EP0618927A1 - Cytokines dotees d'un residu de cysteine non apparie et leurs produits de conjugaison - Google Patents

Cytokines dotees d'un residu de cysteine non apparie et leurs produits de conjugaison

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Publication number
EP0618927A1
EP0618927A1 EP93901259A EP93901259A EP0618927A1 EP 0618927 A1 EP0618927 A1 EP 0618927A1 EP 93901259 A EP93901259 A EP 93901259A EP 93901259 A EP93901259 A EP 93901259A EP 0618927 A1 EP0618927 A1 EP 0618927A1
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Prior art keywords
cytokine
cysteine residue
cytokines
conjugated
unpaired cysteine
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German (de)
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EP0618927A4 (fr
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Tse Wen Chang
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Tanox Inc
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Tanox Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/212—IFN-alpha
    • 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
    • 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
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/555—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound pre-targeting systems involving an organic compound, other than a peptide, protein or antibody, for targeting specific cells
    • A61K47/557—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound pre-targeting systems involving an organic compound, other than a peptide, protein or antibody, for targeting specific cells the modifying agent being biotin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • 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
    • C07K14/555—Interferons [IFN]

Definitions

  • the invention relates to cytokines which are site-specifically mutated to have one unpaired cysteine residue located apart from the receptor-binding site, and
  • conjugates thereof in particular, conjugates where a lipophilic group is conjugated to the unpaired cysteine residue, and to such conjugates of cytokines which have an unpaired cysteine residue located apart from the receptor-binding site in the native form.
  • Cytokines act on cells to regulate cell growth, maturation or cellular activity, by either stimulating or inhibiting growth or maturation or activity of neighboring, functionally related cell types. Cytokines are synthesized and secreted by lymphocytes, macrophages, fibroblasts, neuronal cells, or other cell types.
  • cytokines are generally very potent factors which act at very low concentrations.
  • Each cytokine can have many different effects (pleiotropicity), and act locally on several functionally related cell types.
  • a cytokine is generally involved in a loop of regulatory processes.
  • the cytokine regulates not only the maturation and activity of the target cells, but the production of the cytokine itself is also regulated by certain local factors.
  • the macrophages are activated and induced to secrete interleukin-1 ("IL-1").
  • IL-1 activates the neighboring antigen-specific T cells. These activated T cells then secrete
  • IL-2 interleukin-2
  • CIFN-7 7-interferon CIFN-7
  • cytokines The localized function of cytokines is to be contrasted with hormones of the endocrine system.
  • a hormone is synthesized by a localized tissue or organ and carried by the blood circulation to act on a tissue or organ systemically, or one which is located in a different part of the body.
  • Cytokines generally are small proteins, and are made in small quantities. They are generally not detectable in the circulation even with very sensitive immunochemical assays. Cytokines have very high affinity for their receptors on the target cells, and need only bind to a small portion of the receptors on the surface of the target cells to trigger the subsequent biological events.
  • Many cytokines have been identified, and the genes of many cytokines have been cloned and expressed in various systems. Recombinant cytokines have been produced, purified, and used for various in vitro and in vivo therapeutic purposes.
  • EPO erythropoietin
  • GCSF granulocyte colony stimulation factor
  • GMCSF granulocyte macrophage colony stimulation factor
  • IL-2 is approved for enhancing immunity in patients with certain types of cancer
  • ⁇ -interferon is approved for patients with certain viral infections (such as hepatitis C) or with certain types of cancer.
  • Cytokines which target cells in the blood circulation or bone marrow such as
  • EPO and the aforementioned colony stimulation factors are safer and more efficacious (or at least have been easier to
  • cytokines such as IL-2, IL-1, tumor necrosis factor (“TNF”), IFN- ⁇ , and IFN-7, are extremely toxic when administered systemically.
  • TNF tumor necrosis factor
  • IFN- ⁇ IFN-7
  • IFN-7 IFN-7
  • cytokines which do not target cells in the blood or circulation seem to be toxic when systemically administered because they cannot act on target cells over short distances or locally, or in a microenvironment in tissues, as they would in vivo. Systemic administration results in the cytokine diffusing throughout the circulation and elsewhere, rather than localizing it to the target site.
