Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
• • 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
• • 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
  • A61P37/08—Antiallergic agents
• • 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]
  • C07K14/5406—IL-4
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• the present invention relates to antagonists of human interleukin 4 (IL4) and/or human interleukin 13 (IL13) for the treatment of conditions resulting from undesirable actions of IL4 and/or IL13 such as certain IgE mediated allergic diseases, T cell mediated autoimmune conditions and inappropriate immune responses to infectious agents.
• IL4 human interleukin 4
• IL13 human interleukin 13
• Interleukins are secreted peptide mediators of the immune response. Each of the known interleukins has many effects on the development, activation, proliferation and differentiation of cells of the immune system.
• IL4 has a physiological role in such functions, but can also contribute to the pathogenesis of disease.
• IL4 is associated with the pathway of B lymphocyte development that leads to the generation of IgE antibodies that are the hallmark of allergic diseases such as extrinsic asthma, rhinitis, allergic conjunctivitis, atopic dermatitis and anaphylaxis.
• D 4 can also act as a general growth and differentiation factor for T lymphocytes that may contribute to tissue damage in certain autoimmune conditions such as insulin dependent diabetes, multiple sclerosis and rheumatoid arthritis and in graft rejection. D 4 can also suppress the generation of cell-mediated responses required for the control of infectious disease. Antagonism of the effect of IL4 on T or B lymphocytes can therefore be expected to have beneficial effects on such diseases.
• IL13 has been recently identified and shares similarity in many of the biological properties of IL4 (Minty, A. et al (1993), Nature 362, 248-250) including some aspect(s) of receptor structure/function (Aversa, G. et al (1993), J. Exp. Med. 178, 2213-2218).
• Human IL4 consists of a single polypeptide chain of 129 amino acids with 2 possible N-glycosylation sites and 6 cysteines involved in 3 disulphide bridges (Le, H.V. et. al., (1988), J. Biol. Chem. 263, 10817-10823).
• the amino acid sequence of IL4 and the positions of these disulphide bridges are known (Carr, C. et al, (1991) Biochemistry 30, 1515-1523).
• the disulphide bridges are between residues 3 and 127, 24 and 65, and 46 and 99.
• the molecular weight of IL4 varies with the extent of glycosylation from 15KDa (no glycosylation) to 60KDa or more (hyperglycosylated IL4).
• the DNA sequence for human IL4 has also been described by Yokota, T. et. al., P.N.A.S. 1986 83 5894-5898.
• WO 93/10235 describes certain mutants of IL4 which are EL4 antagonists or partial antagonists.
• EP-A-0464 533 discloses fusion proteins comprising various portions of the constant region of immunoglobulin molecules together with another human protein or part thereof.
• the present invention provides a soluble protein having IL4 and/or IL13 antagonist or partial antagonist activity, comprising an IL4 mutant or variant fused to least one human immunoglobulin constant domain or fragment thereof.
• mutant or variant encompasses any molecule such as a truncated or other derivative of the IL4 protein which retains the ability to antagonise IL4 and/or IL13 following internal administration to a human.
• Such other derivatives can be prepared by the addition, deletion, substitution, or rearrangement of amino acids or by chemical modifications thereof.
• DNA polymers which encode mutants or variants of EL4 may be prepared by site-directed mutagenesis of the cDNA which codes for IL4 by conventional methods such as those described by G.
• IL4 and/or IL13 antagonist or partial antagonist activity means that, in the assay described by Spits et al (J. Immunology 139, 1142 (1987)), LL4- stimulated T cell proliferation is inhibited in a dose-dependent manner.
• Suitable IL4 mutants are disclosed in WO 93/10235, wherein at least one amino acid, naturally occuring in wild type IL4 at any one of positions 120 to 128 inclusive, is replaced by a different natural amino acid.
• the tyrosine naturally occurring at position 124 may be replaced by a different natural amino acid, such as glycine or, more preferably, aspartic acid.
• the immunoglobulin may be of any subclass (IgG, IgM, IgA, IgE), but is preferably IgG, such as IgGl, IgG3 or IgG4.
• the said constant domain(s) or fragment thereof may be derived from the heavy or light chain or both.
• the invention encompasses mutations in the immunoglobulin component which eliminate undesirable properties of the native immunoglobulin, such as Fc receptor binding and or introduce desirable properties such as stability. For example, Angal S., King D.J., Bodmer M.W., Turner A., Lawson A.D.G., Roberts G., Pedley B.
• the constant domain(s) or fragment thereof is preferably the whole or a substantial part of the constant region of the heavy chain of human IgG, most preferably IgG4.
