Classifications

• • 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
• • 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]
  • C07K14/5428—IL-10
• • 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
  • A61K38/20—Interleukins [IL]
• • 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
  • C07K14/54—Interleukins [IL]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• This invention relates to agonists and antagonists of human interleukin-10, and to compositions and methods for making and using them. These agonists and antagonists are produced by introducing amino acid substitutions or deletions at the carboxyl and/or amino terminus of mature human interleukin-10.
• Interleukin 10 is a cytokine capable of mediating a number of actions or effects.
• IL-10 has been isolated from both mouse and human cells and is involved in controlling the immune responses of different classes or subsets of CD4+ T helper (Th) cells. These Th cells can be divided into different subsets that are distinguished by their cytokine production profiles. Two of these subsets are called Thl and Th2 cells.
• Thl T cell clones produce interleukin-2 (IL-2) and gamma interferon (IFN- ⁇ ), whereas Th2 cell clones secrete
• IL-2 interleukin-2
• IFN- ⁇ gamma interferon
• Th cell clones also produce cytokines such as tumor necrosis factor- ⁇ (TNF- ⁇ ), interleukin-3 (IL-3), and granulocyte-macrophage colony stimulating factor (GM-CSF).
• TNF- ⁇ tumor necrosis factor- ⁇
• IL-3 interleukin-3
• GM-CSF granulocyte-macrophage colony stimulating factor
• ThO Th cells
• Thl cells are involved in successful cell-mediated responses to a variety of intracellular pathogens. They are also involved in delayed-type hypersensitivity reactions. Th2 cells are associated with humoral responses, which are characterized by antibody production. In most situations, the immune system develops the Th response that is most effective to eliminate a particular antigen or pathogen, but this is not always the case.
• leishmaniasis is characterized by a defective Thl response.
• This defect can be demonstrated using in vitro assays such as an assay described by Clerici et al. [J. Clin. Invest. 54:1892 (1989)].
• in vitro assays such as an assay described by Clerici et al. [J. Clin. Invest. 54:1892 (1989)].
• the Thl response defect is attributable to endogenous levels of IL-10, because Thl function can be restored in the in vitro assay by the addition of neutralizing antibodies against IL-10.
• the present invention fills this need by providing compositions and methods for providing or inhibiting the biological activity of human IL-10.
• this invention provides antagonists of human IL-10 which comprise mature human IL-10 modified by replacement of the lysine residue at position 157 with an acidic amino acid residue or deletion of one or more amino acid residues in the region containing about the 12 carboxyl- terminal residues.
• the amino acid sequences of three such embodiments are defined in the Sequence Listing by SEQ ID NOs: 1, 2 and 3.
• the present invention further provides nucleic acids encoding an antagonist of human IL-10 which comprises mature human IL-10 modified by replacement of the lysine residue at position 157 with an acidic amino acid residue or deletion of one or more amino acid residues in the region containing about the 12 carboxyl-terminal residues.
• Recombinant vectors comprising such nucleic acids and host cells comprising such recombinant vectors are also provided by this invention.
• This invention still further provides a method for producing an antagonist of human IL-10 which comprises mature human IL-10 modified by replacement of the lysine residue at position 157 with an acidic amino acid residue or deletion of one or more amino acid residues in the region containing about the 12 carboxyl-terminal residues, which method comprises culturing one of the above-mentioned host cells under conditions in which the nucleic acid encoding the antagonist is expressed.
