JPH10503371A - New compound - Google Patents

Abstract

  (57) [Summary] A soluble protein having IL4 and / or IL13 antagonist or partial antagonist activity, consisting of an IL4 variant or variant fused to at least one human immunoglobulin constant region or fragment thereof.

Description

DETAILED DESCRIPTION OF THE INVENTION                               New compound   The present invention is directed to certain IgE-mediated allergic diseases, T-cell mediated autoimmune conditions and Hope for IL4 and / or IL13, such as Human interleukin 4 (IL4) for treating conditions resulting from unwanted effects ) And / or antagonists of human interleukin 13 (IL13) I do.   Interleukins are secretory peptide mediators of the immune response. Known Interleukins are each involved in the development, activation, proliferation and differentiation of cells of the immune system. It has many effects. Although IL4 has a physiological role in such functions, And may contribute to the onset of the disease. In particular, IL4 is associated with exogenous asthma, rhinitis, Allergies such as allergic conjunctivitis, atopic dermatitis and anaphylaxis Pathway of B lymphocyte development leading to the formation of IgE antibodies, a hallmark of sexually transmitted diseases Are linked. IL4 is also used in insulin-dependent diabetes, multiple sclerosis and rheumatoid arthritis. In certain autoimmune conditions, such as osteoarthritis, and in transplant rejection Proliferative and differentiation factors for T lymphocytes contributing to tissue damage It can also act as a key. IL4 is also a cell required to control infectious diseases The formation of an intervening response can be suppressed. Thus, IL4 is not able to respond to T or B lymphocytes. The antagonism of this effect is considered to have an effective effect on such diseases. IL No. 13 which was recently identified and has a mode of receptor structure / function (A versa, G. et al. (1993), J. Exp. Med. 178, 2213 to 2218). Share many similarities with the biological properties of IL4 (Minty, A. et al. (19) 93), Nature 362, 248-250).   Human IL4 has two N-glycosylatable sites and three disulfides. A single 129 amino acid polypeptide with six cysteines involved in binding (Le, HV et al. (1988), J. Biol. Chem. 263, 10817). -10823). The amino acid sequence of IL4 and the location of these disulfide bonds Known (Carr, C. et al., (1991) Biochemistry 30, 1515-1). 523).   A disulfide bond is formed between residues 3 and 127, 24 and 65, and 46 and 99. is there. The molecular weight of IL4 is 15 kDa (not glycosylated) in glycosylation degree. To 60 kDa (IL4 for hyperglycosylation).   The DNA sequence for human IL4 has also been described by Yokota, T. et al. .1986 83 5894-5898.   WO 93/10235 is an IL4 antagonist or partial antagonist Certain variants of IL4 are described.   EP-A-0464533 describes the constant regions of various immunoglobulin molecules separately. And a fusion protein comprising the human protein or a portion thereof.   The present invention provides for fusion of at least one immunoglobulin constant region or fragment thereof. IL4 and / or IL13 ant consisting of a modified IL4 mutant or variant Provides a soluble protein having gonist or partial antagonist activity is there.   The term "variant or variant" is intended for IL4 and / or Are defective derivatives of the IL4 protein or other derivatives that retain the ability to antagonize IL13. Include any molecule, such as a derivative. Such other derivatives add amino acids , By deletion, substitution or transposition, or by chemical modification thereof. Wear.   A DNA polymer encoding a mutant or mutant strain of IL4 can be prepared by a conventional method. For example, G. Winter et al., Nature 1982, 299, 756-758. Or Zoller and Smith 1982; Nucl. Acids Res., 10, 6487-6. 500, the site-directed protrusion of the cDNA encoding IL4 Mutations, or by Chan and Smith, Nucl. Acids Res., 1984, 12 Biochem. Soc. Trans., 2407-2419, or by G. Winter et al. 1984, 12, 224-225. Nucleic Acids Research, 1992 by Mikaelian and Sergeant. 20, 376.   As used herein, “IL4 and / or IL13 antagonists and Or have partial antagonist activity "by Spits et al. Immunology 139, 1142 (1987). It means that vesicle growth is inhibited in a dose-dependent manner.   Naturally occurring in wild-type IL4 at any one position from 120 to 128 At least one amino acid has been replaced with a different natural amino acid IL4 variants are disclosed in WO93 / 10235. In particular, natural at 124 Tyrosine present in glycine, more preferably aspartic acid May be substituted with a natural amino acid.   Immunoglobulins are of any subclass (IgG, IgM, IgA, IgE). Preferably, it is an IgG such as IgG1, IgG3 or IgG4. The constant region or fragment thereof is derived from a heavy or light chain or both Is done. The present invention is directed to the desirability of natural immunoglobulins, such as Fc receptor binding. Remove missing properties and / or introduce desirable properties such as stability, Includes mutations in immunoglobulin components. For example, Angal S., King DJ, Bodmer MW, Turner A., Lawson ADG, Roberts G., P edley B. And Adair R., Molecular Immunology, vol. 30, 105-. At page 108, 1993, residue 241 (Kabat numbering) is changed from serine to pro- Described are IgG4 molecules that have been replaced by phosphorus. This change is due to the serum half of the IgG4 molecule. Increase the life. Canfield SM and Morrison SL are the Journal of E xperimental Medicine, vol. 173, p. Glutamate from leucine at 3 and glutamate at mouse IgG2b The change of residue 248 (Kabat numbering) from G to Leucine is described. To the former Substitution of leucine with glutamate in immunoglobulins associated with the FcγRI receptor Decreases the affinity of the roblin molecule, with the latter glutamate leucine Substitution increases its affinity. EP 0307434 states that the remaining IgG Various mutations, including the L to E mutation of group 248 (Kabat numbering) Is disclosed.   