• the IgG component consists of the CH2 and CH3 domains and the hinge region of IgGl including cysteine residues contributing to inter-heavy chain disulphide bonding, for example residues 11 and 14 of the IgGl hinge region (Frangione B. and Milstein C, Nature vol216pp939-941, 1967).
• the IgGl component consists of amino acids corresponding to residues 1-4 and 6-15 of the hinge, 1-110 of CH2 and 1-107 of CH3 of IgGl described by Ellison J., Berson B. and Hood L.
• the hinge is changed from cysteine in the published IgGl sequence to alanine by alteration of TGT to GCC in the nucleotide sequence.
• the IgG component is derived from IgG4, comprising the CH2 and CH3 domains and the hinge region including cysteine residues contributing to inter-heavy chain disulphide bonding, for example residues 8 and 11 of the IgG4 hinge region (Pinck J.R. and Milstein C, Nature vol216pp941-942, 1967).
• the IgG4 component consists of amino acids corresponding to residues 1-12 of the hinge, 1-110 of CH2 and 1-107 of CH3 of IgG4 described by Ellison J., Buxbaum J. and Hood L., DNA vollppl 1-18, 1981.
• residue 10 of the hinge (residue 241, Kabat numbering) is altered from serine (S) in the wild type to proline (P) and residue 5 of CH2 (residue 248, Kabat numbering) is altered from leucine (L) in the wild type to glutamate (E).
• Fusion of the IL4 mutant or variant to the Ig constant domain or fragment is by C-terminus of one component to N-terminus of the other.
• the IL4 mutant or variant is fused via its C-terminus to the N-terminus of the Ig constant domain or fragment.
• the amino acid sequence of the fusion protein of the invention is represented by SEQ ID No:4, SEQ ID No:7 or SEQ ID No: 10.
• the invention provides a process for preparing a compound according to the invention which process comprises expressing DNA encoding said compound in a recombinant host cell and recovering the product.
• the DNA polymer comprising a nucleotide sequence that encodes the compound also forms part of the invention.
• the DNA polymer comprises or consists of the sequence of SEQ ID No:3, SEQ ID No:6 or SEQ ID No:9.
• the process of the invention may be performed by conventional recombinant techniques such as described in Maniatis et. al., Molecular Cloning - A Laboratory Manual; Cold Spring Harbor, 1982 and DNA Cloning vols I, II and III (D.M. Glover ed., IRL Press Ltd).
• the process may comprise the steps of: i) preparing a replicable expression vector capable, in a host cell, of expressing a DNA polymer comprising a nucleotide sequence that encodes said compound; ii) transforming a host cell with said vector; iii) culturing said transformed host cell under conditions permitting expression of said DNA polymer to produce said compound; and iv) recovering said compound.
• the invention also provides a process for preparing the DNA polymer by the condensation of appropriate mono-, di- or oligomeric nucleotide units.
• the preparation may be carried out chemically, enzymatically, or by a combination of the two methods, in vitro or in vivo as appropriate.
• the DNA polymer may be prepared by the enzymatic ligation of appropriate DNA fragments, by conventional methods such as those described by D. M. Roberts et al in Biochemistry 1985, 24, 5090-5098.
• the DNA fragments may be obtained by digestion of DNA containing the required sequences of nucleotides with appropriate restriction enzymes, by chemical synthesis, by enzymatic polymerisation on DNA or RNA templates, or by a combination of these methods.
• Digestion with restriction enzymes may be performed in an appropriate buffer at a temperature of 20°-70°C, generally in a volume of 50 ⁇ l or less with 0.1-10 ⁇ g DNA.
• Enzymatic polymerisation of DNA may be carried out in vitro using a DNA polymerase such as DNA polymerase I (Klenow fragment) in an appropriate buffer containing the nucleoside triphosphates dATP, dCTP, dGTP and dTTP as required at a temperature of 10°-37°C, generally in a volume of 50 ⁇ l or less.
• Enzymatic ligation of DNA fragments may be carried out using a DNA ligase such as T4 DNA ligase in an appropriate buffer at a temperature of 4°C to ambient, generally in a volume of 50 ⁇ l or less.
• the chemical synthesis of the DNA polymer or fragments may be carried out by conventional phosphotriester, phosphite or phosphoramidite chemistry, using solid phase techniques such as those described in 'Chemical and Enzymatic Synthesis of Gene Fragments - A Laboratory Manual' (ed. H.G. Gassen and A. Lang), Verlag Chemie, Weinheim (1982),or in other scientific publications, for example M.J. Gait, H.W.D. Matthes, M. Singh, B.S. Sproat, and R.C. Titmas, Nucleic Acids Research, 1982, 10, 6243; B.S. Sproat and W. Bannwarth, Tetrahedron Letters, 1983, 24, 5771; M.D. Matteucci and M.H Caruthers, Tetrahedron Letters, 1980, 21, 719; M.D.
