000-03-31 2012-01-31 Purdue Research Foundation Method of treatment using ligand-immunogen conjugates
US20150086506A1 (en) * 2000-06-30 2015-03-26 University Of Louisville Research Foundation, Inc. Alteration of cell membrane for new functions

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Journal of Bacteriology, Volume 169, No. 11, issued November 1987, W. R. BISHAI et al., "High-Level Expression of a Proteolytically Sensitive Diphtheria Toxin Fragment in Escherichia Coli", pages 5140-5151, see Abstract. *
Nucleic Acids Research, Volume 16, No. 22, issued 1988, D. P. WILLIAMS et al., "Design, Synthesis and Expression of a Human Interleukin-2 Gene Incorporating the Codon Usage Bias found in Highly Expressed Escherichia Coli Genes", pages 10457-10467. see Abstract. *
Science, Volume 238, issued 23 October 1987, L. GREENFIELD et al., Mutations in Diphtheria Toxin Separate Binding from Entry and Amplify Immunotoxin Selectively", pages 536-539, see Abstract. *
See also references of EP0433442A4 *
The Journal of Biological Chemistry, Volume 248, No. 11, issued 10 June 1973, T. UCHIDA et al., "Diphtheria Toxin and Related Proteins", pages 3838-3844, see Abstract. *
The Journal of Biological Chemistry, Volume 263, No. 35, issued 15 December 1988, H. LORBER BOUM-GALSKI et al., "Interleukin 2 (IL2) PE40 is Cytotoxic to Cells Displaying Either the p55 or p70 Subunit of the IL2 Receptor", pages 18650-18656, see Abstract. *

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US5556946A (en) * 1989-07-07 1996-09-17 Takeda Chemical Industries, Ltd. Interleukin-2/viral antigen protein chimers
US5728552A (en) * 1989-07-07 1998-03-17 Takeda Chemical Industries, Ltd. DNA encoding a fusion protein comprising a viral antigen and lymphokine
US5645835A (en) * 1990-01-22 1997-07-08 Oncogen Therapeutic antibody based fusion proteins
US5314995A (en) * 1990-01-22 1994-05-24 Oncogen Therapeutic interleukin-2-antibody based fusion proteins
US5349053A (en) * 1990-06-01 1994-09-20 Protein Design Labs, Inc. Chimeric ligand/immunoglobulin molecules and their uses
US6096320A (en) * 1990-08-22 2000-08-01 University Of Saskatchewan Vaccines with chimeric protein comprising gamma-interferon and leukotoxin derived from pasteurella haemolytica
US5594107A (en) * 1990-08-22 1997-01-14 University Of Saskatchewan Chimeric protein comprising an RTX-family cytotoxin and interferon-2 or interferon
US5883230A (en) * 1990-08-29 1999-03-16 Genetics Institute, Inc. Multidomain hematopoiesis stimulators
WO1992006116A1 (fr) * 1990-09-28 1992-04-16 Ortho Pharmaceutical Corporation Facteurs de croissance hybride
AU705327B2 (en) * 1991-03-07 1999-05-20 Seragen, Inc. Use of cell surface receptor targeted molecules for the treatment of viral diseases
WO1992015318A1 (fr) * 1991-03-07 1992-09-17 Seragen, Inc. Utilisation de molecules ciblees sur des recepteurs de surface de cellules pour le traitement des maladies virales
US6074636A (en) * 1991-03-07 2000-06-13 Seragen, Inc. Method of using cell surface receptor targeted molecules for the treatment of viral diseases
US5326559A (en) * 1991-05-16 1994-07-05 Miller D Douglas Treatment of accelerated atheosclerosis with interleukin-2 receptor targeted molecules
EP0584251A4 (fr) * 1991-05-17 1995-08-02 Seragen Inc Molecules ciblees sur un recepteur de cytokine par le traitement de la croissance de cellules neoplasiques.
EP0584251A1 (fr) * 1991-05-17 1994-03-02 Seragen, Inc. Molecules ciblees sur un recepteur de cytokine par le traitement de la croissance de cellules neoplasiques
US6210661B1 (en) * 1992-03-13 2001-04-03 Aventis Pharma Deutschland Gmbh IL-4 receptor for the therapy, prophylaxis and diagnosis of allergic, viral, and bacterial diseases and of fungal infections
US6328954B1 (en) 1992-08-31 2001-12-11 Aventis Pharma Deutschland Gmbh Use of the IL-4 receptor for the therapy prophylaxis and diagnosis of allergic viral parasitic and bacterial diseases and of fungal infections
US5783181A (en) * 1994-07-29 1998-07-21 Smithkline Beecham Corporation Therapeutic uses of fusion proteins between mutant IL 4/IL13 antagonists and immunoglobulins
US6482927B1 (en) * 1995-11-27 2002-11-19 Millennium Pharmaceuticals, Inc. Chimeric proteins comprising the extracellular domain of murine Ob receptor
US6620413B1 (en) 1995-12-27 2003-09-16 Genentech, Inc. OB protein-polymer chimeras
US6541604B1 (en) 1996-01-08 2003-04-01 Genentech, Inc. Leptin receptor having a WSX motif
US7074397B1 (en) 1996-01-08 2006-07-11 Genentech, Inc. Method for enhancing proliferation or differentiation of a cell using ob protein
US7524937B2 (en) 1996-01-08 2009-04-28 Genentech, Inc. WSX receptor agonist antibodies
US8105608B2 (en) 2000-03-31 2012-01-31 Purdue Research Foundation Method of treatment using ligand-immunogen conjugates
US20150086506A1 (en) * 2000-06-30 2015-03-26 University Of Louisville Research Foundation, Inc. Alteration of cell membrane for new functions
US9255133B2 (en) * 2000-06-30 2016-02-09 University Of Louisville Research Foundation, Inc. Alteration of cell membrane for new functions using IL-2 and streptavidin

Also Published As

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EP0433442A1 (fr) 1991-06-26
CA2035868A1 (fr) 1991-01-07
JPH04501808A (ja) 1992-04-02
EP0433442A4 (en) 1992-08-05

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