The constant region or a fragment thereof comprises the entire constant region of the heavy chain of human IgG or Is preferably a substituent, most preferably IgG4. In one aspect The IgG component is a disulfide bond between the CH2 and CH3 regions and the heavy chain. Hinge region of IgG1 containing cysteine residues that contribute to Consisting of residues 11 and 14 of the region (Frangione B. and Milstein C., Nature, vol 216, pages 939-941, 1967). Preferably, the IgG1 component is Ell ison J., Berson B. and Hood LE., Nucleic Aids Research, vol. 1 0, pp. 4071-4079, 1982, residues 1-4 of the hinge of IgG1. And 6-15, amino corresponding to 1-110 of CH2 and 1-107 of CH3 Consists of acids. Residue 5 of the hinge contains the nucleotide sequence TGT to GCC The change changes the cysteine to alanine in the published IgG1 sequence. ing. In another embodiment, the IgG component comprises a CH2 and a CH3 region and a heavy chain. Hinge region encompassing cysteine residues that contribute to disulfide bonds between, for example, It is derived from IgG4 consisting of residues 8 and 11 of the IgG4 hinge region (Pin ck JR and Milstein C., Nature, vol 216, pages 941-942, 19 67). Preferably, the IgG4 component comprises Ellison J., Buxbaum J. and Hood  L., DNA, vol1, pages 11-18, 1981, IgG4 hinge residues. 1-12, amino acids corresponding to 1-110 of CH2 and 1-107 of CH3 Become. An example of a suitable mutation in IgG4 is hinge residue 10 (residue 2 41, Kabat numbering) from wild-type serine (S) to proline (P) Residue 5 of H2 (residue 248, Kabat numbering) was derived from wild-type leucine (L). Change to Lumate (E).   Fusion of an IL4 variant or variant to an Ig constant region or fragment may involve one This is accomplished by fusing the C-terminus of one component to the N-terminus of another component. Preferably Describes the use of an IL4 mutant or variant via its C-terminus in an Ig constant region or flag. Fusion to the N-terminus of the   In a preferred embodiment, the amino acid sequence of the fusion protein of the present invention is SEQ ID NO: 4 , SEQ ID NO: 7 or SEQ ID NO: 10.   In a further aspect, the present invention relates to a process for the preparation of a compound according to the invention, comprising a recombinant host. Expression of DNA encoding the compound in a main cell and collection of the product And provide a method for achieving this.   The present invention also provides a DNA polymer comprising a nucleotide sequence encoding the compound. Form part of   In a preferred embodiment, the DNA polymer is SEQ ID NO: 3, SEQ ID NO: 6 or Consists of the sequence of SEQ ID NO: 9.   The method of the present invention is described in Maniatis et al., Molecular Cloning-A Laboratory Manu. al: Cold Spring Harbor, 1982 and DNA Cloning, Volumes I and II. And Volume III (Edited by DM Glover, IRI Press Ltd.) This can be done by any conventional recombination technique.   In particular, the method comprises: i) DNA comprising a nucleotide sequence encoding a compound of the present invention in a host cell Preparing a replicable expression vector capable of expressing the polymer; ii) transforming a host cell with the vector; iii) Conditions under which the transformed host cells are capable of expressing the DNA polymer. To produce the compound; and iv) recovering the compound Process.   The present invention also provides suitable mono-, di- or oligomeric nucleotide units. A method for preparing a DNA polymer by fusing is provided.   The preparation can be made chemically, enzymatically or by a combination of the two methods. Can be performed in vitro or in vivo as appropriate. Thus, the DNA Rimmer has been described by DM Roverts et al., Biochemistry, 1985, 24, 5090-5. In accordance with a conventional method as described in No. 098, an appropriate DNA fragment is It is prepared by ligating together.   A DNA fragment is obtained by chemically synthesizing DNA having a required nucleotide sequence. By enzymatic polymerization with a DNA or RNA template, or by these methods Can be obtained by digesting with an appropriate restriction enzyme.   Digestion with restriction enzymes is carried out at a temperature of 20-70 ° C., generally 50 μl or less. It can be performed with 0.1 to 10 μg of DNA in the following volume of a suitable buffer. Wear.   The enzymatic polymerization of DNA is carried out at a temperature of 10 to 37 ° C., generally 50 μl or less. Lower volumes of nucleoside triphosphates dATP, dCTP, dGTP and DNA polymerase I (Klenow) in a suitable buffer containing Fragmentation) can be performed in vitro using DNA polymerases such as Wear.   Enzymatic ligation of DNA fragments is performed at 4 ° C. to ambient temperature, typically 50 μl. Or a smaller volume of DNA, such as T4 DNA ligase, in a suitable buffer It can be performed using ligase.   Chemical synthesis of DNA polymers or fragments is described in Chemical and Enzyma. tic Synthesis of Gene Fragments-A Laboratory Manual '(HG Gas en and A. Lang), Verlag Chemie, Weinheim (1982) or other families. Academic publications, e.g. J. Gait, HWD Matthes, M. Singh, BS Spr oat and RC Titmas, Nucleic Acids Research, 1982, 10, 62. 43; BS Sproat and W. Bannwarth, Tetrahedron Letters, 1983. , 24, 5771; MD Matteucci and MH Caruthers, Tetrahedron. Letters, 1980, 21, 719; MD Matteucci and MH Caruthers , Journal of the American Chemical Society, 1981, 103, 318. 5; SP Adams et al., Journal of the American Chemical Society, 198. 3, 105, 661; ND Sinha, J. Biernat, J. McMannus and H. Ko. ester, Nucleic Acids Research, 1984, 12, 4539; and HW. D. Matthes et al., EMBO Journal, 1984, 3, 801. Using conventional phosphotriester, phosphorous or phosphoryl It can be performed by midite chemistry. Preferably, an automatic DNA synthesizer is used. I have.   The DNA polymer is preferably a DNA sequence that encodes the compound together. It is prepared by ligating two or more DNA molecules constituting The present invention According to the first DNA encoding the IL4 variant or variant. A second molecule encoding the molecule and its immunoglobulin region or fragment thereof Ligating DNA molecules.   A DNA molecule is prepared by appropriate restriction enzymes on a vector having the necessary coding sequences. Obtained by digestion or by using the polymerase chain reaction synthesis method. Can be   The exact structure of the DNA molecule and the method for obtaining it will depend on the structure of the desired product. You. Designing an appropriate method for constructing a DNA molecule encoding a compound Is conventional for those skilled in the art.   Expression of the DNA polymer encoding the compound in a recombinant host cell can occur in the host cell. Alternatively, it may be carried out using a replicable expression vector capable of expressing a DNA polymer. The Expression vectors are novel and further form part of the present invention.   A replicable expression vector cleaves a vector that is compatible with the host cell, and Obtaining a linear DNA segment having a precon; One or more of the compounds encoding the compound under ligation conditions together with the By combining DNA molecules, they can be prepared according to the present invention.   