  • cytokines have been or are being investigated for treating diseases affecting local or regional areas.
  • Epidermal growth factor (“EGF”), fibroblast growth factor (“FGF”), insulin-like growth factor (“IGF”), and platelet-derived growth factor (“PDGF”) have been studied in clinical trials for accelerating healing of wounds resulting from accidents or surgery. Some of these cytokines are also being studied
  • cytokine could be localized to maintain sufficient concentration for an appropriate period at the target site, they could be more therapeutically effective. Also, because the toxic effects associated with systemic administration of cytokines would be eliminated, high doses of cytokine could be administered to the diseased tissue site. Some potential applications of localized cytokine administration would include treating solid tumors with TNF, IFN- ⁇ , IFN-7, or IL-2. An injured part of the spine could possibly be treated with nerve growth factor (“NGF”) or ciliary nerve trophic factor ("CNTF").
  • NNF nerve growth factor
  • CNTF ciliary nerve trophic factor
  • the administration IFN- ⁇ , IFN-j8, IFN-7, or IL-2 to these tissues may be effective.
  • the local administration of PDGF, EGF, FGF, or IGF to the areas of the injured tissues may be effective.
  • EGF fibroblast growth factor
  • FGF fibroblast growth factor
  • IGF in combination with anti- allergy medication or antibiotics.
  • Injured gums caused, for example, by orthodontic surgery may be treated with EGF or IGF.
  • EGF or IGF could also be used to treat eye trauma caused by ophthalmic surgery or injury. Baldness could be treated by
  • ricin A chain (which is a toxin) after modification by fatty acid conjugation, has greatly increased non-specific cytotoxicity on various cell types. Kabanov, A.N.
  • T ⁇ F has been fatty acylated in order to enhance its ability to incorporate into liposomes.
  • Utsumi T. et al. Cancer Res. 51:3362 (1991).
  • the liposomes were to be administered intravenously into patients to deliver the T ⁇ F to cells of the reticuloendothelial system.
  • the fatty acyl group was conjugated by coupling the e amino group of the lysine residues, or the ⁇ - amino group of the first amino acid residue of the T ⁇ F polypeptide chain, with the ⁇ -hydroxysuccinimide ester of the fatty acid. Since they are 6 lysine residues per T ⁇ F polypeptide chain, the final products varied in terms of number of fatty groups per molecule and the particular lysine residues to which the fatty acids were conjugated.
  • acylated proteins contain fatty acid linked to the polypeptide by an O-ester or thiol (SH) ester bond. These acylated proteins are localized to cell membranes and are acylated primarily by saturated palmitic acid, a 16-carbon residue fatty acid.
  • acylated proteins contain fatty acids linked to the polypeptide by an amide bond. These proteins are acylated with myristic acid, a 14- carbon saturated fatty acid, which is linked to an amino terminal glycine residue. In contrast to proteins acylated with palmitate, myristate-containing proteins have been shown to be both soluble and membrane-bound.
  • This isoform of IL-1 is membrane-bound on the surface of monocytes and macrophages. Bakouche, O. et al. /. Immunol. 147:2164 (1991). The O-ester and
  • S-ester linkages to the fatty acyl group are carried out enzymatically in the cis-Golgi apparatus and in the transitional elements of the endoplasmic reticulum, where enzymes recognize certain structural features of the proteins which are to be acylated.
  • the invention includes cytokines which are site-specifically mutated to contain one unpaired cysteine residue located apart from the receptor-binding site. More specifically, a cytokine molecule is site-specifically mutated by recombinant DNA
  • binding site This can be achieved, for example, by substituting an unpaired cysteine residue for another amino acid residue, such as serine, at a site apart from the receptor binding site.
  • AUC cytokines modifying group, whether the unpaired residue is native or introduced by site-specific mutation, are referred to as AUC cytokines, (AUC denotes "allosteric unpaired cysteine").
  • the invention further includes site-specific lipophilization of such AUC cytokines.
  • Lipophilization increases the lipophilicity (affinity for lipid), which aids the resulting product in attaching to cell membranes or in localization to a target site, and thereby reduces its clearance rate.
  • the lipophilized cytokine can attach to the cells at the site of administration, which slows the rate of diffusion into the vascular space or into the mucous fluid and hence retains the product at the target site for longer duration.
