US20030064481A1

US20030064481A1 - Novel compounds

Info

Publication number: US20030064481A1
Application number: US10/050,227
Authority: US
Inventors: Michael Browne; Peter Young; Allan Shatzman; Kay Murphy; Conrad Chapman; Helen Clinkenbeard
Original assignee: SmithKline Beecham Corp
Current assignee: SmithKline Beecham Corp
Priority date: 1994-07-29
Filing date: 2002-01-16
Publication date: 2003-04-03
Also published as: GB9415379D0; US5783181A; ZA956253B

Abstract

A soluble protein having IL4 and/or IL13 antagonist or partial antagonist activity comprises an IL4 mutant or variant fused to least one human immunoglobulin constant domain or fragment thereof.

Description

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.

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. In particular 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. IL4 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. IL4 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).


10
HIS-LYS-CYS-ASP-ILE-THR-LEU-GLN-GLU-ILE-ILE-LYS-THR-LEU-ASN-

20	30
SER-LEU-THR-GLU-GLN-LYS-THR-LEU-CYS-THR-GLU-LEU-THR-VAL-THR-

40
ASP-ILE-PHE-ALA-ALA-SER-LYS-ASN-THR-THR-GLU-LYS-GLU-THR-PHE-

50	60
CYS-ARG-ALA-ALA-THR-VAL-LEU-ARG-GLN-PHE-TYR-SER-HIS-HIS-GLU-

70
LYS-ASP-THR-ARG-CYS-LEU-GLY-ALA-THR-ALA-GLN-GLN-PHE-HIS-ARG-

80	90
HIS-LYS-GLN-LEU-ILE-ARG-PHE-LEU-LYS-ARG-LEU-ASP-ARG-ASN-LEU-

100
TRP-GLY-LEU-ALA-GLY-LEU-ASN-SER-CYS-PRO-VAL-LYS-GLU-ALA-ASN-

110	120
GLN-SER-THR-LEU-GLU-ASN-PHE-LEU-GLU-ARG-LEU-LYS-THR-ILE-MET-

129
ARG-GLU-LYS-TYR-SER-LYS-CYS-SER-SER

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 15 KDa (no glycosylation) to 60 KDa 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 IL4 antagonists or partial antagonists.

EP-A-0 464 533 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.

The term “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 IL4 may be prepared by site-directed mutagenesis of the cDNA which codes for IL4 by conventional methods such as those described by G. Winter et al in Nature 1982, 299, 756-758 or by Zoller and Smith 1982; Nucl. Acids Res., 10, 6487-6500, or deletion mutagenesis such as described by Chan and Smith in Nucl. Acids Res., 1984, 12, 2407-2419 or by G. Winter et al in Biochem. Soc. Trans., 1984; 12. 224-225 or polymerase chain reaction such as described by Mikaelian and Sergeant in Nucleic Acids Research, 1992, 20, 376.

As used herein. “having IL4 and/or IL13 antagonist or partial antagonist activity” means that, in the assay described by Spits et al (J. Immunology 139, 1142 (1987)), IL4-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. In particular, 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 IgG1, 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. and Adair R., Molecular Immunology vol30pp105-108, 1993, describe an IgG4 molecule where residue 241 (Kabat numbering) is altered from serine to proline. This change increases the serum half-life of the IgG4 molecule. Canfield S. M. and Morrison S. L., Journal of Experimental Medicine vol173pp1483-1491, describe the alteration of residue 248 (Kabat numbering) from leucine to glutamate in IgG3 and from glutamate to leucine in mouse IgG2b. Substitution of leucine for glutamate in the former decreases the affinity of the immunoglobulin molecule concerned for the FcγRI receptor, and substitution of glutamate for leucine in the latter increases the affinity. EP0307434 discloses various mutations including an L to E mutation at residue 248 (Kabat numbering) in IgG.