• This invention still further provides methods for inhibiting the biological activity of IL-10 comprising contacting cells bearing receptors for IL-10 with an effective amount of an antagonist of human IL-10 which comprises mature human IL-10 modified by replacement of the lysine residue at position 157 with an acidic amino acid residue or deletion of one or more amino acid residues in the region containing about the 12 carboxyl-terminal residues.
• the present invention still further provides agonists of human IL-10 which comprise mature human IL-10 modified by deletion of from one to eleven of the amino-ter minal amino acid residues.
• Nucleic acids encoding such agonists, recombinant vectors and transformed host cells comprising such nucleic acids, methods for making the antagonists, and pharmaceutical compositions comprising one or more of the IL-10 agonists or antagonists and a pharmaceutically acceptable carrier are also provided by this invention.
• the antagonists of this invention are useful for treating diseases such as leishmaniasis which are characterized by a defective Thl response attributable to endogenous IL-10. They may also be useful for treatment of diseases related to IL-10-mediated immunosuppression or overproduction of IL-10, such as B-cell lymphomas. Moreover, the antagonists are useful for studies elucidating the mechanism of action of IL-10 and for rational drug design, because they display strong receptor binding which is uncoupled from effector function. Immobilized on a solid support, the antagonists can be used for the affinity purification of soluble forms of the
• IL-10 receptor in which the transmembrane region has been deleted.
• Epstein Bar Virus (EBV) viral IL-10 protein (BCRFI, or vIL-10) also possesses the biological activity of IL-10 and presumably binds to IL-10 receptors. Expression of vIL-10 activity by EBV presumably confers some survival advantage to the virus in terms of its ability to infect, replicate and/or maintain itself within a host.
• the ability of vIL-10 to down-regulate IFN- ⁇ production by both T cells and NK cells, together with its B-cell viability enhancing effects suggests that vIL-10 can suppress antiviral immunity while at the same time enhancing the potential of EBV to transform human B cells.
• the IL-10 antagonists of this invention may therefore also be useful for effectively boosting antiviral immunity against EBV, and possibly other viruses. For more on the potential uses of IL-10 antagonists, see, e.g., Howard et al, J. Clin. Immunol. 12:239 (1992).
• the mutant IL-10 antagonists of this invention are disclosed in the Example below.
• the lysine residue at position 157 of the sequence of mature human IL-10 is replaced by a glutamic acid residue (SEQ ID NO: 1).
• three (SEQ ID NO: 2) or four (SEQ ID NO: 3) amino acid residues are deleted from the carboxyl terminus of human IL-10.
• These antagonists are referred to below as the K157E, C ⁇ 3 and C ⁇ 4 antagonists, respectively.
• mature human IL-10 is defined as a protein lacking a leader sequence which (a) has an amino acid sequence substantially identical to the sequence defined by SEQ ID NO: 4 and (b) has biological activity that is common to native IL-10.
• conservative substitutions involve groups of synonymous amino acids, e.g., as described in U.S. patent No. 5,017,691 to Lee et al.
• cysteine residue at position 12 is essential for biological activity.
• deletion of the first 12 residues including this cysteine residue produced a variant which had no biological activity. Therefore, deletions at the amino terminus are limited to deletion of one or more of the first 11 residues.
• Such amino-terminal deletions can be combined with the above-mentioned carboxyl-terminal modifications to produce antagonists having the characteristics described below, but possibly different pharmacokinetic properties. These antagonists are also a part of this invention.
• Nucleic acids encoding the IL-10 agonists and antagonists are also a part of this invention. Of course those skilled in the art are well aware that, due to the degeneracy of the genetic code, there are many different nucleic acids that could encode each of the agonists and antagonists. The particular codons used can be selected for convenient construction and optimal expression in prokaryotic or eukaryotic systems.