  • the biological activity and binding properties of the lipophilized cytokine are substantially the same as the native form.
  • the preferred fatty acyl groups may range from 6-18 carbon atoms in length.
  • the invention further includes AUC cytokines conjugated to an antibody or another type of binding molecule, horseradish peroxidase, alkaline phosphatase, fiuorescein substances, or biotin.
  • AUC cytokines such as IL-2, IL-4, and EGF, may also be conjugated with cytotoxins, such as ricin A chain or pseudomonus exotoxin, for targeting tumor cells expressing high densities of the respective receptors.
  • the invention further includes pharmaceutical compositions of lipophilized cytokines, or the other cytokine conjugates described above.
  • the pharmaceutical composition may include suitable adjuvants, diluents and solvents.
  • cytokines include EGF, FGF, IGF, PDGF, TNF, IFN- ⁇ , IFN-/S, IFN-7, IL-2 IL-1,
  • NGF, and CNTF can be lipophilized with the techniques of the invention to make them suitable for such treatment.
  • Certain enzymes such as DNAase, which can be taken by inhalation and is potentially useful in dissolving DNA in alveolar mucus in patients with cystic fibrosis, and superoxide dismutase, which can be administered to the inflamed joints of patients with osteoarthritis, can also be lipophilized with the techniques of the invention to make it suitable for such treatment.
  • All of these cytokines are relatively small proteins, and the human genes expressing them have been cloned, characterized, and expressed in host cells. Therefore, recombinant cytokines can be readily produced in quantities suitable for site-specific mutation and lipophilization is feasible.
  • the site-specific mutation of the cytokine which permits conjugation of the lipophilic substance apart from the binding site, can be accomplished in a number of ways, including by recombinant techniques.
  • the lipophilic substance can be conjugated to the cytokine by a number of techniques, including a chemical reaction
  • the lipophilic group itself, and the final lipophilized product should be substantially resistant to enzyme cleavage.
  • the lipophilized product should have substantially the same receptor-binding activity and biological activity as the native form.
  • the lipophilic substance can be a number of agents, including fatty acid groups, and lipophilic and uncharged peptides.
  • the lipophilic group should be nonimmunogenic, nonantigenic, and not so large as to affect biological activity or receptor binding.
  • the cytokine can be conjugated to other chemical modifier groups, including an antibody or another type of binding molecule, horseradish peroxidase, alkaline phosphatase, fiuorescein substances, biotin, or a cytotoxin molecule.
  • a preferred lipophilized product is conjugated to a fatty acyl group.
  • the length of the fatty acyl group determines the lipophilicity of the final product, and the lipophilicity of the cytokine itself determines the equilibrium/distribution of it between the cellular plasma membrane and the extracellular space.
  • greater lipophilicity is provided by a longer fatty acyl group.
  • the greater lipophilicity renders a final product which will be distributed in a more limited area, and will be less diffusive, and will remain longer at the site of administration.
  • a cytokine conjugated with a 16 or 18-carbon acyl group will attach well to the cellular plasma membrane and have limited diffusibility. However, greater lipophilicity is not necessarily more desirable, because some solubility and diffusibility are required to achieve maximum receptor binding and biological effect.
  • the fatty acyl groups providing optimal pharmacokinetics for a typical cytokine are 8-14 carbon long, saturated, and unbranched.
  • a cytokine is suitable for localized as opposed to systemic administration, based on the results for the in vivo human clinical studies, if it has one or more of the following properties:
  • the substance has a rapid clearance rate, i.e. , a serum half life of less than 3-4 hours;
  • the substance is systemically administered, the therapeutic effect on the targeted disease is marginal;
  • the affected site which is to be targeted by the substance is appropriately localized such that the delivery of the substance by a local, nonsystemic administration is feasible; e.g., a nonmetastatic solid tumor or an eye infection is appropriately
  • histologically localized is not appropriately localized.
  • Lipophilization of a cytokine should provide a product with a higher affinity for the cellular plasma membrane than the native cytokine, thereby providing a lipophilized product which will remain at the target site longer than the native product.
  • the lipophilized product should stay attached to the cells for periods of time and not rapidly diffuse into the capillaries to be carried away in the blood stream. Similarly, when a lipophilized product is administered to a mucosal surface, it should be able to attach to the cells and avoid being rapidly washed away by the mucous fluids.