The connection of the linear segment and one or more DNA molecules can be simultaneous or, if desired, consecutive. May be performed.   That is, the DNA polymer may be preformed or, if desired, vector. May be formed during the construction of the reactor.   The choice of vector depends on prokaryotic cells, such as E. coli, or eukaryotic cells. Vesicles, such as mouse C127, mouse myeloma, Chinese hamster ovary or HeLa cells, fungi such as filamentous fungi or unicellular yeast or insect cells such as mud Some will depend on the host cell being Sofira. The host cell also It may be a transformed organism. Suitable vectors include plasmids, bacteriological Phage, cosmid and e.g. baculovirus, vaccinia or semlii Includes recombinant viruses derived from forest fever virus.   The preparation of a replicable expression vector is performed, for example, by the procedure described above by Maniatis et al. Performed routinely using appropriate enzymes for DNA restriction, polymerization and ligation. . Polymerization and ligation can be performed as described above for the preparation of DNA polymers. it can. Digestion with restriction enzymes is generally carried out at a temperature of Should be performed with 0.1 to 10 μg of DNA in a smaller volume of appropriate buffer. Can be.   According to the present invention, a recombinant host cell is used to transform a host cell under transformation conditions of the present invention. It is prepared by transforming with a replicable expression vector. Suitable transformation Conditions are conventional and are described, for example, in Maniatis et al., Supra, or in "DNA Cloning, Vol. II, edited by DM Glover, IRL Press Ltd, 1985. Have been.   The choice of transformation conditions is determined by the host cell. In other words, E coli Such as bacterial host_TwoAcad. Sci., 1973 (Cohen et al., Proc. Nat. Acad. Sci.). , 69, 2110) or RbCl, MnCl._Two, Potassium acetate and glycero Of 3- [N-morpholino] -propane -May be treated with sulfonic acid, RbCl and glycerol. Feeding in culture Cells are transformed by calcium co-precipitation of vector DNA onto the cells. Is also good.   The present invention also relates to a host cell transformed with the replicable expression vector of the present invention. Reach.   Culturing the transformed host cells under conditions that allow expression of the DNA polymer. Nutrition is described, for example, in Maniatis et al., "DNA Cloning," above. As is customary. Thus, preferably, the cells are provided with nutrients, Incubate at a temperature below ° C.   The expression product is recovered by conventional means depending on the host cell. That is, the host cell If is a bacterium like E. coli, it can be physically, chemically or enzymatically Alternatively, the protein product may be chemically lysed and the protein product may be isolated from the resulting lysate. The product is a bacterium If secreted by cells, collect it from the peripheral cavity or nutrient medium. You may. If the host cell is a mammalian cell, the product is isolated from the nutrient medium. It is common to use   The DNA polymer can be isolated from a stable transformed mammalian cell line expressing the product. A vector designed to be released; for example, a bovine papillomavirus vector or May be incorporated into an amplification vector for Chinese hamster ovary cells (DNA  Cloning, Volume II, edited by DM Glover, IRL Press 1985; Kaufman, R. J. et al., Molecular and Cellular Biology 5, 1750-1759, 198. 5; Pavlakis GN and Hamer, DH, Proceedings of the National Academy of Science (USA) 80, 397-401, 1983; Goeddel, DV et al., European Patent Application No. 0093619, 1983).   The compounds of the present invention have IL4 and / or IL13 antagonist activity, Conditions resulting from the unwanted effects of IL4 and / or IL13, For example, IgE-mediated allergic diseases and T-cell mediated autoimmune symptoms or chronic micro- It may be useful in treating biological infections.   Accordingly, the present invention further relates to compounds of the present invention, including pharmaceutically acceptable carriers. A pharmaceutical composition comprising:   In use, the compounds are typically used in human pharmaceutical carriers, diluents and / or excipients. It is used in the form of a pharmaceutical composition in combination with a form, but the exact form of the composition is Depends on the method of administration. The compound may be, for example, an aerosol or spray solution for inhalation. Or used in the form of a sterile solution for parenteral administration.   When administering the compound of the present invention, the dose may be IL4 and / or IL13 mediated. Confers the desired effect on existing symptoms, thereby reducing IgE antibody-mediated symptoms Or the amount at which the progression of the autoimmune disease is stopped or regressed. The dosage is Generally varies with age, medical condition, and contraindications, if any, or severity I do. Unit dosages can vary from less than 1 mg to 300 mg, but typically Range from 1 to 20 mg per dose, daily dose of 0.02 to 40 mg / kg Is administered once or more times a day, for example, 1 to 6 times.   Compositions suitable for injection include solutions, suspensions or emulsions, or It is in the form of a dry powder to be dissolved or suspended in a suitable vehicle.   Fluid unit dosage forms are prepared using sterile, compound- and pyrogen-free vehicles. It is. Depending on the vehicle and concentration used, either dissolve the compound in the vehicle or Can be suspended. Solution may be used in any form for parenteral administration Can be used, especially for intravenous infections. In preparing solutions, compound Dissolve in vehicle and make the solution isotonic by adding sodium chloride if necessary Use aseptic technique before filling and sealing suitable sterile vials or ampoules. And sterilized by filtering through a sterilizing filter. Alternatively, secure the solution. If the qualification is sufficient, the solution in the sealed container can be sterilized in an autoclave. Yo No. Advantageously, buffers, solubilizers, stabilizers, preservatives or bactericides, sedimentation preventives Dispersants or additives such as emulsifiers and / or local anesthetics in the vehicle Is also good.   