• nucleic acids encoding the agonists and antagonists are made using the polymerase chain reaction (PCR) [Saiki et al., Science 239:481 (1988)], as exemplified by Daugherty et al. [Nucleic Acids Res. 79:2471 (1991 )] to modify cDNA encoding human IL-10.
• PCR polymerase chain reaction
• Such cDNA is well known in the art and can be prepared using standard methods, as described, e.g., in International Patent Application Publication No.
• Clones comprising sequences encoding human IL-10 have also been deposited with the American Type Culture Collection (ATCC), Rockville, Maryland, under Accession Numbers 68191 and 68192.
• the DNA can be modified using well known techniques of site-directed mutagenesis. See, e.g., Gillman et al., Gene 5:81 (1979); Roberts et al., Nature 325:731 (1987) or Innis (Ed.), 1990, PCR Protocols: A Guide to Methods and Applications, Academic Press, New York, NY.
• nucleic acids of the present invention can also be chemically synthesized using, e.g., the phosphoramidite solid support method of Matteucci et al. [J. Am. Chem. Soc. 703:3185 (1981)], the method of Yoo et al. [J. Biol. Chem. 7(54: 17078 (1989)], or other well known methods.
• Recombinant vectors comprising the foregoing nucleic acids are also a part of this invention, as are host cells transformed with such vectors, and methods for making the agonists and antagonists. Insertion of DNA encoding one of the agonists and antagonists into one of the many known expression vectors is easily accomplished when the termini of both the DNAs and the vector comprise compatible restriction sites. If this cannot be done, it may be necessary to modify the termini of the DNAs and/or vector by digesting back single-stranded DNA overhangs generated by restriction endonuclease cleavage to produce blunt ends, or to achieve the same result by filling in the single-stranded termini with an appropriate DNA polymerase.
• any site desired may be produced by ligating nucleotide sequences (linkers) onto the termini.
• linkers may comprise specific oligonucleotide sequences that define desired restriction sites.
• the cleaved vector and the DNA fragments may also be modified if required by homopolymeric tailing or PCR.
• the antagonists of this invention are characterized by human IL-10 receptor binding affinities that are similar to that of human IL-10 itself but are essentially devoid of biological activity. Preferably they will have less than about 10% of the biological of human IL-10 in a standard assay, more preferably less than about 1 %.
• the antagonists typically produce at least about 25% inhibition of a biological activity of IL-10 in cells bearing IL-10 receptors.
• the degree of inhibition will be at least about 50% and, more preferably, at least about 75%.
• the actual degree of inhibition may vary with the particular biological activity measured.
• the agonists and antagonists can also be chemically synthesized by a suitable method such as by exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis.
• Chemically synthesized polypeptides are preferably prepared by solid phase peptide synthesis as described, e.g., by Merrifield [J. Am. Chem. Soc. 55:2149 (1963); Science 232:341 (1986)] and Atherton et al. (Solid Phase Peptide Synthesis: A Practical Approach, 1989, IRL Press, Oxford).
• the agonists and antagonists can be purified, e.g., using HPLC, gel filtration, ion exchange and partition chromatography, countercurrent distribution and/or other well known methods.
• compositions can be prepared by admixing one or more of the I -10 agonists or antagonists, or a pharmaceutically acceptable salt thereof, and a physiologically acceptable carrier.
• Useful pharmaceutical carriers can be any compatible, non-toxic substance suitable for delivering the compositions of the invention to a patient.
• Sterile water, alcohol, fats, waxes, and inert solids may be included in a carrier.
• Pharmaceutically acceptable adjuvants may also be incorporated into the pharmaceutical composition.