  • C. Diseases Suitable for Treatment with Lipophilized Cytokines and Methods Thereof A number of diseases and conditions can be treated with local administration of lipophilized cytokines. If a disease condition is appropriately localized and the affected tissue site is accessible for drug delivery, the local administration of a lipophilized cytokine is desirable, as relatively high doses can be administered without the toxicity associated with systemic administration. The mode of administration should be such that it delivers the drug to all of the affected tissue. "High-density" injection, where small volumes of the therapeutic solution are injected into a large number of sites per unit volume of tissue, is the preferred mode of administration.
  • injections of 10 ⁇ l of therapeutic solution into 10 sites of 1 ml of solid tissue would be a typical protocol for administration.
  • Solid tumors are one example of a condition suitable for treatment with lipophilized cytokine.
  • This treatment would be especially attractive when the tumor has not gone into metastasis, or has only limited metastasis.
  • This direct injection can also be administered in conjunction with surgery, in order to provide an even more direct access to the tumor site. Such injection near the excision site also ensures the optimal immune response to aid in destroying the residual tumor cells.
  • a lipophilized cytokine may be effective.
  • IFN- ⁇ IFN-jS, or IFN-7. Again, there is direct access to the affected site with direct injection.
  • Spinal injuries can be treated with direct injection of lipophized NGF or CNTF.
  • Lipophilized superoxide dismutase may be administered to the inflamed joints of patients with osteoarthritis.
  • Infections affecting the lungs such as an influenza or bacterial, viral, or parasitic pneumonia, can be treated with inhalation of lipophilized IFN- ⁇ , JFN- ⁇ , or
  • DNase can dissolve DNA in the alveolar mucus, thus reducing the viscosity of the fluid.
  • a preferred inhalation device is a metered dose inhaler, which ensures administration of a measured dosage.
  • Localized viral, bacterial or yeast infections of the vagina, rectum, mout throat, nasal linings, eyes, and/or ears can be treated with application of lipophilize
  • IFN- ⁇ , TFN- ⁇ , or IFN-7 to the mucosal surface, preferably with a dropper o spraying device.
  • a dropper o spraying device Such topical application of lipophilized EGF, FGF, or IGF can als
  • lipophilized EGF, FG or IGF can be applied by intradermal or subcutaneous injection, or by topica
  • Fatty acyl groups which are modified to contain active linkin groups include N-hydroxysuccinimide esters of fatty acid, and can be prepare according to the method of Lapidot, Y. et al. J. Lipid Res. 8:142 (1976). Some o these active esters are also available from commercial sources, such as Sigm Chemical Co. (St. Louis, MO) and Matreya, Inc. (Pleasant Gap, PA).
  • the coupling of the N-hydroxysuccinimide ester to the fatty acid to th cytokine can be performed according to the techniques described in Utsumi, T. et al.
  • fatty acyl group be conjugated to each cytokin molecule.
  • the preferred stoichiometry can be achieved by controlling the ratio o reactants during the coupling reaction.
  • the lipohilized cytokines which have one fatty acyl group per molecule can be purified by a routine procedure.
  • the fatty acyl groups are conjugated via the e-amino groups of lysine residues on the cytokine, one cannot ensure the coupling of one fatty acyl group to the e-amino group of a particular lysine residue on the cytokine. Therefore, the final lipophilized cytokine will be heterogenic in terms of the number of fatty acyl groups per cytokine molecule and the location of the fatty acyl groups. This type of conjugation creates the possibility that some of the modified cytokine molecules will lose receptor-binding activity, because the fatty acyl groups may couple to amino groups at or near the cytokine receptor binding sites.
  • the preferred method of conjugation is to first modify the cytokine in a site-specific manner so that the lipophilic group will bind to the site-specific modification rather than at other locations on the cytokine molecule.
  • the cytokine genes are site-specifically mutated by recombinant DNA methods so that the mutated cytokine has an unpaired cysteine residue far enough away from (allosteric to) the receptor binding sites to prevent it from interfering in receptor binding or biological activity.