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. In one aspect the IgG component consists of the CH2 and CH3 domains and the hinge region of IgG1 including cysteine residues contributing to inter-heavy chain disulphide bonding, for example residues 11 and 14 of the IgG1 hinge region (Frangione B. and Milstein C., Nature vol216pp939-941, 1967). Preferably the IgG1 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 IgG1 described by Ellison J., Berson B. and Hood L. E., Nucleic Acids Research vol10, pp4071-4079, 1982. Residue 5 of the hinge is changed from cysteine in the published IgG1 sequence to alanine by alteration of TGT to GCC in the nucleotide sequence. In another aspect 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). Preferably 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 vol1pp11-18, 1981. In one example of a suitable mutation in IgG4. 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. Preferably the IL4 mutant or variant is fused via its C-terminus to the N-terminus of the Ig constant domain or fragment.

In a preferred aspect, 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.

In a further aspect, 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.

In a preferred aspect 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).

In particular, 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. Thus, 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 μl or less with 0.1-10 μg 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 μl 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 μl 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. Matteucci and M. H. Caruthers, Journal of the American Chemical Society, 1981, 103, 3185; S. P. Adams et al., Journal of the American Chemical Society,1983, 105, 661; N. D. Sinha, J. Bierat, J. McMannus, and H. Koester, Nucleic Acids Research, 1984, 12, 4539; and H. W. D. Matthes et al., EMBO Journal, 1984, 3, 801. Preferably an automated DNA synthesizer is employed.

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 react ion 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.

Thus, 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 et 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 μl or less with 0.1-10 μg 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. II, D. M. Glover ed., IRL Press Ltd, 1985.

The choice of transforming conditions is determined by the host cell. Thus, 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. Thus, preferably 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. Thus, where 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. et 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).

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 comprising a compound of the invention and a pharmaceutically acceptable carrier.

In use 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 1 mg to 300 mg, but typically will be in the region of 1 to 20 mg per dose, in one or more doses, such as one to six doses per day, such that the daily dosage is in the range 0.02-40 mg/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. Alternatively 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. Advantageously, 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.

In a further aspect there is provided 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 IL4 and/or IL13.

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.

No unexpected toxicological effects are expected when compounds of the invention are administered in accordance with the present invention.

The following Examples illustrate the invention.

EXAMPLE 1

IL4.Y124D/IgG1 fusion protein [0062]
The construction of an IIA.Y124D/IgGl chimeric cDNA, the expression of the corresponding protein in a mammalian expression system and its activity are described. [0063]
1. Construction of DNA coding for fusion protein [0064]
(a) Construction of IL4.Y124D coding region [0065]
A variant of the human IL4 gene, which has been described (Kruse, N., Tony, H-P and Sebald, W. EMBO Journal 11: 3237 [1992]) in which residue 124 in the protein has been mutated from tyrosine in the wild type to aspartic acid, was produced by PCR mutagenesis of the human IL4 cDNA (purchased from British Biotechnology). The IL4.Y124D cDNA was inserted into the expression vector pTR312, using the HindIII and BglII 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. Robinson. Journal of Biological Chemistry 263: 1599, [1988]) to form the plasmid pDB906. [0066]
To amplify the IL4.Y124D molecule and add convenient restriction sites at each end for subcloning, a PCR reaction was performed using 20 ng of the pDB906 plasmid as the substrate. 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: [0067]
1) 5′ CGA ACC ACT GAA TTC CGC ATT GCA GAG ATA 3′ (includes an EcoRI restriction site, GAATTC) [0068]
2) 5′ CAC AAA GAT CCT TAG GTA CCG CTC GAA CAC TTT GA 3′ (includes a KpnI restriction site, GGTACC) [0069]
Primers were used at a final concentration of 5 ng/μl, and dNTPs were added at a final concentration of 0.2 mM in a total reaction volume of 100 μl. 31 cycles of PCR were performed. Cycles consisted of a denaturation step of 1 minute at 94° C., an annealing step of 1 minute 30 seconds at 50° C., and an elongation step of 1 minute 30 seconds at 72° C. On cycle 1 denaturation was extended to 5 minutes and on the final cycle elongation was extended to 7 minutes. 2.5 units of the Taq polymerase enzyme from Advanced Biotechnologies were used in the PCR reaction. A PCR product of 587 bp was produced. This was purified using the Promega “Magic PCR cleanup” kit, and then digested with EcoRI and KpnI in react buffer 4 (all restriction enzymes were obtained from GibcoBRL.), to generate ‘sticky ends’. After 4 hours 30 minutes at 37° C., the reaction was heated to 70° C. for 10 minutes and then ethanol precipitated. Analysis of the resulting DNA by agarose gel electrophoresis showed the presence of three bands of approximately 570 bp, 463 bp and 100 bp . The 570 bp 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™ procedure, and ligated into Bluescript KS[0070] ⁺™ which was prepared by digestion with EcoRI and KpnI followed by Geneclean™. 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 SmaI and KpnI. 20 μg recombinant DNA was incubated with 25 units SmaI in react buffer 4, at 30° C. overnight. 25 units of KpnI were then added to the digest, which was incubated at 37° C. for 5 hours. The resulting fragment of approximately 580 bp was purified by Geneclean™ to generate an IL4.Y124D/SmaI/KpnI fragment.
(b) Construction of IgG1 coding region [0071]
The COSFcLink vector (Table 1) contains human IgG1 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 vol10, pp4071-4079, 1982. Residue 5 of the hinge is changed from cysteine in the published IgG1 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][0072]
The construction of COSFc began with a pUC18 vector containing the human IgG1 cDNA above (pUC18-Fc), which was digested with KpnI and SacII, deleting the CH1, 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: [0073]