• the DNA polymer is preferably prepared by ligating two or more DNA molecules which together comprise a DNA sequence encoding the compound.
• a particular process in accordance with the invention comprises ligating a first DNA molecule encoding a said IL4 mutant or variant and a second DNA molecule encoding a said immunoglobulin domain or fragment thereof.
• the DNA molecules may be obtained by the digestion with suitable restriction enzymes of vectors carrying the required coding sequences or by use of polymerase chain reaction technology.
• the precise structure of the DNA molecules and the way in which they are obtained depends upon the structure of the desired product.
• the design of a suitable strategy for the construction of the DNA molecule coding for the compound is a routine matter for the skilled worker in the art.
• the expression of the DNA polymer encoding the compound in a recombinant host cell may be carried out by means of a replicable expression vector capable, in the host cell, of expressing the DNA polymer.
• the expression vector is novel and also forms part of the invention.
• the replicable expression vector may be prepared in accordance with the invention, by cleaving a vector compatible with the host cell to provide a linear DNA segment having an intact replicon, and combining said linear segment with one or more DNA molecules which, together with said linear segment, encode the compound, under ligating conditions.
• the ligation of the linear segment and more than one DNA molecule may be carried out simultaneously or sequentially as desired.
• the DNA polymer may be preformed or formed during the construction of the vector, as desired.
• the choice of vector will be determined in part by the host cell, which may be prokaryotic, such as E. coli, or eukaryotic, such as mouse C127, mouse myeloma, Chinese hamster ovary or Hela cells, fungi e.g. filamentous fungi or unicellular yeast or an insect cell such as Drosophila.
• the host cell may also be a transgenic animal.
• Suitable vectors include plasmids, bacteriophages, cosmids and recombinant viruses derived from, for example, baculoviruses, vaccinia or Semliki Forest virus.
• the preparation of the replicable expression vector may be carried out conventionally with appropriate enzymes for restriction, polymerisation and ligation of the DNA, by procedures described in, for example, Maniatis ej al., cited above. Polymerisation and ligation may be performed as described above for the preparation of the DNA polymer. Digestion with restriction enzymes may be performed in an appropriate buffer at a temperature of 20°-70°C, generally in a volume of 50 ⁇ l or less with 0.1- lO ⁇ g DNA.
• the recombinant host cell is prepared, in accordance with the invention, by transforming a host cell with a replicable expression vector of the invention under transforming conditions.
• Suitable transforming conditions are conventional and are described in, for example, Maniatis et al., cited above, or "DNA Cloning" Vol. ⁇ , D.M. Glover ed., IRL Press Ltd, 1985.
• a bacterial host such as E. coli may be treated with a solution of CaCl2 (Cohen et al, Proc. Nat. Acad. Sci., 1973, 69, 2110) or with a solution comprising a mixture of RbCl, MnCl2, potassium acetate and glycerol, and then with 3-[N-morpholino]- propane-sulphonic acid, RbCl and glycerol.
• Mammalian cells in culture may be transformed by calcium co-precipitation of the vector DNA onto the cells.
• the invention also extends to a host cell transformed with a replicable expression vector of the invention.
• Culturing the transformed host cell under conditions permitting expression of the DNA polymer is carried out conventionally, as described in, for example, Maniatis et al and "DNA Cloning" cited above.
• the cell is supplied with nutrient and cultured at a temperature below 45°C.
• the expression product is recovered by conventional methods according to the host cell.
• the host cell is bacterial, such as E. coli it may be lysed physically, chemically or enzymatically and the protein product isolated from the resulting lysate. If the product is to be secreted from the bacterial cell it may be recovered from the periplasmic space or the nutrient medium. Where the host cell is mammalian, the product may generally be isolated from the nutrient medium.
• the DNA polymer may be assembled into vectors designed for isolation of stable transformed mammalian cell lines expressing the product; e.g. bovine papillomavirus vectors or amplified vectors in Chinese hamster ovary cells (DNA cloning Vol.II D.M. Glover ed. IRL Press 1985; Kaufman, R.J. £1 aL, Molecular and Cellular Biology 5, 1750-1759, 1985; Pavlakis G.N. and Hamer, D.H., Proceedings of the National Academy of Sciences (USA) 80, 397-401, 1983; Goeddel, D.V. et al., European Patent Application No. 0093619, 1983).
• bovine papillomavirus vectors or amplified vectors in Chinese hamster ovary cells
• Compounds of the present invention have IL4 and/or IL13 antagonist activity and are therefore of potential use in the treatment of conditions resulting from undesirable actions of IL4 and or IL13 such as IgE mediated allergic diseases and T cell mediated autoimmune conditions or chronic microbial infection.