Dry powder which can be dissolved or suspended in a suitable vehicle before use Sterilize pre-sterilized drugs and other ingredients using aseptic techniques in the field of sterilization It may be prepared by filling a container. In addition, drugs and other ingredients Solution, sterilize the solution by filtration, and use aseptic techniques in the field of sterilization. And may be distributed into suitable containers. The product is then lyophilized and the container is sterile. And sealed.   Parenteral suspensions suitable for intramuscular, subcutaneous, or intradermal injection can be used for sterile compounds in sterile Suspension instead of dissolving in vehicle, sterilization cannot be achieved by filtration. And prepared in substantially the same way, except Isolate the compound under sterile conditions or Alternatively, it may be sterilized by, for example, gamma irradiation after isolation. Advantageously Is a compound containing a suspending agent, for example, polyvinylpyrrolidone, in the composition. Promotes even distribution.   Compositions suitable for administration via the respiratory tract include aerosols for inhalation, nebulized solutions or Includes fine powder. In the latter case, less than 50 microns, especially less than 10 microns Is preferred. Such compositions are manufactured in conventional manner and are compatible with conventional administration devices. Can be used together.   In a further aspect, the undesirable effects of IL4 and / or IL13 A method of treating a condition of interest, comprising administering to a patient an effective amount of a compound of the invention. Providing a method comprising:   The invention furthermore relates to use as an active therapeutic substance, in particular for IL4 and / or Provides compounds for treating conditions resulting from unwanted effects of IL13 You.   The present invention also relates to diseases resulting from the undesired effects of IL4 and / or IL13. There is provided the use of a compound of the invention in the manufacture of a medicament for treating a condition.   When a compound of the present invention is administered in accordance with the present invention, unexpected toxic effects are expected. Absent.   Hereinafter, the present invention will be described with reference to examples. Example 1 IL4. Y124D / IgG1 fusion protein   Construction of IL4.Y124D / IgG1 chimeric cDNA in mammalian expression system The expression of the corresponding protein and its activity are described. 1. Construction of DNA encoding fusion protein (A) Construction of IL4.Y124D coding region   Note that residue 124 of the protein was mutated from wild-type tyrosine to aspartic acid. (Kruse, N, Tony, HP and Sebald, W. EMBO Journal  11: 3237 [1992]), a human IL4 gene mutant was DNA (purchased from British Biotechnology) was produced by PCR mutagenesis. I was born. The IL4.Y124D cDNA was constructed using HindIII and Bg1II sites. It was inserted into the expression vector pTR312 (MJ Browne, JE Carey, CG Ch apman, AWR Tyrrell, C Entwise, GMP Lawrence, B Reavy, I  Dodd, AEsmail & JH Robinson. Journal of Biochemistry263: 1 599, [1988]), plasmid pDB906 was formed.   Amplify the IL4.Y124D molecule and add convenient restriction sites to each of the subclones. 20 ng of pDB90 using PCR reaction as substrate for addition at the end of Performed using 6 plasmids. PCR primers should have restriction sites Designed, flanked by 10-15 nucleotide base pairs, terminated with primer Was "anchor". The primer sequences were as follows:   The primers were used at a final concentration of 5 ng / μl, and dNTPs were reacted in a total of 100 μl. Added at a final concentration of 0.2 mM in volume. 31 cycles of PCR were performed. Cycling A denaturation step at 94 ° C. for 1 minute, an annealing step at 50 ° C. for 1 minute 30 seconds, , 72 ° C for 1 minute and 30 seconds. In cycle 1, denaturation in 5 minutes The extension was extended to 7 minutes in the last cycle. Advanced Biotechnologies 2.5 units of Taq polymerase enzyme from the source were used in the PCR reaction. 587b p PCR product was produced. This is called Promega “Magic PCR Cleanup ( Magic PCR cleanup) "kit and then in reaction buffer 4 , EcoRI and KpnI (all restriction enzymes were purchased from GibcoBRL) To generate "sticky ends". After 4 hours 30 minutes at 37 ° C., the reaction was The mixture was heated at 10 ° C. for 10 minutes and then subjected to ethanol precipitation. Agaro Analyzed by gel electrophoresis, about 570 bp, 463 bp and 100 bp The presence of three bands was observed. Since the digest was incomplete, there were 57 The 0 bp fragment represents the full length IL4.Y124D variant of IL4. Two yo The smaller fragment has an EcoRI site in the IL4.Y124D cDNA. Produced for. Geneclean 570 bp band^TMPurified by operation, EcoR I and KpnI followed by Geneclean^TMBluescr prepared by digestion with iptKS^{+ TM}Connected to. Bluescript KS⁺/IL4.Y124D recombinant Formed. This large amount of recombinant DNA was used for promega "Magic Maxiprep ( Magic Maxiprep) "method. The IL4.Y124D insert was It was excised from the Bluescript recombinant using I and KpnI. 20 μg recombination The DNA was incubated overnight at 30 ° C. with 25 units of SmaI in reaction buffer 4. Incubated. Then add 25 units of KpnI to the digest and add it Incubate at 37 ° C for 5 hours. The obtained flag of about 580 bp Mention the Genclean^TMAnd purified by IL4.Y124D / SmaI / KpnI fragment Event was generated. (B) Construction of IgG1 coding region   The COSFcLink vector (Table 1) was obtained from Ellison J., Berson B. and Hood.  LE, Nucleic Acids Research, Vol. 10, pp. 4071-4079, 19 82, amino acids 1-4 and 6-15 of the hinge, 1-110 and CH2 of CH2. And human IgG1 cDNA encoding CH3 1-108. Hinge Was changed by changing the TGT of the nucleotide sequence to GCC. Change the IgG1 sequence from cysteine to alanine. This is called RT-PCR Human IgG plasma cell leukemia ARH-77 (Amersham-type tissue collection (Amer ican Type Tissue Collection), cloned completely, sequenced and published Identity with the sequence [Patent Application Publication WO92 / 00985].   