• compositions useful for parenteral administration of such drugs are well known; e.g. Remington's Pharmaceutical Science, 18th Ed. (Mack Publishing Company, Easton, PA, 1990). Single-dose packaging will often be preferred, e.g., in sterile form.
• Administration of the agonists and antagonists is preferably parenteral by intraperitoneal, intravenous, subcutaneous or intramuscular injection or infusion, or by any other acceptable systemic method.
• the antagonists may be administered by an implantable or injectable drug delivery system [see, e.g., Urquhart et al., Ann. Rev. Pharmacol. Toxicol. 24:199 (1984); Lewis, Ed., Controlled Release of Pesticides and Pharmaceuticals, 1981 , Plenum Press, New York, New York; U.S. patents Nos. 3,773,919 and 3,270,960].
• Oral administration may also be carried out, using well known formulations which protect the antagonists from gastrointestinal proteases. See also Langer, Science 249: 1521 ( 1990).
• the agonists and antagonists can also be delivered by standard gene therapy techniques, including, e.g., direct nucleic acid injection into tissues, the use of recombinant viral vectors or liposomes and implantation of transfected cells. See, e.g., Rosenberg, J. Clin. Oncol. 70:180 (1992).
• the agonists and antagonists can be administered alone or in combination with one or more of the other agents commonly used to treat conditions characterized by a defective Th response.
• drugs such as interleukin-12 (IL-12) or gamma interferon (IFN- ⁇ ) can be co-administered with the antagonists.
• Insulin, cyclosporin, prednisone or azathioprine can be co-administered with the agonists, e.g., if they are used to replace IL-10 for the treatment or prevention of insulin-dependent diabetes mellitus (see co-pending U.S. application Serial No. 07/955,523, filed October 1 , 1992).
• Such co-administration of one or more other agents can be concomitant (together with) or sequential (before or after) administration of the agonist or antagonist. All of the administered agents should be present in the patient at sufficient levels to be therapeutically effective. Typically, if a second agent is administered within about the half-life of the first agent, the two agents are considered to be co-administered.
• Determination of the appropriate dosage of an agonist or antagonist for a particular situation is within the skill of the art. Generally, treatment is initiated with smaller dosages that are less than optimum. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day if desired.
• An effective amount will be a dose that produces a demonstrable improvement in one or more clinical parameters and/or a statistically significantly improved response in one or more of the known Th functions, some of which such as IL-2 production are described above.
• This response can be measured in vitro using blood cells taken from the patient, e.g., as described by Clerici et al., supra. Such an in vitro assay can be carried out prior to the onset of therapy, to provide a reference baseline to which an improved response can be compared.
• the actual amount and frequency of administration of the agonists and antagonists and the pharmaceutically acceptable salts thereof for a particular patient will be regulated according to the judgment of the attending clinician, taking into account such factors as age, condition and size of the patient and severity of the symptom(s) being treated.
• hIL-10 human IL-10
• Tissue culture medium, fetal bovine serum, and glutamine were purchased from Gibco-BRL (Gaithersburg, MD).
• Oligonucleotide primers were synthesized by standard methods using an Applied Biosystems 380A, 380B or 394 DNA Synthesizer (Foster City, CA).
• Transient expression was carried out as follows. COS cells (ATCC CRL 1651 ) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 6 mM glutamine, and penicillin/streptomycin. Transfection was carried out by electroporation using a BioRad GENE PULSER® (Richmond, CA).
• DMEM Dulbecco's modified Eagle's medium
• Cells were detached from culture dishes by trypsin-EDTA treatment and suspended in fresh culture medium. About 5 x 10 6 cells in a volume of 250 ⁇ l were mixed with 5 ⁇ g of plasmid DNA and then electroporated, with voltage and capacitance set at 0.2 volts and 960 mFD, respectively.