• the invention therefore further provides a pharmaceutical composition
• a pharmaceutical composition comprising a compound of the invention and a pharmaceutically acceptable carrier.
• the compound will normally be employed in the form of a pharmaceutical composition in association with a human pharmaceutical carrier, diluent and/or excipient, although the exact form of the composition will depend on the mode of administration.
• the compound may, for example, be employed in the form of aerosol or nebulisable solution for inhalation or sterile solutions for parenteral administration.
• the dosage ranges for administration of the compounds of the present invention are those to produce the desired effect on the IL4 and/or IL13 mediated condition, for example whereby IgE antibody mediated symptoms are reduced or progression of the autoimmune disease is halted or reversed.
• the dosage will generally vary with age, extent or severity of the medical condition and contraindications, if any.
• the unit dosage can vary from less than lmg to 300mg, but typically will be in the region of 1 to 20mg per dose, in one or more doses, such as one to six doses per day, such that the daily dosage is in the range 0.02-40mg/kg.
• compositions suitable for injection may be in the form of solutions, suspensions or emulsions, or dry powders which are dissolved or suspended in a suitable vehicle prior to use.
• Fluid unit dosage forms are prepared utilising the compound and a pyrogen-free sterile vehicle.
• the compound depending on the vehicle and concentration used, can be either dissolved or suspended in the vehicle. Solutions may be used for all forms of parenteral administration, and are particularly used for intravenous infection. In preparing solutions the compound can be dissolved in the vehicle, the solution being made isotonic if necessary by addition of sodium chloride and sterilised by filtration through a sterile filter using aseptic techniques before filling into suitable sterile vials or ampoules and sealing. Alternatively, if solution stability is adequate, the solution in its sealed containers may be sterilised by autoclaving. Advantageously additives such as buffering, solubilising, stabilising, preservative or bactericidal, suspending or emulsifying agents and or local anaesthetic agents may be dissolved in the vehicle.
• Dry powders which are dissolved or suspended in a suitable vehicle prior to use may be prepared by filling pre-sterilised drug substance and other ingredients into a sterile container using aseptic technique in a sterile area.
• the drug and other ingredients may be dissolved in an aqueous vehicle, the solution is sterilised by filtration and distributed into suitable containers using aseptic technique in a sterile area. The product is then freeze dried and the containers are sealed aseptically.
• Parenteral suspensions, suitable for intramuscular, subcutaneous or intradermal injection are prepared in substantially the same manner, except that the sterile compound is suspended in the sterile vehicle, instead of being dissolved and sterilisation cannot be accomplished by filtration.
• the compound may be isolated in a sterile state or alternatively it may be sterilised after isolation, e.g. by gamma irradiation.
• a suspending agent for example polyvinylpyrrolidone is included in the composition to facilitate uniform distribution of the compound.
• Compositions suitable for administration via the respiratory tract include aerosols, nebulisable solutions or microfine powders for insufflation. In the latter case, particle size of less than 50 microns, especially less than 10 microns, is preferred. Such compositions may be made up in a conventional manner and employed in conjunction with conventional administration devices.
• a method of treating conditions resulting from undesirable actions of IL4 and/or IL13 which comprises administering to the sufferer an effective amount of a compound of the invention.
• the invention further provides a compound of the invention for use as an active therapeutic substance, in particular for use in treating conditions resulting from undesirable actions of EL4 and/or EL 13.
• the invention also provides the use of a compound of the invention in the manufacture of a medicament for treating conditions resulting from undesirable actions of IL4 and or IL13.
• the IL4.Y124D cDNA was inserted into the expression vector pTR312, using the Hindlll and Bglll sites, (M J Browne, J E Carey, C G Chapman, A W R Tyrrell, C Entwisle, G M P Lawrence, B Reavy, I Dodd, A Esmail & J H
• PCR primers were designed to include restriction enzyme sites, flanked by 10-15 nucleotide base pairs to "anchor" the primers at each end.
• the primer sequences were as follows:
• the 570bp fragment represents the full-length IL4.Y124D variant of IL4 and was present because the digest was incomplete. The two smaller fragments were produced due to the presence of an EcoRI site within the IL4.Y124D cDNA.
• the 570bp band was purified by the Geneclean TM procedure, and ligated into Bluescript KS + TM which was prepared by digestion with EcoRI and Kpnl followed by Geneclean TM . A Bluescript KS+/IL4.Y124D recombinant was thus generated. Large amounts of this recombinant DNA were produced using the Promega "Magic Maxiprep" method.
• the IL4.Y124D insert was excised from the Bluescript recombinant using Smal and Kpnl.