Construction of COSFc was performed using the pUC18 vector containing the human IgG1 cDNA described above. -(PUC18-Fc), which was digested with KpnI and SacII, and CH1, The hinge and part of CH2 were deleted. The deletion region was replaced with the following hinge-CH2 region. Region was replaced with a PCR amplified fragment. Use the following PCR primers Was: and   A DNA fragment containing the hinge-CH2 region was amplified from pUC18-Fc, Digest with KpnI and SacII, gel purify, KpnI / SacII digested pUC18-Fc Cloned into the vector. Located at position 230 of the IgG1 heavy chain (Kabat numbering Kabat et al., "Sequence of Proteins of Immunological Interest", 5th edition. Edition, USD Department of Health and Human Services, NIH Publishing, No. 9 1- 3242 (1991)) Replaces Cys with GCC for TGT in the nucleotide sequence. I changed it to Ala. The modified DNA sequence of the PCR primer 'A unique KpnI site at the end was introduced. The resulting plasmid was called pUC18Fcmod. The binding and PCR amplified regions were sequenced for confirmation.   The complete hinge-CH2-CH3 insert in pUC18-Fcmod was replaced with KpnI and And XbaI as a single DNA fragment, gel purified, KpnI and It was ligated to SFcR1Cos4 cut with XbaI to form COSFc.   SFcR1Cos4 is pST4DHFR (Deen, K, McDougal, JS, Inaker , R, Folena-Wasserman, G, Arthos, J, Rosenberg, J, Maddon, PJ, Ax el, R and Sweet, RW, Nature331: 82 [1988]). , Cytomegalovirus (CMV) promoter and bovine growth hormone (BGH) Soluble Fc receptor type I inserted between the polyadenylation regions (sFc R1), a β-globin promoter and SV40 polyadenylase. Dihydrofolate reductase (DHFR) cDNA inserted between the Contains the V40 origin of replication and the ampicillin resistance gene for growth in bacteria I do. By cutting the vector with KpnI and XbaI, the COSFc vector becomes Hinge-CH2-CH inserted between CMV promoter and BGH poly A region The sFcR1 coding region is removed so that it contains three regions.   This EcoRI site was recreated and BstEII, PstI and EcoRV were cloned. The unique EcoRI site of the vector introduces a The COSFc Link vector was prepared from COSFc by inserting Was.   The ligation was sequenced and the orientation in the vector was confirmed. Final vector size Is 6.37 kb. (C) Construction of DNA encoding fusion protein   To insert the IL4.Y124D cDNA, the COSFcLink vector was Prepared by digestion with oRV and KpnI as follows: 5 μg DNA Incubate with 15 units of EcoRV in reaction 2 for 5 hours at 37 ° C. , Followed by ethanol precipitation. The obtained DNA is reacted at 37 ° C. for 3 hours in reaction 4. During that time, it was digested with KpnI and subjected to ethanol precipitation. IL4.Y124D / SmaI / The KpnI and COSFcLink / EcoRV / KpnI fragments were ligated and IL4 Form plasmid pDB951 encoding .Y124D / IgG1 fusion protein did. Reaction using Amersham DNA ligation kit, product code RPN1507 The material was incubated overnight at 16 ° C. to achieve ligation. Transfer the ligation product to Promega  Transformed into JM-capable cells (high capacity), containing 50 μg / ml ampicillin And plated on Luria Broth agar. Transformants are transferred to Luria Broth (Ampisi) Phosphorus at 50 μg / ml) and the DNA was purified using Promega “Magic Maki”. IL4.Y124D / COSFcLink recombinant DN Production of A was confirmed by restriction digest and DNA sequencing. Complete IL4.Y1 The 24D sequence and binding to COSFcLink DNA were confirmed by DNA sequencing. (Table 2). The coding sequence of the recombinant IL4.Y124D / IgG1 DNA is shown in Table 3. The amino acid sequence of the fusion protein is shown in Table 4. IL4.Y124D / COSFcL Ink recombinant DNA is prepared, purified using a cesium chloride gradient, and And transfected HeLa cells temporarily. 2. Expression of fusion protein   HeLa cells in MEMα medium containing 10% fetal bovine serum and 1% glutamine (Gibco). For assay, 1x 4 days before transfection 10⁶75 cm of HeLa cells^Two10% neonatal bovine serum, 1% Contact with 15 ml RPMI-1640 medium containing glutamine ("seed medium") Seeded. One day before transfection, an additional 12.5 ml of seed medium was added to each. Added to flask. On the day of transfection, the medium was replaced with 15 ml Transfection medium "(containing 10% neonatal bovine serum and 1% non-essential amino acids). (MEM medium containing Earle's salt) (time 0). +3 hours Sometimes 0.125M CaCl_Two1x HBS (HEPES buffered saline) The appropriate DNA (25 μg) was added to the cells. At +7 hours, glycero cells Shock (15% v / v) followed by 1 mM containing 5 mM sodium butyrate. Incubate overnight in 2.5 ml of seed medium. The next day, cells Was washed with PBS (Dulbecco's phosphate buffered saline) and 12. 5 ml of "collection medium" (with 2% of 7.5% stock sodium bicarbonate solution) RPMI-1640) was added. After a further 124 hours of incubation, The supernatant is removed and centrifuged at 1000 rpm for 5 minutes to remove cell debris and Stored at either ° C or -20 ° C. 3. Biological activity   To assay supernatants for IL4 antagonist activity: Spits et al. I mmunology139, 1142 (1987). Incubate for 3 days with phytohemagglutinin and T cell mitogen To upregulate the IL4 receptor. Then got The blasts were stimulated with IL4 for another 3 days. Proliferation is achieved by incorporation of 3H thymidine It was measured.   The IL4.Y124D / IgG1 chimera has 133 pM IL in a dose dependent manner. By human peripheral blood T lymphocytes stimulated in 4^ThreeInhibits H-thymidine incorporation Was. Example 2 IL4. Y124D / IgG4 fusion protein 1. Construction of DNA encoding fusion protein   Perform PCR to amplify the IL4.Y124D coding region to form an XhoI site A silent nucleotide substitution was introduced at the 3 'end. Base for PCR reaction As a quality, 20 ng of the linearized pDB951 plasmid (Example 1.1 (c)) was used. Using. The oligonucleotide primers used were as follows:   A second PCR reaction was performed to determine the hinge-CH2-CH3 flag of the human IgG4 heavy chain. Fragment was amplified. The substrate for this is a synthetic human IgG4 heavy chain cDNA, The sequence is given in Table 5 and is based on the Genbank sequence GB: HUMIGCD2. (Ellison J., Buxbaum J. and Hood LE., DNA1: 11 -18, 1981). Many silent substitutions have been made to the published nucleotide sequence. I went. The gene is synthesized by combining two 0.5 kb synthetic DNA fragments. Combined. Each 0.5 kb fragment contains a series of overlapping oligonucleotides. Was prepared by annealing and then filling the gaps with PCR. Two The 0.5 kb fragment was joined at the SacII site and inserted into the pCR2 vector. Complete A 1.0 kb ApaI-BglII fragment containing the entire constant region was isolated and An expression vector containing an IL4 specific variable region was ligated to pCD. This construction Was used as a PCR substrate to amplify the hinge-CH2-CH3 region of IgG4. .   