• the cells were transferred into 10 cm culture dishes and cultured at 37°C in 5% CO2 for 6 hours in 10 ml of serum-containing DMEM. After the cells had attached to the dishes, the medium was removed by aspiration and replaced with serum-free medium. Seventy-two hours later, the conditioned medium was harvested for analysis.
• hIL-10 cDNA was generated by PCR using a pCDSR ⁇ -based hIL-10 vector [Vieira et al., Proc. Natl. Acad. Sci. USA 55: 1172 (1991 ); sequence deposited in GenBank under Accession No. M57627] as a template, although other known sources of the cDNA could have been used.
• B1789CC SEQ ID NO: 5
• a Pstl site and an EcoRl site were added to 5' primer B1789CC and to a 3' primer designated A1715CC (SEQ ID NO: 6), respectively.
• the hIL-10 cDNA was subjected to PCR in a 50 ⁇ l volume reaction mixture with a 50 ⁇ l paraffin oil overlay, in a 0.5 ml Eppendorf tube.
• the reaction mixture typically contained 26.5 ⁇ l of H2O, 5 ⁇ l of Taq (Thermus aquaticus) DNA polymerase buffer [final concentrations in the reaction: 10 mM Tris-HCl, pH 8.8, 50 mM KC1, 1.5 mM MgCl2, 0.001% (w/v) gelatin], 200 ⁇ M dNTPs, 60 ng of template DNA, 10 pmoles each of 5' primer B1789CC and 3' primer A1715CC, and 0.5 ⁇ l of Taq polymerase (2 units).
• the reaction was carried out in a PHC-1 Thermocycler (Techne, Princeton, NJ) with 30 cycles of 95°C, 2 minutes for denaturation; 42°C, 2 minutes for annealing; and 70°C, 1 minute for synthesis. At the end of the 30th cycle, the reaction mixture was incubated another 9 minutes at 72°C for extension.
• the PCR mixture was subjected to electrophoresis in a
• the hIL-10 cDNA-containing vectors were propagated in E. coli strain DH5 ⁇ (Gibco-BRL), and the sequence of the DNA was verified by DNA sequencing.
• the pSV. Sport-based hIL-10 expression vector was used for COS transfection as well as for construction of mutant hIL-10 vectors.
• the resynthesized hIL-10 cDNA retained a unique Bglll site and a unique Bst ⁇ U site, both of which were present in the wild-type cDNA. These two internal restriction sites, the relative positions of which are shown schematically below, were later used to generate mutant hIL-10 cDNAs by cassette replacement.
• mutant cDNA fragments corresponding to the Z. .. tEII/ZscoRI region of wild-type hIL-10 cDNA were synthesized by PCR and used to replace the corresponding region in the pSV.Sport hIL-10 DNA described above.
• the K157E, C ⁇ 3 and C ⁇ 4 mutant antagonists of human IL-10 were produced by PCR using oligonucleotide primers complementary to the sequence of the resynthesized hIL-10 cDNA described above, with designated mutations pre-introduced in the 3'-end primers.
• a 5' primer designated B3351CC having an amino acid sequence defined by SEQ ID NO: 7 was used to produce three mutant antagonists.
• the sequence of this 5' primer was complementary to a human IL-10 cDNA internal sequence encompassing a unique BstEll restriction site of the wild-type hIL-10 cDNA.
• the 3' primers used to make the antagonists had sequences complementary to the 3' end sequence of hIL-10 encoding cDNA.
• human IL-10 cDNA was subjected to PCR in a 50 ⁇ l volume reaction mixture with a 50 ⁇ l paraffin oil overlay, in a 0.5 ml Eppendorf tube.
• the reaction mixture typically contained 26.5 ⁇ l of H2O, 5 ⁇ l of pfu DNA polymerase buffer [final concentrations in the reaction: 20 mM Tris-HCl, pH 8.2, 10 mM KC1, 2 mM MgC_2, 6 mM (NH4)2S 04, 0.1% Triton X-100, and 10 ⁇ g/ml nuclease-free bovine serum albumin (BSA)], 200 ⁇ M dNTPs, 40 ng of template DNA, 10 pmoles each of 5' primer B3351CC and one of the 3' primers, and 0.5 ⁇ l of pfu polymerase (2.5 units).
• BSA bovine serum albumin
• the reaction was carried out in a PHC-1 Thermocycler (Techne, Princeton, NJ) with 22 cycles of: 94°C, 2 minutes for denaturation; 50°C, 2 minutes for annealing; and 72°C, 2 minutes for synthesis. At the end of the 22nd cycle, the reaction mixture was incubated another 7.5 minutes at 72°C for extension.
• the PCR mixture was processed by phenol-CHCl3 extraction and ethanol precipitation and then digested sequentially with Z? _ • tEII and EcoRl.