• the COSFcLink vector (Table 1) contains human IgGl cDNA encoding amino acids 1-4 and 6-15 of the hinge, 1-110 of CH2 and 1-108 of CH3 described by Ellison J., Berson B. and Hood L. E., Nucleic Acids Research vollO, pp4071-4079, 1982. Residue 5 of the hinge is changed from cysteine in the published IgGl sequence to alanine by alteration of TGT to GCC in the nucleotide sequence. This was cloned from the human IgG plasma cell leukemia ARH-77 (American Type Tissue Collection), using RT-PCR and fully sequenced to confirm identity with the published sequence [patent application publication WO 92/00985]
• COSFc The construction of COSFc began with a pUCl ⁇ vector containing the human IgGl cDNA above (pUC18-Fc), which was digested with Kpnl and SacII, deleting the CHI, hinge and part of CH2. The deleted region was replaced with a PCR amplified fragment containing the hinge-CH2 region as follows. Using the following PCR primers:
• a DNA fragment containing the hinge-CH2 region was amplified from pUC18-Fc, digested with Kpnl and SacII, gel purified and cloned into the Kpnl/SacII digested pUC18-Fc vector.
• the Cys which occurs at position 230 (Kabat numbering; Kabat et al., "Sequences of Proteins of Immunological Interest, 5th Edition, US Department of Health and Human Services, NIH Publication No. 91-3242 (1991)) of the IgG 1 heavy chain, was altered to an Ala through a TGT to GCC substitution in the nucleotide sequence.
• the entire hinge-CH2-CH3 insert in pUC18-Fcmod was removed in a single DNA fragment with Kpnl and Xbal, gel purified, and ligated into SFcRlCos4 cut with Kpnl and Xbal to create COSFc.
• SFcRlCos4 is a derivative of pST4DHFR (Deen, K , McDougal, JS, Inacker, R, Folena-Wasserman, G, Arthos, J, Rosenberg, J, Maddon, PJ, Axel, R, and Sweet, RW. Nature 3_3 .
• sFcRl soluble Fc receptor type I
• BGH bovine growth hormone
• DHFR dihydrofolate reductase
• the COSFcLink vector was made from COSFc by inserting an oligonucleotide linker at the unique EcoRI site of the vector, which recreates this EcoRI site, and also introduces BstEII. Pstl and EcoRV cloning sites.
• the oligonucleotides used were:
• the COSFcLink vector was prepared by digesting with EcoRV and Kpnl as follows: 5 ⁇ g DNA was incubated with 15 units EcoRV in react 2 at 37°C for 5 hours, followed by ethanol precipitation. The resulting DNA was digested with Kpnl in react 4 at 37°C for 3 hours, and ethanol precipitated. The IL4.Y124D/SmaI/KpnI and the COSFcLink/EcoRV/Kpnl fragments were ligated together to form plasmid pDB951, which encodes the IL4.Y124D/IgGl fusion protein.
• the ligation was achieved using an Amersham DNA ligation kit, product code RPN 1507, the reactions being incubated at 16°C overnight.
• the ligation reaction products were transformed into Promega JM109 competent cells (high efficiency) and plated onto Luria Broth agar containing ampicillin at 50 ⁇ g/ml. Transformants were cultured in Luria Broth (containing ampicillin at 50 ⁇ g/ml) and DNA prepared using Promega "Magic Minipreps”. Production of an IL4.Y124D/COSFcLink recombinant DNA was verified by restriction digests and DNA sequencing. The complete IL4.Y124D sequence and the junctions with the COSFcLink DNA were confirmed by DNA sequencing (Table 2).
• the coding sequence of the recombinant IL4.Y124D/IgGl DNA is shown in Table 3 and the amino acid sequence of the fusion protein is shown in Table 4.
• the IL4.Y124D/COSFcLink recombinant DNA was prepared and purified using caesium chloride gradients and the DNA used to transiently transfect HeLa cells.
• HeLa cells were grown in MEM ⁇ medium (Gibco) with 10% foetal calf serum and 1% glutamine.
• MEM ⁇ medium Gibco
• 1 x 10 ⁇ HeLa cells were seeded in 15mls RPMI-1640 medium with 10% newborn calf serum, 1% glutamine ("seeding medium"), in a 75cm ⁇ flask, four days prior to transfection. On the day prior to transfection, a further 12.5mls seeding medium was added to each flask. On the day of transfection, the medium was changed to 15mls of "transfection medium” (MEM medium with Earle's salts containing 10% newborn calf serum and 1% non essential amino acids), at time zero.
• transfection medium MEM medium with Earle's salts containing 10% newborn calf serum and 1% non essential amino acids
• the IL4.Y124D/IgGl chimera inhibited ⁇ H thymidine incorporation by human peripheral blood T lymphocytes stimulated with 133pM IL4 in a dose dependent manner.