Oligonucleotides used to amplify the IgG4 hinge-CH2-CH3 region The primers were as follows:   The conditions for both PCR reactions were described for the induction of pDB951. It was. Briefly, primers were used at 5 ng / μl and dNTPs were It was added at a final concentration of 0.2 mM in a reaction volume of 0 μl. 2.5 units Advanced 31 cycles of PCR using Taq polymerase enzyme from Biotechnologies The reaction was performed. The cycle is a denaturation step at 94 ° C. for 1 minute, It consisted of an annealing step and an extension step at 72 ° C. for 1 minute and 30 seconds. Cycling The denaturation was extended to 5 minutes in the first cycle and the extension to 7 minutes in the final cycle.   About 700 bp (IgG4 hinge-CH2-CH3) and 400 bp (IL 4. Y124D) PCR product was obtained and Promega “Magic PCR Cleaner The purified PCR reaction was then digested with the following enzymes. To form "sticky ends": XhoI and XbaI for IgG4 and IL 4. EcoRV and XhoI for Y124D. Incubate the digest at 37 ° C for 3 hours. And then subjected to ethanol precipitation. The obtained DNA is subjected to gel electrophoresis. 690 bp (Hinge of IgG4—CH2-CH3) and 370 bp The size of p (IL4.Y124D) was obtained.   pDB951 (IL4.Y124D at COSFcLink) was transferred to EcoRV and And XbaI digestion to give the hinge-CH2-CH3 and I The L4.Y124D fragment was ligated and most of the IL4.Y124D / IgG 1. Prepare a vector to remove the fusion molecule. The slightly remaining part is I About 75 bp at the 5 'end of L4, which is IL4.Y12 produced by PCR amplification It was not present on the 4D EcoRV / XhoI fragment. 5 μg of pDB95 One DNA was prepared using Reaction Buffer 2 (GibcoBRL) in a total volume of 30 μl. Digested. The obtained 5.8 kb DNA fragment was subjected to Geneclean.^TMUsing operations And purified.   The three described fragments (IL4.Y124D EcoRV / XhoI, IgG4 XhoI / XbaI hinge-CH2-CH3 and EcoRV of pDB951 /5.8 kb fragment obtained by XbaI digestion) and ligated together to form IL 4. Plasmid pDB952 encoding Y124D / IgG4 fusion protein Done. The ligation reaction was performed using a DNA ligation kit (product code RPN150) manufactured by Amersham. The reaction was performed by incubating the reaction at 16 ° C. overnight using 7). Ligation reaction Transform the product into Promega JM109 capable cells (high capacity) Plated on Luria Broth agar containing 50 μg / ml. Transformants Culture in Luria Broth (containing 50 μg / ml of ampicillin) and DNA Was prepared using Promega "Magic Minipreps". IL4.Y124D / IgG4 recombinant DNA production was confirmed with restriction digests and complete IL4.Y124D and And the hinge-CH2-CH3 IgG4 region was confirmed by DNA sequencing. Table 6 Describes only the sequence of the coding region of the IL4.Y124D / IgG4 fusion molecule and Table 7 Contains the amino acid sequence of the fusion protein. IL4.Y124D / IgG4 recombinant DNA Prepared, purified on a cesium chloride gradient, and used to temporarily transfuse HeLa cells. Was transfected. 2. Expression of fusion protein   HeLa cells were cultured in MEMα medium containing 10% fetal bovine serum and 1% glutamine ( Gibco). 1x1 4 days before transfection for assay 0⁶Pieces of HeLa cells in a flask (75 cm^Two), 10% newborn bovine serum, 1% Inoculate 15 ml of RPMI-1640 containing glutamine ("seed medium") Was. One day before transfection, an additional 12.5 ml of seed medium was added to each flask. Added to Sco. On the day of transfection, the medium was replaced with 15 ml of “trans "Fection medium" (with 10% neonatal bovine serum and 1% non-essential amino acids) (MEM medium containing Yale salt) (time 0). At +3 hours, 0.125 M CaCl_Two1x HBS (HEPES buffered saline) with appropriate DNA (25 μg) was added to the cells. At +7 hours, cells were subjected to glycerol shock (1 5% v / v) and then 12.5 ml seed containing 5 mM sodium butyrate The mixture was left to stand overnight in the medium. The next day, cells were removed from PBS (Dulbet). Wash with Co-Phosphate Buffered Saline and add 12.5 ml of “Collecting Medium” (7.5% RPMI-1640 containing 2% stock sodium bicarbonate solution) was added. After an additional 24 hours of incubation, the supernatant was removed and 5 minutes at 1000 rpm. Centrifugation to remove cell debris and store at either 4 ° C or -20 ° C. Was. 3. Biological activity   To assay supernatants for IL4 antagonist activity: Spits et al., J. Im munology1391142 (1987) using the method described in Incubate for 3 days with phytohemagglutinin and T cell mitogen To upregulate the IL4 receptor. Then got The blasts were stimulated with IL4 for another 3 days. Proliferation is achieved by incorporation of 3H thymidine It was measured.   The IL4.Y124D / IgG4 chimera was injected with 133 pM IL4 in a dose dependent manner. Intensified by human peripheral blood T lymphocytes^ThreeH-thymidine incorporation was inhibited. Example 3 IL4.Y124D / IgG4 PE fusion protein 1. Construction of DNA encoding fusion protein   Perform PCR to amplify the IL4.Y124D coding region, as described in Example 2. To introduce a silent nucleotide substitution at its 3 'end forming an XhoI site I do.   A second PCR reaction was performed and the human IgG4 heavy chain PE variant hinge-CH2-C Amplify the H3 fragment. In IgG4 PE, residue 10 (K abat numbering, changing residue 241) from wild-type serine (S) to proline (P) , CH5 residue 5 (Kabat numbering, residue 248) was replaced with wild-type leucine (L) To glutamate (E). Angal S., King DJ, Bodmer MW. Turner A., Lawson ADG, Roberts G., Pedley B. and Adair. R., Molecular Immunology, 130, 105-108, 1993, IgG4 in which 241 residues (Kabat numbering) have been changed from serine to proline Describe the molecule. This change increases the serum half-life of the IgG4 molecule.   PCR of the IgG4 PE mutant with the synthetic human IgG4 heavy chain cDNA shown in Table 5 It was formed by mutagenesis and then ligated into the pCD expression vector. this Is a PCR reaction for amplifying the hinge-CH2-CH3 fragment of IgG4PE Was a brassmid used as a substrate for. The sequence of the IgG4 PE mutant is shown in Table 8. Put on. Residues of an IgG4 nucleotide sequence that are modified to prepare a PE variant Is as follows: With reference to Table 8:     Changing residue 322 from wild-type "T" to "C" with a PE variant;     Changing residue 333 from wild-type "A" to "G" with a PE variant; and     Residues 343-344 changed from wild-type "CT" to "GA" by PE mutant Do   Oligonucleotide primers are described for derivatization of pDB952 Used to amplify the hinge-CH2-CH3 region of an IgG4 PE mutant You.   