  • native cytokine molecules are single chain polypeptides and contain even numbers of cysteine residues. These cysteine residues form disulfide bonds between pairs of cysteine residues. The specific pairing of the cysteine residues is determined by the 3-dimensional folding of the polypeptide chain, which is determine by the sequence of the polypeptide. The disulfide bonds are not usually exposed o the surface of the protein molecule, and their function is to hold the protein in a rigi
  • cytoplasm secreted proteins, such as cytokines, have disulfide bonds, wherea proteins which remain in the cytoplasm or on the inner surface of the plasm membrane do not have disulfide bonds.
  • a cysteine residue can be introduced onto the cytokine at a particular site t provide a docking site for fatty acyl group conjugation.
  • the residue should be on the surface of the protein molecule and accessible fo fatty acylation.
  • a serine residue which is in or near a peptide stretch that is highl hydrophilic is most suited for replacement with a cysteine residue.
  • Cysteine and serine residues are structurally highly homologous. The close proximity to or the location in a hydrophilic peptide stretch will ensure that the residue will be on th surface of the protein molecule, so as to be available for chemical conjugation after substitution.
  • Other residues which can be substituted are those which are polar or
  • the first step is to determine the amino acid sequence.
  • the sequences are available from the literature, and sequencing is not necessary.
  • sequencing can be performed by nucleotide sequencing of the cDNA clones of the mRNA of the cytokines.
  • the deduced amino acid sequences can be confirmed by N-terminal amino acid sequence analysis and from a molecular weight
  • indices in relation to the linear amino acid sequence are available and can be used.
  • MicroGenie sequence analysis package distributed by Beckman Instruments, Inc. Palo, Alto, CA. provides a softwar program for performing hydrophilicity plots.
  • the next step is to identify the hydrophilic regions in the polypeptide chain an
  • the preferred residue for substitution is a serine residue.
  • mutant constructs (a many as ten) each having only one substitution per mutant construct. Eventually, using the procedures described further below, the mutant constructs are screened t determine which have a substitution allosteric to the binding site.
  • PCR Polymerase chain reaction
  • oligonucleotide primers that correspond to the 5' and 3' end of the mRNA of th cytokine, and that contain proper cloning sequences.
  • RNA preparation from activated lymphocytes, leukocytes, fibroblasts, or other cell lines producing the particular cytokines, from which the cDNA are to be cloned.
  • the cloned cDNA after sequencing confirmation, is inserted into a plasmid, such as pUC19, for subsequent procedures!
  • oligonucleotide primers of about 25 nucleotides which contain the triplet condon of a cysteine residue in place of the triplet condon of the serine (or other) residue which is to be replaced. These primers with the installed mutations permit the synthesis of full length DNA genes with the site- directed mutations.
  • a convenient method was developed by Kunkel, T.A., Proc.
  • a preferred method to construct the entire family of native genes and mutant constructs is to synthesize the complete genes with a DNA synthesizer.
  • Overlapping oligonucleotides of 60-80 nucleotides from the positive and negative stands which are complementary among the adjacent oligonucleotides at their 3 ' ends can be synthesized with one of the commercial DNA synthesizers, such as one from Applied Biosystems, Inc.
  • the oligonucleotides provide both the templates and primers (mutually primed synthesis) to generate the desired sequence in one-single step. After elongation is performed with T7 DNA polymerase, the segments are linked by a ligase.
  • the oligonucleotides at the two ends of the genes are properly designed to include restriction enzyme sites, so that the synthesized genes can be inserted into the proper expression vector.
  • the reagents to be prepared and the stepwise procedure is described by Moore, D.D., Current ⁇ Protocols in Molecular Biology, Supp. 6 ⁇ 8.2.8,
  • the one with the specific mutation may be shared for the individual constructs.
  • (v) Expression The next step is to express the wild type and the mutated sets of cDNA in a eukaryotic or prokaryotic expression system and produce the native cytokine and the mutant cytokines, and then to purify the cytokines to produce sufficient amounts of each.
  • the eukaryotic expression system is preferred because it assures disulfide bond formation and the proper folding of the polypeptide chains.