• the restriction digestion products were subjected to electrophoresis in a 1% agarose/Tris-acetate gel containing 0.5 ⁇ g/ml ethidium bromide. DNA fragments having the expected sizes were excised from the gel and recovered by phenol-CHCl3 extraction and ethanol precipitation.
• the BstEll/EcoRl restriction fragments of the hIL-10 mutants were used to replace the corresponding region of the wild-type hIL-10 DNA in the pSV.Sport vector.
• the pSV. Sport-based hIL-10 mutant cDNAs were propagated in E. coli strain DH5 ⁇ and verified by DNA sequencing. The same expression vectors were used to transfect COS cells as described above.
• modified cDNA fragments corresponding to the PstllBglll region of the wild-type hIL-10 cDNA were synthesized by PCR using pairs of primers without a DNA template. The resulting fragments were used to replace the corresponding region of the wild-type hIL-10 DNA in the pSV.Sport vector. Variants were thus produced in which 7 (variant N ⁇ 7), 10 (variant N ⁇ 10), 11 (variant N ⁇ 11 ) or 12 (variant N ⁇ 12) residues were deleted from the N-terminus of wild-type hIL-10.
• the pairs of primers used to make each variant, followed by the SEQ ID NOs defining their sequences, were as follows:
• PCR was carried out using 10 pmoles of each primer of the indicated primer pairs, as described above for synthesis of the C-terminal mutant antagonists.
• the PCR mixture was processed by phenol-CHCl3 extraction and ethanol precipitation and then digested sequentially with Bglll and Pstl.
• the restriction digestion products were subjected to electrophoresis in an agarose gel as described above, and DNA fragments having the expected sizes were excised from the gel and recovered by phenol-CHCl3 extraction and ethanol precipitation.
• the PstllBglll restriction fragments of the hIL-10 variants were used to replace the corresponding region of the wild-type hIL-10 DNA in the pSV.Sport vector, after excision of that region by PstllBglll digestion and ligation of the replacement fragment.
• the pSV. Sport-based hIL-10 mutant cDNAs were propagated, verified and used as described above.
• N ⁇ 7, N ⁇ 10, N ⁇ 11 and N ⁇ 12 variants had amino acid sequences defined by residues 8-160, 11-160, 12-160 and 13-160, respectively, of the sequence of SEQ ID NO: 4.
• Antagonists having modifications at both the amino and carboxyl termini can readily be prepared by combining the foregoing methods.
• an N ⁇ 7/K157E antagonist can be made by producing pSV.Sport containing cDNA encoding the K157E antagonist, by carrying out PCR using 5' primer B3351CC and 3' primer C3481CC as described above. Following preparation and isolation of the N ⁇ 7 variant fragment using 5' primer C3352CC and 3' primer C3355CC as described above, the PstllBglll restriction fragment of the variant is used to replace the corresponding region of the K157E mutant DNA in the pSV.Sport vector, after excision of that region by PstllBgll digestion and ligation of the replacement fragment.
• COS cells were transfected as described above with expression vector pSV.Sport bearing cDNA inserts encoding human IL-10; antagonists K157E, C ⁇ 3 or C ⁇ 4; or agonist variants N ⁇ 7, N ⁇ 10, N ⁇ 11 or N ⁇ 12. The cells were then incubated in 10 cm culture dishes in serum-containing culture medium for 48 to 72 hours. Following this incubation, the culture dishes were washed twice with phosphate-buffered saline (PBS) and incubated at 37°C in 5% CO2 for 30 minutes, with 8 ml/dish of methionine-free DMEM medium supplemented with dialyzed FBS and glutamine.
• PBS phosphate-buffered saline
• the medium in each dish was removed by aspiration and replaced with 500 ⁇ l of methionine-free medium containing 250-300 ⁇ Ci of 35 S-methionine (DuPont NEN, Boston, MA; specific activity 43.3 mCi/ml).
• the cells were incubated at 37°C in 5% CO2 for 5 hours, after which 10 ⁇ l of 1.5 mg/ml L-methionine stock solution was added to the dishes and a 30 minute chase was carried out.
• the labeled conditioned medium was collected and subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis [SDS PAGE; Laemmli, Nature 227:680 (1970)] in 10-20% gels under non-reducing conditions, and the gels were dried and autoradiographed using standard methods and Kodak XAR film.