5′ TCG AGC TCG GTA CCG AGC CCA AAT CGG CCG ACA AAA

CTC ACA C 3′

and

5′ GTA CTG CTC CTC CCG CGG CTT TGT CTT G 3′
A DNA fragment containing the hinge-CH2 region was amplified from pUC18-Fc, digested with KpnI and SacII, gel purified and cloned into the KpnI/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 IgG1 heavy chain, was altered to an Ala through a TGT to GCC substitution in the nucleotide sequence. An altered DNA sequence in one of the PCR primers introduced a unique KpnI site at the 5′ end of the hinge. The resulting plasmid was called pUC18Fcmod, and the junctions and PCR amplified region were sequenced for confirmation. [0074]
The entire hinge-CH2-CH3 insert in pUC18-Fcmod was removed in a single DNA fragment with KpnI and XbaI, gel purified, and ligated into SFcR1Cos4 cut with KpnI and XbaI to create COSFc. [0075]
SFcR1Cos4 is a derivative of pST4DHFR (Deen, K, McDougal. J S, Inacker, R. Folena-Wassermann, G., Arthos, J., Rosenberg, J., Maddon, P. J., Axel, R., and Sweet, R. W. Nature 331: 82 [1988]) and contains the soluble Fc receptor type I (sFcR1) inserted between the cytomegalovirus (CMV) promoter and bovine growth hormone (BGH) polyadenylation regions, and also contains the dihydrofolate reductase (DHFR) cDNA inserted between the β-globin promoter and SV40 polyadenylation regions, an SV40 origin of replication, and an ampicillin resistance gene for growth in bacteria. Cutting the vector with KpnI and XbaI removes the sFcR1 coding region, so that the COSFc vector contains the hinge-CH2-CH3 region inserted between the CMV promoter and BGH polyA regions. [0076]
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, PstI and EcoRV cloning sites. The oligonucleotides used were: [0077]