• PCR was performed to amplify the IL4.Y124D coding region and introduce a silent nucleotide substitution at the 3' end which creates a Xhol site.
• substrate for the PCR reaction 20ng of linearised pDB951 plasmid (Example 1.1(c)) was used.
• the oligonucleotide primers used were as follows:
• a second PCR reaction was performed to amplify the hinge-CH2-CH3 fragment of the human IgG4 heavy chain.
• the substrate for this was a synthetic human IgG4 heavy chain cDNA, the sequence of which is described in Table 5, and is based on the Genbank sequence GB:HUMIGCD2 (Ellison J., Buxbaum J. and Hood L.E., DNA ill 1-18, 1981). Numerous silent substitutions were made to the published nucleotide sequence.
• the gene was assembled by combining two 0.5Kb synthetic DNA fragments. Each 0.5Kb fragment was made by annealing a series of overlapping oligonucleotides and then filling in the gaps by PCR.
• the two 0.5Kb fragments were joined at the SacII site and inserted into the pCR2 vector.
• a 1.0Kb Apal-Bglll fragment containing the entire constant region was isolated and ligated into an expression vector, pCD, containing a humanized IL4 specific variable region. This construct was used as the PCR substrate to amplify the hinge-CH2-CH3 region of IgG4.
• the oligonucleotide primers used for amplification of the IgG4 hinge- CH2-CH3 region were as follows:
• PCR products of approximately 700b ⁇ (hinge-CH2-CH3 of IgG4) and 400bp (IL4.Y124D) were obtained and purified using the Promega "Magic PCR cleanup" kit.
• the purified PCR reactions were then digested with the following enzymes to create "sticky ends": Xhol and Xbal for IgG4 and EcoRV and Xhol for H .Y124D.
• the digests were incubated at 37°C for 3 hours and then ethanol precipitated.
• the resulting DNAs were analysed by gel electrophoresis and gave sizes of approximately 690bp (hinge-CH2-CH3 of IgG4) and 370bp (IL4.Y124D).
• a vector was prepared into which to ligate the hinge-CH2-CH3 of IgG4 and IL4.Y124D PCR fragments by digesting pDB951 (IL4.Y124D in COSFcLink) with EcoRV and Xbal to remove most of the IL4.Y124D/IgGl fusion molecule. The only part remaining is approximately 75bp at the 5' end of IL4, which is not present in the IL4.Y124D EcoRV/XhoI fragment produced by PCR amplification. 5 ⁇ g of pDB951 DNA was digested in a total volume of 30 ⁇ l using react 2 buffer (GibcoBRL). The resulting 5.8Kb DNA fragment was purified using the Geneclean procedure.
• the three fragments described (IL4.Y124D EcoRV/XhoI, hinge-CH2- CH3 of IgG4 Xhol/Xbal and the 5.8Kb fragment resulting from EcoRV/Xbal digestion of pDB951) were ligated together to form plasmid pDB952, which encodes the IL4.Y124D/IgG4 fusion protein.
• the ligation was carried out using a DNA ligation kit from Amersham (product code RPN 1507), incubating the reactions at 16° C overnight.
• the ligation reaction products were transformed into Promega JM109 competent cells (high efficiency) and plated onto Luria Broth agar containing ampicillin at 50 ⁇ g/ml.
• Transformants were cultured in Luria Broth (containing ampicillin at 50 ⁇ g/ml) and DNA prepared using Promega "Magic Minipreps”. Production of an IL4.Y124D ⁇ gG4 recombinant DNA was verified by restriction digests, and the complete IL4.Y124D and hinge-CH2-CH3 IgG4 regions were verified by DNA sequencing. Table 6 describes the sequence of the coding region only of the FL4.Y124D/IgG4 fusion molecule, and Table 7 contains the amino acid sequence of the fusion protein. The IL4.Y124D/IgG4 recombinant DNA was prepared and purified using caesium chloride gradients and the DNA used to transiently transfect HeLa cells.
• HeLa cells were grown in MEM ⁇ medium (Gibco) with 10% foetal calf serum and 1% glutamine.
• MEM ⁇ medium Gibco
• 1 x 10" HeLa cells were seeded in 15mls RPMI-1640 medium with 10% newborn calf serum, 1% glutamine ("seeding medium"), in a 75cm ⁇ flask, four days prior to transfection. On the day prior to transfection, a further 12.5mls seeding medium was added to each flask. On the day of transfection, the medium was changed to 15mls of "transfection medium” (MEM medium with Earle's salts containing 10% newborn calf serum and 1% non essential amino acids), at time zero.