  • a preferred system for the expression of the foreign cytokine genes uses the expression vector derived from nuclear polyhedron protein gene of the insect virus baculovirus (Autographa Californica). The virus can be grown in the insect cells. Sprodoptera frugiperda cells (sf9 cells). Luckow, V.A. and Summers,
  • cytokine proteins which do not contain a disulfide bond, such as IFN-7, and for which a carbohydrate moiety either does not exist or is not involved in the binding to the cytokine receptor, the expression may be carried out in prokaryotic cells.
  • E. coli expression system the expressed cytokine proteins need to be solubilized, reduced to unfold the polypeptide chain, and allowed to renature to form the most favorable 3-dimensional structure.
  • a preferred system is the FLAG Biosystem kit, offered by International Biotechnologies of Kodak (New Haven, CT.). This system also contains the reagents for the detection and purification of the non- fused protein. Monoclonal antibodies for most cytokines have been developed and are commercially available. These monoclonal antibodies can be used to affinity-purify the respective cytokines. fvi) Conjugation
  • the next step is to conjugate the purified native and mutant cytokines with fatty acyl groups, preferably of about 8-14 carbons in length.
  • fatty acyl groups preferably of about 8-14 carbons in length.
  • the first step is to create
  • the mild reducing conditions do not reduce the disulfide bonds buried inside the molecular backbone of the cytokine.
  • the reducing agent is removed by gel filtration or ion exchange chromatography.
  • treated cytokine is then reacted with the fatty acyl groups, which have been previously modified with an active group.
  • the native cytokine likely will not conjugate with the fatty acyl groups, as the native cytokine usually does not have any accessible, unpaired cysteine residues. However, for those native cytokines which do have accessible unpaired cysteine residues, they will also be conjugated to fatty acyl groups by the procedure described above. Thereafter, they can be analyzed for receptor binding/biological activity as described immediately below, to determine whether they are AUC cytokines. If this
  • the unpaired cysteine residue may be replaced by a serine residue (to ensure that it does not conjugate with the fatty acyl group), and another residue at another location can be replaced with a cysteine residue.
  • This substitution of a serine for a cysteine will not affect the receptor binding or biological activity.
  • the conjugation reaction will only create fatty acylation of such cytokines at the one unpaired cysteine residue, and not elsewhere. fvi ⁇ .
  • the last step is to analyze and compare the receptor-binding and biological activity of the native cytokine and the fatty acyl-conjugated mutant cytokine.
  • those mutant cytokine molecules with particular cysteine residue substitutions which have substantially the same receptor-binding and biological activity as the native cytokines are referred to as AUC cytokines.
  • IFN- ⁇ 2 is a member of the family of interferon cytokines, and Table 1 below summarizes the pertinent key properties of this family, and more particularly includes information about the cysteine residues thereof.
  • IFN- ⁇ There are 19 forms of IFN- ⁇ , one form of IFN-3, and one form of IFN-7.
  • IFN- ⁇ has 4 cysteine residues, forming 2 disulfide bonds, between residue Nos. 1 and 99 (or 98 or 100) and between Nos. 29 and 39. Only IFN- ⁇ l and IFN- ⁇ D have an unpaired cysteine residue (No. 86). JFN- ⁇ has 1 disulfide bond
  • IFN-7 has no cysteine residue. TABLE 1. CYSTEINE RESIDUES OF HUMAN INTERFERONS
  • amino acid residue No. 86 is a serine or tyrosine residue in all IFN- ⁇ except IFN- ⁇ l and IFN- ⁇ D suggests that the cysteine residue No. 86 in IFN- ⁇ l and IFN- ⁇ D is not crucial.
  • IFN- ⁇ 2 has four cysteine residues forming two disulfide bonds.
  • the nucleotide sequence of the cDNA gene and the deduced amino acid sequence are known and published. Using a hydrophicility analysis program provided by MicroGenie, which adopts the principles of Hopp, T.P. and Wood, K.R. Mol.
  • the amino acid residues selected for site- directed mutagenesis are: serine No. 11, arginine No. 22, lysine No. 31, glutamic acid No. 42, glutamic acid No. 51, serine No. 72, glutamic acid No. 113, serine No. 115, lysine No. 133, serine No.160, and Serine No. 163 (creating eleven mutant constructs in total).
  • the preferred method for preparing the native IFN- ⁇ 2 gene and the 11 mutuant gene constructs is with the oligonucleotide synthesis method as discussed above in
  • cytokines As analyzed above, most native cytokines have even numbers of cysteine residues and do not contain an unpaired cysteine residue.