About 700 bp (hinge-CH2-CH3 of IgG4 PE mutant) and 400 bp (IL4.Y124D) PCR product, Promega "Magic PCR Clear" The purified PCR reaction is then purified using the "Sticky ends": XhoI and XbaI for IgG4 PE and IL4.Y124D forms EcoRV and XhoI. Digest 3 g at 37 ° C Incubate for hours and then subject to ethanol precipitation. The obtained DNA is about 69 0 bp (hinge of IgG4 PE-CH2-CH3) and 370 bp (IL4. Y124D).   High amount of hinge-CH2-CH3 fragment of IgG4 PE mutant and IL 4. Purify and digest the PCR product to obtain the Y124D fragment And XbaI and XbaI for the hinge-CH2-CH3 of the IgG4 PE fragment. Alternatively, for the IL4.Y124D fragment, either EcoRV or XhoI Bluescript KS prepared by digestion^{+ TM}Next, Geneclean^TMNire Tie. Thus, Bluescript KS⁺/ CH4 of IgG4 PE recombinant 2-CH3 and Bluescript KS⁺/IL4.Y124D recombinant is produced. Many The amount of these DNAs is in Promega "Magic Maxiprep." Using the IgG4 PE hinge-CH2-CH3 fragment. It is excised from the Bluescript recombinant using XhoI and XbaI. About 6 obtained 90 bp fragment from Geneclean^TMAnd a large amount of IgG4 PE hinge- Generate a CH2-CH3 XhoI / XbaI fragment. IL4.Y124D The fragment was excised from the Bluescript recombinant using EcoRV and XhoI, The obtained fragment of about 370 bp was subjected to Geneclean.^TMTo purify.   As described in Derivation of pDB952, pDB951 was transformed with EcoRV and Xba Digestion with IgG4 PE hinge-CH2-CH3 and IL4. Prepare a vector to ligate the Y124D fragment.   The three described fragments (IL4.Y124D EcoRV / XhoI, Hinge-CH2-CH3 and pDB951 of IgG4 PE mutant XhoI / XbaI 5.8 kb fragment obtained by EcoRV / XbaI digestion of Then, using a DNA ligation kit (product code RPN1507) manufactured by Amerscham, Incubate the reaction overnight at 16 ° C. to form plasmid pDB953 . The ligation product was transformed into Promega JM109 competent cells (high competence) and Plate on Luria Broth agar containing μg / ml ampicillin. Trait The transformant was cultured on Luria Broth (containing 50 μg / ml of ampicillin). DNA is prepared using Promega "Magic Miniplex". IL4.Y The production of the recombinant DNA of the 124D / IgG4PE mutant was confirmed by restriction digest, and IL4.Y124D and hinge-CH2-CH3 IgG4 PE mutant regions Confirm by NA sequencing. Table 9 shows the IL4.Y124D / IgG4PE fusion fraction. Only the sequence of the offspring coding region is listed, and Table 10 contains the amino acid sequence of the fusion protein. . Prepare IL4.Y124D / IgG4PE recombinant DNA and use cesium chloride gradient And the DNA is used to transiently transfect HeLa cells. 2. Expression of fusion protein   HeLa cells were cultured in MEMα medium containing 10% fetal bovine serum and 1% glutamine ( Gibco). For assay, 1x 4 days before transfection 10⁶Pieces of HeLa cells in a flask (75 cm^Two), 10% newborn bovine serum, 1 Inoculate 15 ml of RPMI-1640 ("seed medium") containing 5% glutamine did. One day before transfection, an additional 12.5 ml of seed medium was Added to Lasco. On the day of transfection, the medium was transferred to 15 ml Sfection medium "(with 10% neonatal bovine serum and 1% non-essential amino acids (MEM medium containing ale salt) (time 0). +12 hours at 3 hours 5M CaCl_Two1x HBS (HEPES buffered saline) with the appropriate DNA (2 5 μg) was added to the cells. At +7 hours, cells were shocked with glycerol shock ( 15% v / v) and then 12.5 ml of a sheet containing 5 mM sodium butyrate. And incubated overnight in the medium. The next day, cells were removed from PBS (Dulbe Wash with 12.5 ml of “Collecting medium” (7.5%) RPMI-1640 containing 2% of stock sodium bicarbonate solution from After an additional 24 hours of incubation, the supernatant was removed and 5 minutes at 1000 rpm. Centrifugation to remove cell debris and store at either 4 ° C or -20 ° C. Was. 3. Biological activity   To assay supernatants for IL4 antagonist activity: Spits et al. I mmunology1391142 (1987) using the method described in Incubate for 3 days with phytohemagglutinin and T cell mitogen To upregulate the IL4 receptor. Then got The blasts were stimulated with IL4 for another 3 days. Proliferation is achieved by incorporation of 3H thymidine It was measured.   The IL4.Y124D / IgG4 PE chimera produced 133 pM IL in a dose dependent manner. By human peripheral blood T lymphocytes stimulated with^ThreePrevents incorporation of H-thymidine Harmed. Embodiment 4. FIG. Mammal animals containing DNA encoding IL4.Y124D / IgG4PE Product expression vector 1. DNA construction   pCDN vectors (Aiyar, N., Baker, E., Wu, HL, Nambi, P., Edw ards, RM, Trill, JJ, C. Bergsma, D.E. Molecular and Cellular Bi ochemistry 131: 75-86, 1994) is the CMV promoter, It contains an anchor cloning region and a BGH polyadenylation region. This By Geneticin^TMΒ-globin promoter and SV40 to select Bacterial neomycin phosphotransfer inserted between polyadenylation regions. Β-globin to amplify the ATPase gene (NEO) and methotrexate (MTX) DHFR selection kit inserted between the bin promoter and the BGH polyadenylation region Set, ampicillin resistance gene for growing bacteria and SV40 initiation of replication Contains points.   To insert the IL4.Y124D / IgG4 PE cDNA, pCDN vector Was prepared by digesting with Nde1 and BstX1 as follows: 15 μg DNA in Reaction 2 with 30 units of BstX1 (Gibco-BRL) at 55 ° C. Incubated for hours and subjected to ethanol precipitation. The obtained DNA is used in reaction 2. Using Nde1, digestion was performed at 37 ° C. for 1 hour, followed by ethanol precipitation. pDB953 (Example 3.1) was digested with BstX1 and Nde1 as follows to obtain IL4. The .Y124D / IgG4 PE fragment was prepared: reacting 15 μg of DNA Incubate with 30 units of BstX1 at 55 ° C. for 1 hour in ethanol 2 Subjected to settling. The obtained DNA is digested at 37 ° C. for 1 hour using Nde1 in reaction 2. And subjected to ethanol precipitation.   IL4.Y124D / IgG4PE Nde1 / BstX1 and pCDN Nde1 / BstX1 fragment and the plasmid pCDN-IL4.Y124D / An IgG4 PE was formed. Ligation was performed using 2 units of T4 DNA ligase (Gibco- BRL) and T4 DNA ligase buffer together. 16 reactants Incubated overnight at ° C. Transfer the ligation product to Gibco-BRL DH5a-capable cells (Subcloning ability), and Lur containing 75 μg / ml ampicillin. Plated on ia Broth agar. Transformants were transformed with Luria Broth (50 μg / (containing ampicillin) and DNA was prepared by alkaline lysis . Production of pCDN-IL4.Y124D / IgG4PE DNA was confirmed by restriction digest. I accepted. The complete sequence of the recombinant IL4.Y124D / IgG4PE DNA was sequenced. Confirmed by constant. pCDN-IL4.Y124D / IgG4 PE recombinant DNA Was prepared, purified using a Qiagen column, and temporarily transposed to COS cells using DNA. And CHO cells are electroporated to form stable clones. Was completed. 