5′ AATTCGGTTACCTGCAGATATCAAGCT 3′

3′ GCCAATGGACGTCTATAGTTCGATTAA 5′
The junction was sequenced to confirm orientation in the vector. The size of the final vector is 6.37 kb. [0078]
(c) Construction of DNA coding for fusion protein. [0079]
To insert the IL4.Y124D cDNA, the COSFcLink vector was prepared by digesting with EcoRV and KpnI as follows: 5 μg 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 KpnI in react 4 at 37° C. for 3 hours, and ethanol precipitated. The IL4.Y124D/SmaI/KpnI and the COSFcLink/EcoRV/KpnI fragments were ligated together to form plasmid pDB951, which encodes the IL4.Y124D/IgG1 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 μg/ml. Transformants were cultured in Luria Broth (containing ampicillin at 50 μg/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/IgG1 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. [0080]
2. Expression of the fusion protein [0081]
HeLa cells were grown in MEMA medium (Gibco) with 10% foetal calf serum and 1% glutamine. For the assay, 1×10[0082] ⁶HeLa cells were seeded in 15 mls RPMI-1640 medium with 10% newborn calf serum, 1% glutamine (“seeding medium”), in a 75 cm²flask, four days prior to transfection. On the day prior to transfection, a further 12.5 mls seeding medium was added to each flask. On the day of transfection, the medium was changed to 15 mls of “transfection medium” (MEM medium with Earle's salts containing 10% newborn calf serum and 1% non essential amino acids), at time zero. At time +3 hours, 25 kg of the appropriate DNA in 0.125M CaCl₂, 1×HBS (HEPES buffered saline) was added to the cells. At time +7 hours, the cells were subjected to a glycerol shock (15%v/v) and then left to incubate overnight in 12.5 mls seeding medium containing 5 mM sodium butyrate. The next day the cells were washed with PBS (Dulbecco's phosphate buffered saline) and 12.5 mls “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 1000 rpm for 5 minutes to remove cell debris and stored at either 4° C. or −20° C.
3. Biological Activity [0083]
For assay of supernatant for IL4 antagonist activity: using the method described in Spits et al., J. Immunology 139, 1142 (1987), human peripheral blood lymphocytes were incubated for three days with phytohaemagluttinin, a T cell mitogen, to upregulate the IL4 receptor. The resultant blast cells were then stimulated for a further three days with IL4. Proliferation was measured by the incorporation of 3H thymidine. [0084]
The IL4.Y124D/IgG1 chimera inhibited [0085] ³H thymidine incorporation by human peripheral blood T lymphocytes stimulated with 133 pM IL4 in a dose dependent manner.