• transfection medium MEM medium with Earle's salts containing 10% newborn calf serum and 1% non essential amino acids
• the IL4.Y124D/IgG4 chimera inhibited ⁇ H thymidine incorporation by human peripheral blood T lymphocytes stimulated with 133pM IL4 in a dose dependent manner.
• PCR is performed to amplify the IL4.Y124D coding region and introduce a silent nucleotide substitution at the 3' end which creates a Xhol site as described in Example 2.
• a second PCR reaction is performed to amplify the hinge-CH2-CH3 fragment of the human IgG4 heavy chain PE variant.
• residue 10 of the hinge is altered from serine (S) in the wild type to proline (P) and residue 5 of CH2 (residue 248, Kabat numbering) is altered from leucine (L) in the wild type to glutamate (E).
• Angal S. King D.J., Bodmer M.W., Turner A., Lawson A.D.G., Roberts G., Pedley B. and Adair R., Molecular
• the IgG4 PE variant was created using PCR mutagenesis on the synthetic human IgG4 heavy chain cDNA described in Table 5, and was then ligated into the pCD expression vector. It was this plasmid which was used as the substrate for the PCR reaction amplifying the hinge-CH2-CH3 fragment of IgG4 PE.
• the sequence of the IgG4 PE variant is described in Table 8.
• residues of the IgG4 nucleotide sequence which were altered to make the PE variant are as follows: referring to Table 8: residue 322 has been altered to "C” in the PE variant from “T” in the wild type; residue 333 has been altered to "G” in the PE variant from "A” in the wild type; and residues 343-344 have been altered to "GA” in the PE variant from "CT” in the wild type.
• Oligonucleotide primers are used for amplification of the IgG4 PE variant hinge-CH2-CH3 region as described for the derivation of pDB952.
• PCR products of approximately 700b ⁇ (hinge-CH2-CH3 of IgG4 PE mutant) and 400bp (IL4.Y124D) are obtained and purified using the Promega "Magic PCR cleanup" kit.
• the purified PCR reactions are then digested with the following enzymes to create "sticky ends": Xhol and Xbal for IgG4 PE and EcoRV and Xhol for IL4.Y124D.
• the digests are incubated at 37°C for 3 hours and then ethanol precipitated.
• the resulting DNAs are of sizes of approximately 690bp (hinge-CH2- CH3 of IgG4 PE) and 370bp (IL4.Y124D).
• the purified and digested PCR products are ligated into Bluescript KS + TM which is prepared by digestion with either Xhol and Xbal for the hinge-CH2-CH3 of IgG4 PE fragment or EcoRV and Xhol for the IL4.Y124D fragment, followed by GenecleanTM.
• Bluescript KS + /hinge-CH2- CH3 of IgG4 PE recombinant and a Bluescript KS + /TL4.Y124D recombinant are thus generated. Large amounts of these DNAs are produced using the Promega "Magic Maxiprep" method.
• the IgG4 PE hinge-CH2-CH3 fragment is excised from the Bluescript recombinant using Xhol and Xbal. The resulting fragment of approximately 690bp is purified by GenecleanTM to generate large amounts of the IgG4 PE hinge-CH2-CH3 Xhol/Xbal fragment.
• the IL4.Y124D fragment is excised from the Bluescript recombinant using EcoRV and Xhol and the resulting fragment of approximately 370bp is purified by GenecleanTM.
• a vector is prepared into which to ligate the hinge-CH2-CH3 of IgG4 PE and HAY124D fragments by digesting pDB951 with EcoRV and Xbal as described for the derivation of pDB952.
• EcoRV/Xbal digestion of pDB951 are ligated together to form plasmid pDB953 using a DNA ligation kit from Amersham (product code RPN 1507), incubating the reactions at 16°C overnight.
• the ligation reaction products are transformed into Promega JM109 competent cells (high efficiency) and plated onto Luria Broth agar containing ampicillin at 50 ⁇ g/ml. Transformants are cultured in Luria Broth (containing ampicillin at 50 ⁇ g/ml) and DNA prepared using Promega "Magic Minipreps".
• IL4.Y124D/IgG4 PE variant recombinant DNA is verified by restriction digests, and the complete IL4.Y124D and hinge-CH2-CH3 IgG4 PE variant regions are verified by DNA sequencing.
• Table 9 describes the sequence of the coding region only of the IL4.Y124D/IgG4 PE fusion molecule, and Table 10 contains the amino acid sequence of the fusion protein.
• the IL4.Y124D/IgG4 PE recombinant DNA is prepared and purified using caesium chloride gradients and the DNA used to transiently transfect HeLa cells. 2. Expression of the fusion protein HeLa cells were grown in MEM ⁇ medium (Gibco) with 10% foetal calf serum and 1% glutamine.