• the autoradiography revealed distinct labeled bands for human IL-10; antagonists K157E, C ⁇ 3 and C ⁇ 4; and for agonists N ⁇ 7 and N ⁇ 10, all of which migrated with apparent molecular weights of about 16 to 18 kilodaltons.
• all three human IL-10 carboxyl-terminal mutant antagonists were expressed at somewhat reduced levels — about 2 to 4 fold less than that of IL-10.
• Expression of the amino-terminal agonist variant N ⁇ 7 was comparable to that of IL-10, while the N ⁇ 10 variant was expressed at a level about 4 fold less than the level of IL-10. Expression of variants N ⁇ 11 and N ⁇ 12 was too low to be detected by this method.
• ELISA enzyme-linked immunosorbent assay
• the detection limit of this assay was about 1 ng/ml, and IL-10 levels in the range of 100 to 300 ng/ml were typically measured in culture media following a 72-hour incubation. It was thereby found that the relative levels of the IL-10 and the antagonists correlated well with the results obtained by metabolic labeling, suggesting that the epitopes recognized by the monoclonal antibodies used were not in the mutated regions.
• expression levels measured for human IL-10, K157E, C ⁇ 3, C ⁇ 4, N ⁇ 7, N ⁇ 10, N ⁇ 11 and N ⁇ 12 were 133, 80, 63, 48, 139, 28, 23 and 6.5 ng/ml, respectively.
• Human IL- 10 and the representative IL-10 mutant antagonists were examined for activity using mouse mast cells and human peripheral mononuclear cells (PBMCs).
• PBMCs peripheral mononuclear cells
• a mast cell stimulation assay was performed essentially as described by O'Garra et al. [Int. Immunol. 2: 821 (1990) and Thompson-Snipes et al. [J. Exp. Med. 773:507
• PBMC peripheral mononuclear cells
• PBMCs PBMCs were transferred to wells (10 5 cells/well in 200 ⁇ l of RPMI-1640 medium containing 5% FBS, penicillin/streptomycin, non-essential amino acids, sodium pyruvate and 2 mM glutamine) of 96-well microtiter plates.
• Human IL-10 was added to some of the wells at a fixed
• the plates were incubated at 37°C in a humidified atmosphere of 5% C02 for 24 hours, after which the supernatant fluids were collected and stored at -20°C for later analysis.
• Levels of IL-6, IL-l ⁇ and TNF ⁇ were measured in the collected samples using ELISA kits (R & D Systems,
• a mixed lymphocyte response (MLR) assay was performed.
• Human PBMCs were isolated as described above.
• Stimulator PBMCs were prepared by treating the cells with 50 mg/ml mitomycin C (Sigma, St. Louis, MO) for 20 minutes at 37°C.
• each of responder PBMCs and stimulator cells were mixed in each well of a 96-well microtiter dish, along with varying amounts of human IL-10 or one of the K157E, C ⁇ 3 or C ⁇ 4 antagonists, in a total volume of 200 ⁇ l (in triplicate).
• the cells were incubated at 37°C with 5% CO2 for 6 days, after which the cultures were pulsed with 1 ⁇ Ci of tritiated thymidine ([ 3 H]-TdR; 15.6 Ci/mmol, NEN, Boston, MA) per well for 16 hours. Lysates were harvested onto a filter using a 96-well cell harvester (Skatron, Inc., Sterline, VA) and counted in a ⁇ -counter (Pharmacia LKB Nuclear Inc., Gaithersburg, MD).
• Purified human IL-10 (about 99% pure) was radioiodinated by the ENZYMOBEAD® method (BioRad, Richmond, CA), following the manufacturer's instructions. Approximately 4 x 10 5 transfected COS cells expressing human IL-10 receptor cDNA were pelleted by centrifugation at 200 x g for 10 minutes, washed in binding buffer (PBS, 10% fetal calf serum, 0.1% NaN3), and resuspended in 200 ⁇ l of binding buffer containing [ 125 1] -human IL-10 (specific radioactivity 225 ⁇ Ci/ ⁇ g) at a concentration of 150 pM, with serially diluted conditioned medium from COS cells expressing cDNA encoding human IL-10 or one of the mutant antagonists of the invention.
• binding buffer PBS, 10% fetal calf serum, 0.1% NaN3
• Human IL-10 100 K157E 136 ⁇ 65 C ⁇ 3 172 ⁇ 28 C ⁇ 4 120 ⁇ 9
• NAME Lunn, Paul, G.
• GGCATCTCGG AGATCTCGAA
• GCATGTTAGG CAGGTTGCCT GGGAAGTGGG TGCAGGCCCT 60
• GGCATCTCGG AGATCTCGAA
• GCATGTTAGG CAGGTTGCCT GGGAAGTGGG TGGCCCTCAC 60
• CCCAGTCAGG AGGAC 75