EXAMPLE 2

IL4.Y124D/IgG4 fusion protein [0086]
1. Construction of DNA coding for fusion protein [0087]
PCR was performed to amplify the IL4.Y124D coding region and introduce a silent nucleotide substitution at the 3′ end which creates a XhoI sire. As substrate for the PCR reaction 20 ng of linearised pDB951 plasmid (Example 1.1(c)) was used. The oligonucleotide primers used were as follows: [0088]
1) 5′ CAC AAG TGC GAT ATC ACC TTA CAG GAG ATC 3′ (includes an EcoRV restriction site, GATATC) [0089]
2) 5′ CTC GGT ACC GCT CGA GCA CTT TGA GTC TTT 3′ (includes a XhoI restriction site, CTCGAG). [0090]
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 1:11-18, 1981). Numerous silent substitutions were made to the published nucleotide sequence. The gene was assembled by combining two 0.5 Kb synthetic DNA fragments. Each 0.5 Kb fragment was made by annealing a series of overlapping oligonucleotides and then filling in the gaps by PCR. The two 0.5 Kb fragments were joined at the SacII site and inserted into the pCR2 vector. A 1.0 Kb ApaI-BglII 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. [0091]
The oligonucleotide primers used for amplification of the IgG4 hinge-CH2—CH3 region were as follows: [0092]
1) 5′ GGT GGA CAA CTC GAG CGA GTC CAA ATA TGG 3′ (includes a XhoI restriction site, CTCGAG) [0093]
2) 5′ TTA CGT AGA TCT AGA CTA CAC TCA TTT ACC 3′ (includes an XbaI site, TCTAGA). [0094]
The conditions for both PCR reactions were as described for the derivation of pDB951. Briefly, primers were used at 5 ng/μl, and dNTPs at a final concentration of 0.2 mM in a total reaction volume of 100 μl. 2.5 Units of Taq polymerase enzyme from Advanced Biotechnologies were used and 31 cycles of PCR performed. Cycles consisted of a denaturation step of 1 minute -at 94° C., an annealing step of 1 minute 30 seconds at 50° C., and an elongation step of 1 minute 30 seconds at 72° C. On cycle 1 denaturation was extended to 5 minutes and on the final cycle elongation was extended to 7 minutes. [0095]
PCR products of approximately 700 bp (hinge-CH2—CH3 of IgG4) and 400 bp (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”: XhoI and XbaI for IgG4 and EcoRV and XhoI for IL4.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 690 bp (hinge-CH2—CH3 of IgG4) and 370 bp (IL4.Y124D). [0096]
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 XbaI to remove most of the IL4.Y124D/IgG1 fusion molecule. The only part remaining is approximately 75 bp at the 5′ end of IL4, which is not present in the IL4.Y124D EcoRV/XhoI fragment produced by PCR amplification. 5 μg of pDB951 DNA was digested in a total volume of 30 μl using react 2 buffer (GibcoBRL). The resulting 5.8 Kb DNA fragment was purified using the Geneclean™ procedure. [0097]
The three fragments described (IL4.Y124D EcoRV/XhoI, hinge-CH2—CH3 of IgG4 XhoI/XbaI and the 5.8 Kb fragment resulting from EcoRV/XbaI 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 μg/ml. Transformants were cultured in Luria Broth (containing ampicillin at 50 μg/ml) and DNA prepared using Promega “Magic Minipreps”. Production of an IL4.Y124D/IgG4 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 IL4.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. [0098]
2. Expression of the fusion protein [0099]
HeLa cells were grown in MEMα medium (Gibco) with 10% foetal calf serum and 1% glutamine. For the assay, 1×10[0100] ⁶HeLa cells were seeded in 15 mls RPMI-1640 medium with 10% newborn calf serum, 1% glutamine (“seeding medium”), in a 75 cm²flask, four days prior to transfection. On the day prior to transfection, a further 12.5 mls seeding medium was added to each flask. On the day of transfection, the medium was changed to 15 mls of “transfection medium” (MEM medium with Earle's salts containing 10% newborn calf serum and 1% non essential amino acids), at time zero. At time +3 hours, 25 μg of the appropriate DNA in 0.125M CaCl₂, 1×HBS (HEPES buffered saline) was added to the cells. At time +7 hours, the cells were subjected to a glycerol shock (15%v/v) and then left to incubate overnight in 12.5 mls seeding medium containing 5 mM sodium butyrate. The next day the cells were washed with PBS (Dulbecco's phosphate buffered saline) and 12.5 mls “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 1000 rpm for 5 minutes to remove cell debris and stored at either 4° C. or −20° C.
3. Biological Activity [0101]
For assay of supernatant for IL4 antagonist activity: using the method described in Spits et al., J. Immunology 139, 1142 (1987), human peripheral blood lymphocytes were incubated for three days with phytohaemagluttinin, a T cell mitogen, to upregulate the IL4 receptor. The resultant blast cells were then stimulated for a further three days with IL4. Proliferation was measured by the incorporation of 3H thymidine. [0102]
The IL4.Y124D/IgG4 chimera inhibited [0103] ³H thymidine incorporation by human peripheral blood T lymphocytes stimulated with 133 pM IL4 in a dose dependent manner.