2. Expression of fusion protein a) Transient expression in COS   COS-1 cells were grown in DMEM medium with 10% fetal calf serum. Twenty-four hours prior to transfection, transfer cells to 35 mm tissue culture dishes x10^FiveCells were inoculated. 1 μg DNA in 100 μl DMEM without serum Was added to 6 μl of LIPOFE in 100 μl of DMEM without serum. Add to the solution containing CTAMINE reagent (Gibco-BRL) and mix gently. Incubate at room temperature for 45 minutes. Wash the cells once with serum-free DMEM You. 0.8 ml of serum-free DMEM in DNA-LIPOFECTAMINE solution And mix gently and overlay the diluted solution on the cells. Cells at 37 ° C For 5 hours and then 1 ml of DMEM containing 20% fetal calf serum. Add. Cells are assayed 48-72 hours later to determine expression levels. b) Electroporation into CHO cells   CHO cells, ACC-098 (CHO DG-44 derived suspension cell line, Urlaub G., Kas, E., Carothers, A.M. and Chasin, L.A. Cell, 33.405 -412, 1983) was grown in serum-free growth medium (WO 92/0524). 6). 15 μg of pCDN-IL4.Y124D / IgG4PE plasmid was The plasmid was linearized by digestion with 0 units of Not1 at 37 ° C. for 3 hours. And precipitated with ethanol. The obtained DNA was mixed with 50 μl of 1 × TE (10 mM Tris, pH 8.0, 1 mM EDTA). DNA to 38 Using a Bio Rad Gene Pulser set to 0 V and 25 μFd, 1 × 10⁷ ACC-098 cells were electroporated. 2.5x1 cells in growth medium 0^FourResuspend cells / ml and add 200 μl of cell suspension to 96-well plate Was plated in each well. After 48 hours, the medium was supplemented with 400 μg / ml G418 ( Geneticin). After 24 hours have passed since the selection, The conditioned medium from knee was screened by ELISA assay. Highest expression Ronnie was transferred to a 24-well plate for scaling up. 1

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI C12P 21/02 9051-4C A61K 37/02 ABF // (C12P 21/02 C12R 1:91) (81) Designation Country EP (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM) , GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, MW, SD, SZ, UG), AM, AT, AU, BB, BG, BR, BY, CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, IS, JP, KE, KG, KP, KR, KZ, LK, LR, LT, LU, LV, MD, M , MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TT, UA, UG, US, UZ, VN Brown, Michael Joseph United Kingdom, CM 19.5 Aidi, Essex, Harlow, The Pinnacles, Cold Harbor Road (No Address) Smith Kline Beecham Pharmaceuticals (72) Inventor Murphy , Kay Elizabeth UK, CM 19.5 Aidi, Essex, Harlow, The Pinnacles, Cold Harbor Road (without address) Smith Kline Beecham Pharmaceuticals (72) Inventor Chapman, Conrad Gerald UK, CM 19.5 Ady, Essex, Harlow, The Pinnacles, -Led Harbor Road (without address) Smith Kline Beecham Pharmaceuticals (72) Inventor Clinkenbad, Helen Elizabeth UK, CM 19.5 AD, ESEX, Harlow, The Pinnacles , Cold Harbor Road (no address) Smith Kline Beecham Pharmaceuticals (72) Inventor Young, Peter Ronald United States 19406 No. 709, Swedland Road, King of Prussia, Pennsylvania Smithkline Bee Cham Pharmaceuticals, Research and Development (72) Inventor Schatzman, Alan Richard United States 19406 No. 709, Swedland Road, King of Prussia, Pennsylvania United States Down-Beecham Pharmaceuticals, Lisa over Chi and Development

Claims (1)

[Claims]   1. At least one human immunoglobulin constant region or fragment thereof IL4 and / or IL13A comprising a fused IL4 variant or variant. A soluble protein having an antagonist or partial antagonist activity.   2. Native to wild-type IL4 at any one position from position 120 to position 128 Wherein at least one amino acid present in the plant is replaced with a different natural amino acid The compound according to claim 1.   3. The naturally occurring tyrosine at position 124 has been replaced with a different natural amino acid. The compound according to claim 2, wherein   4. Confirmation that the naturally occurring tyrosine at position 124 has been replaced with aspartic acid. A compound according to claim 3.   5. Any of the preceding claims wherein the immunoglobulin is of the subclass IgG. A compound according to any one of the preceding claims.   6. The constant region (s) or fragments thereof comprise the constant region of the heavy chain of human IgG. 6. The compound according to claim 5, which is a whole or a substantial part of   7. The constant region (s) or fragments thereof are invariant in the heavy chain of human IgG4. The compound according to claim 5, which is a whole or a substantial part of the region.   8. Amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 7 or SEQ ID NO: 10 The compound of claim 1 having a sequence.   9. A method for producing a compound according to any one of the preceding claims, The DNA encoding the compound is expressed in a recombinant host cell, and the product is recovered. A method consisting of:   Ten. i) a nucleic acid encoding a compound according to any one of the preceding claims in a host cell. A replicable expression vector capable of expressing a DNA polymer comprising a nucleotide sequence To prepare; ii) transforming a host cell with the vector; iii) Conditions under which the transformed host cells are capable of expressing the DNA polymer. To produce the compound; and iv) recovering the compound The method of claim 9 comprising:   11． Nucleotide encoding the compound according to any one of claims 1 to 8. A DNA polymer consisting of a DNA sequence.   12． 2. The method according to claim 1, comprising the sequence of SEQ ID NO: 3, SEQ ID NO: 6 or SEQ ID NO: 9. 2. The DNA polymer according to 1.   13. A replicable expression vector comprising the DNA polymer according to claim 11.   14. A host cell transformed with the replicable expression vector according to claim 13.   15． A compound according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising a body.   16． Treatment of conditions resulting from unwanted effects of IL4 and / or IL13 A method comprising administering to a patient an effective amount of a compound according to claim 1. Method.   17． 9. A compound according to any one of claims 1 to 8 for use in therapy.   18． Treatment of conditions resulting from unwanted effects of IL4 and / or IL13 A compound according to any one of claims 1 to 8 for use in.   19. Treatment of conditions resulting from unwanted effects of IL4 and / or IL13 9. The method according to claim 1 in the manufacture of a medicament for use in Use of compounds.