• the cells were subjected to a glycerol shock (15%v/v) and then left to incubate overnight in 12.5mls seeding medium containing 5mM sodium butyrate. The next day the cells were washed with PBS (Dulbecco's phosphate buffered saline) and 12.5mls "harvest medium” (RPMI-1640 with 2% of a 7.5% stock sodium bicarbonate solution) was added. After a further 24 hour incubation, the supernatants were removed, centrifuged at lOOOrpm for 5 minutes to remove cell debris and stored at either 4°C or -20°C.
• PBS Dulbecco's phosphate buffered saline
• 12.5mls "harvest medium” RPMI-1640 with 2% of a 7.5% stock sodium bicarbonate solution
• the IL4.Y124D/IgG4 PE chimera inhibited ⁇ H thymidine incorporation by human peripheral blood T lymphocytes stimulated with 133pM IL4 in a dose dependent manner.
• Example 4 Mammalian Expression vector containing DNA coding for IL4.Y124D/IgG4 PE.
• the pCDN vector (Aiyar, N.. Baker, E., Wu, H-L., Nambi, P., Edwards, R. Trill, J.J., Ellis, C, Bergsma, D. Molecular and Cellular Biochemistry 131:75-86, 1994 contains the CMV promoter, a polylinker cloning region, and the BGH polyadenylation region.
• This vector also contains a bacterial neomycin phosphotransferase gene (NEO) inserted between the ⁇ -globin promoter and SV40 polyadenylation region for GeneticinTM selection, the DHFR selection cassette inserted between the ⁇ -globin promoter and BGH polydenylation region for methotrexate (MTX) amplification, an ampicillin resistance gene for growth in bacteria, and a SV40 origin of replication.
• NEO bacterial neomycin phosphotransferase gene
• the pCDN vector was prepared by digesting with Ndel and BstXl as follows: 15 ⁇ g of DNA was incubated with 30 units of BstXl in react 2 (Gibco-BRL) at 55°C for 1 hour, and ethanol precipitated. The resulting DNA was digested with Ndel in react 2 at 37°C for 1 hour, and ethanol precipitated.
• the IL4.Y124D/IgG4 PE fragment was prepared from pDB953 (Example 3.1) by digesting with BstXl and Ndel as follows: 15 ⁇ g of DNA was incubated with 30 units of BstXl in react 2 at 55°C for 1 hour, and ethanol precipitated. The resulting DNA was digested with Ndel in react 2 at 37°C for 1 hour, and ethanol precipitated.
• the IL4.Y124D/IgG4 PE Ndel/BstXl and pCDN Ndel/BstXl fragments were ligated together to form the plasmid pCDN-IL4.Y124D/IgG4 PE.
• the ligation was achieved using 2 units of T4 DNA Ligase (Gibco BRL) with T4 DNA Ligase buffer. The reactions were incubated at 16°C overnight.
• the ligation reaction products were transformed into Gibco-BRL DH5a competent cells (subcloning efficiency) and plated onto Luria Broth agar containing 75 ug/ml ampicillin. Transformants were cultured in Luria Broth (containing ampicillin at 50 ug/ml) and DNA prepared by alkaline lysis. Production of a pCDN-
• IL4.Y124D/IgG4 PE DNA was confirmed by restriction digests. The complete sequence of the recombinant LL4.Y124D/IgG4 PE DNA was confirmed by sequencing. The pCDN- EL4.Y124D/IgG4 PE recombinant DNA was prepared and purified using Qiagen columns and the DNA was used to transiently infect COS cells and electroporated into CHO cells to create stable clones.
• CHO cells ACC-098 (a suspension cell line derived from CHO DG-44, Urlaub, G., Kas, E., Carothers, A.M. and Chasin, L.A. Cell, 33. 405-412, 1983) were grown in serum free growth medium WO 92/05246.
• 15 ⁇ g of the pCDN-IL4.Y124D/IgG4 PE plasmid was digested using 30 units of Notl at 37°C for 3 hours to linearize the plasmid, and precipitated with ethanol.
• the resulting DNA was resuspended in 50ul of 1 X TE (lOmM Tris, pH 8.0, ImM EDTA).
• the DNA was electroporated into 1 X 10 7 ACC-098 cells, using a Bio Rad Gene Pulser set at 380V and 25 ⁇ Fd.
• the cells were resupended into growth medium at 2.5 X 10 ⁇ cells/ml, and 200 ⁇ l of the cell suspension was plated into each well of a 96 well plate. 48 hours later the medium was switched to growth medium containing 400 ⁇ g/ml G418 (Geneticin). Twenty one days post selection, conditioned medium from the colonies which arose were screened by Elisa assay. The highest expressing colonies were transferred to 24 well plates in order to be scaled up.

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