  • human EGF polypeptide is 53 amino acid residue in length containing 6 cysteine residues (3 disulfide bonds); human basic FGF polypeptide is 155 amino acid residue in length containing 4 cysteine residues (2 disulfide bonds), human IGF polypeptide is 70 amino acid residue in length containin
  • cysteine residues 3 disulfide bonds
  • human TNF- ⁇ polypeptide is 157 amino aci residue in length containing 2 cysteine residues (1 disulfide bond)
  • human /8-NG polypeptide is 188 amino acid residue in length containing 6 cysteine residues ( disulfide bonds).
  • Relatively few cytokines have odd numbers of cysteine residues an thus an unpaired cysteine residue.
  • human IL-2 polypeptide which is 153 amino acid residue in length containing 3 cysteine residues (1 disulfide bond).
  • the unpaired cysteine residue No. 125 can be substituted to a non-cysteine residue without losing IL-2's receptor-binding and biological activity. E.P.A. application 136489. Thus, this cysteine residue (No. 125) provides a very likely site for conjugation.
  • the present invention focuses mainly on the fatty acyl derivatives of AUC cytokines for local or mucosal therapeutic administration. However, these uses are not the only therapeutic applications of AUC cytokines. Cytokines, such as IL-2, IL-
  • cytotoxins such as pseudomonus exotoxin
  • EGF EGF
  • the coupling of cytotoxins to the native cytokines by the conventional methods will yield heterogeneous conjugates.
  • the coupling of cytotoxins to AUC cytokines via the unpaired cysteine residue will yield improved conjugates for the targeting of cytotoxins to cells.
  • Therapeutic applications are not the only applications of AUC cytokines.
  • Several applications of AUC cytokines for providing useful reagents for research and diagnosis may also be developed.
  • studying cytokine function it is important to quantitate the levels of the cytokine in culture or in body fluids, the expression levels of the cytokine receptors on target cells, and/or the levels of receptor-expressing cells in a tissue. In certain diseases or conditions, the levels of these biological parameters
  • Monitoring these levels therefore, can offer a means to determine or monitor disease status.
  • cytokines conjugated with indicator substances such as antibodies, horseradish peroxidase, alkaline phosphatase, fiuorescein substances, or biotin, for use in
  • conjugated cytokine molecules may be affected by the coupling of the modifier group to a lysine residue which is in or close to the binding site. Conjugation of these modifier groups via the single unpaired cysteine residue of an AUC cytokine, using the methods described above, can minimize or eliminate these problems and thus provide improved reagents for research and diagnosis. Cytokines are better than receptor-specific antibodies for studying the receptors, because cytokines usually have higher affinities than antibodies for their respective receptors. Cytokines are smaller

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Abstract

On décrit des cytokines dont des sites spécifiques font l'objet d'une mutation de manière à présenter un résidu de cystéine non apparié placé hors du site de liaison du récepteur. On décrit aussi leur produits de conjugaison et en particulier ceux où un groupe lipophile se conjugue avec le résidu de cystéine non apparié et avec des produits de conjugaison de cytokines dotés d'un résidu de cystéine non apparié placé hors du site de liaison du récepteur dans leur forme originelle. Toutes ces cytokines sont d'un type tel que la conjugaison d'un groupe lipophile avec le résidu de cystéine non apparié ne perturbe pas significativement la liaison du récepteur ou l'activité biologique du produit conjugué. On décrit aussi des produits de conjugaison où un groupe lipophile, un anticorps, la peroxydase de raifort, la phosphatase alcaline, des substances dérivées de la flurescine, la biotine ou une cytotoxine se conjugue avec le résidu de cystéine non apparié. Ces cytokines conjuguées avec des acyles gras pénètrent mieux et restent plus longtemps dans les sites locaux et on peut les administrer localement pour traiter par exemple des tumeurs solides, des blessures de la colonne vertebrale, la zone génitale externe, la gorge, le nez, les muqueuses nasales, les yeux et la peau.