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Genetics & Genomics (AREA)
• Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Zoology (AREA)
• Molecular Biology (AREA)
• Medicinal Chemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Biomedical Technology (AREA)
• Biophysics (AREA)
• Biochemistry (AREA)
• Gastroenterology & Hepatology (AREA)
• Pharmacology & Pharmacy (AREA)
• Toxicology (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• Animal Behavior & Ethology (AREA)
• Wood Science & Technology (AREA)
• General Engineering & Computer Science (AREA)
• Biotechnology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Epidemiology (AREA)
• Physics & Mathematics (AREA)
• Immunology (AREA)
• General Chemical & Material Sciences (AREA)
• Plant Pathology (AREA)
• Microbiology (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Peptides Or Proteins (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)

Priority Applications (8)

Applications Claiming Priority (2)

Publications (1)

Family

ID=22271670

Family Applications (1)

Country Status (17)

Cited By (25)

Families Citing this family (1)

Citations (2)

Family Cites Families (1)

• 1994
  • 1994-07-22 IL IL11041394A patent/IL110413A0/xx unknown
  • 1994-07-22 JP JP7505238A patent/JPH08507930A/ja active Pending
  • 1994-07-22 SK SK83-96A patent/SK8396A3/sk unknown
  • 1994-07-22 CA CA002168110A patent/CA2168110A1/en not_active Abandoned
  • 1994-07-22 SK SK1505-96A patent/SK150596A3/sk unknown
  • 1994-07-22 HU HU9503983A patent/HUT73463A/hu unknown
  • 1994-07-22 WO PCT/US1994/008052 patent/WO1995003411A1/en not_active Application Discontinuation
  • 1994-07-22 ZA ZA945434A patent/ZA945434B/xx unknown
  • 1994-07-22 EP EP94923956A patent/EP0711346A1/en not_active Withdrawn
  • 1994-07-22 CZ CZ96233A patent/CZ23396A3/cs unknown
  • 1994-07-22 AU AU73996/94A patent/AU681178B2/en not_active Ceased
  • 1994-07-22 CN CN94192880A patent/CN1127529A/zh active Pending
  • 1994-07-22 SG SG1996000969A patent/SG43798A1/en unknown
  • 1994-07-22 KR KR1019960700358A patent/KR960704041A/ko not_active Application Discontinuation
  • 1994-07-22 NZ NZ269663A patent/NZ269663A/en unknown
  • 1994-07-22 PL PL94312718A patent/PL312718A1/xx unknown
• 1996
  • 1996-01-25 NO NO960309A patent/NO960309D0/no unknown
  • 1996-01-26 FI FI960353A patent/FI960353A0/fi unknown

Patent Citations (2)

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events