EP93901259A 1991-12-10 1992-12-10 Cytokines dotees d'un residu de cysteine non apparie et leurs produits de conjugaison. Withdrawn EP0618927A4 (fr)

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US80545291A 1991-12-10 1991-12-10
US805452 1991-12-10
PCT/US1992/010889 WO1993012142A1 (fr) 1991-12-10 1992-12-10 Cytokines dotees d'un residu de cysteine non apparie et leurs produits de conjugaison

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EP0618927A1 true EP0618927A1 (fr) 1994-10-12
EP0618927A4 EP0618927A4 (fr) 1995-05-24

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US6165458A (en) * 1997-12-26 2000-12-26 Pharmaderm Laboratories Ltd. Composition and method for dermal and transdermal administration of a cytokine
US6475796B1 (en) 1999-05-20 2002-11-05 Scios, Inc. Vascular endothelial growth factor variants
AU5026100A (en) * 1999-05-20 2000-12-12 Scios Inc. Vascular endothelial growth factor dimers
DE10144252A1 (de) * 2001-08-31 2003-03-27 Fraunhofer Ges Forschung Nanopartikel mit daran immobilisiertem biologisch aktivem TNF
GB0123262D0 (en) * 2001-09-27 2001-11-21 Adprotech Ltd Polymeric compounds

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0105777A1 (fr) * 1982-09-22 1984-04-18 Etablissement Public dit: CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (CNRS) Lipopeptides, leur obtention et leur application comme émulsifiants
EP0236987A2 (fr) * 1986-03-10 1987-09-16 F. Hoffmann-La Roche Ag Protéines chimiquement modifiées et leur production
EP0354992A2 (fr) * 1988-07-08 1990-02-21 Yeda Research And Development Company Limited Conjugués de VIP et de ses fragments actifs avec des portions hydrophobiques et des préparations pour l'utilisation dans le traitement de l'impuissance masculine
WO1990012874A2 (fr) * 1989-04-21 1990-11-01 Genetics Institute, Inc. Variantes de polypeptides par addition de cysteine, et leurs modifications chimiques
EP0441765A1 (fr) * 1990-02-09 1991-08-14 Washington University Substrats nouveaux pour enzymes à base d'analogues d'acides gras
WO1991012229A1 (fr) * 1990-02-12 1991-08-22 National Research Council Of Canada Procede de preparation de derives acyles de composes acylables
EP0458064A2 (fr) * 1990-05-04 1991-11-27 American Cyanamid Company Stabilisation de la somatotropine et autres protéines à travers une modification des résidus de cystéine

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0105777A1 (fr) * 1982-09-22 1984-04-18 Etablissement Public dit: CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (CNRS) Lipopeptides, leur obtention et leur application comme émulsifiants
EP0236987A2 (fr) * 1986-03-10 1987-09-16 F. Hoffmann-La Roche Ag Protéines chimiquement modifiées et leur production
EP0354992A2 (fr) * 1988-07-08 1990-02-21 Yeda Research And Development Company Limited Conjugués de VIP et de ses fragments actifs avec des portions hydrophobiques et des préparations pour l'utilisation dans le traitement de l'impuissance masculine
WO1990012874A2 (fr) * 1989-04-21 1990-11-01 Genetics Institute, Inc. Variantes de polypeptides par addition de cysteine, et leurs modifications chimiques
EP0441765A1 (fr) * 1990-02-09 1991-08-14 Washington University Substrats nouveaux pour enzymes à base d'analogues d'acides gras
WO1991012229A1 (fr) * 1990-02-12 1991-08-22 National Research Council Of Canada Procede de preparation de derives acyles de composes acylables
EP0458064A2 (fr) * 1990-05-04 1991-11-27 American Cyanamid Company Stabilisation de la somatotropine et autres protéines à travers une modification des résidus de cystéine

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BIOCHEMISTRY, vol.26, 1987, WASHINGTON D.C.,USA pages 1029 - 1036 WILCOX ET AL 'THE MAJORITY OF CELLULAR FATTY ACID ACYLATED PROTEINS ARE LOCALIZED TO THE CYTOPLASMIC SURFACE OF THE PLASMA MEMBRANE' *
See also references of WO9312142A1 *

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AU3323593A (en) 1993-07-19
WO1993012142A1 (fr) 1993-06-24
EP0618927A4 (fr) 1995-05-24

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