EXAMPLE 3

IL4.Y124D/IgG4 PE fusion protein [0104]
1. Construction of DNA coding for fusion protein [0105]
PCR is performed to amplify the IL4.Y124D coding region and introduce a silent nucleotide substitution at the 3′ end which creates a XhoI site as described in Example 2. [0106]
A second PCR reaction is performed to amplify the hinge-CH2—CH3 fragment of the human IgG4 heavy chain PE variant. In IgG4 PE, 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). Angal S., King D. J., Bodmer M. W., Turner A., Lawson A. D. G., Roberts G., Pedley B. and Adair R., Molecular Immunology vol30pp105-108, 1993, describe an IgG4 molecule where residue 241 (Kabat numbering) is altered from serine to proline. This change increases the serum half-life of the IgG4 molecule. [0107]
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. The residues of the IgG4 nucleotide sequence which were altered to make the PE variant are as follows: [0108]
referring to Table 8: [0109]
residue 322 has been altered to “C” in the PE variant from “T” in the wild type; [0110]
residue 333 has been altered to “G” in the PE variant from “A” in the wild type; and [0111]
residues 343-344 have been altered to “GA” in the PE variant from “CT” in the wild type. [0112]
Oligonucleotide primers are used for amplification of the IgG4 PE variant hinge-CH2—CH3 region as described for the derivation of pDB952. [0113]
PCR products of approximately 700 bp (hinge-CH2—CH3 of IgG4 PE mutant) and 400 bp (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”: XhoI and XbaI for IgG4 PE and EcoRV and XhoI 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 690 bp (hinge-CH2—CH3 of IgG4 PE) and 370 bp (IL4.Y124D). [0114]
To obtain larger amounts of the IgG4 PE variant hinge-CH2—CH3 fragment and the IL4.Y124D fragment, the purified and digested PCR products are ligated into Bluescript KS[0115] ⁺™ which is prepared by digestion with either XhoI and XbaI for the hinge-CH2—CH3 of IgG4 PE fragment or EcoRV and XhoI for the IL4.Y124D fragment, followed by Geneclean™. A Bluescript KS+/hinge-CH2—CH3 of IgG4 PE recombinant and a Bluescript KS⁺/IL4.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 XhoI and XbaI. The resulting fragment of approximately 690 bp is purified by Geneclean™ to generate large amounts of the IgG4 PE hinge-CH2—CH3 XhoI/XbaI fragment. The IL4.Y124D fragment is excised from the Bluescript recombinant using EcoRV and XhoI and the resulting fragment of approximately 370 bp is purified by Geneclean™.
A vector is prepared into which to ligate the hinge-CH2—CH3 of IgG4 PE and IL4.Y124D fragments by digesting pDB951 with EcoRV and XbaI as described for the derivation of pDB952. [0116]
The three fragments described (IL4.Y124D EcoRV/XhoI, hinge-CH2—CH3 of IgG4 PE variant XhoI/XbaI and the 5.8Kb fragment resulting from EcoRV/XbaI 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 μg/ml. Transformants are cultured in Luria Broth (containing ampicillin at 50 μg/ml) and DNA prepared using Promega “Magic Minipreps”. Production of an IIA.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. [0117]
2. Expression of the fusion protein [0118]
HeLa cells were grown in MEMα medium (Gibco) with 10% foetal calf serum and 1% glutamine. For the assay, 1×10[0119] ⁶HeLa cells were seeded in 15 mls RPMI-1640 medium with 10% newborn calf serum, 1% glutamine (“seeding medium”), in a 75 cm²flask, four days prior to transfection. On the day prior to transfection, a further 12.5 mls seeding medium was added to each flask. On the day of transfection, the medium was changed to 15 mls of “transfection medium” (MEM medium with Earle's salts containing 10% newborn calf serum and 1% non essential amino acids), at time zero. At time +3 hours, 25[μg of the appropriate DNA in 0.125M CaCl₂, 1×HBS (HEPES buffered saline) was added to the cells. At time +7 hours, the cells were subjected to a glycerol shock (15%v/v) and then left to incubate overnight in 12.5 mls seeding medium containing 5 mM sodium butyrate. The next day the cells were washed with PBS (Dulbecco's phosphate buffered saline) and 12.5 mls “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 1000 rpm for 5 minutes to remove cell debris and stored at either 4° C. or −20° C.
3. Biological Activity [0120]
For assay of supernatant for IL4 antagonist activity: using the method described in Spits et al., J. Immunology 139, 1142 (1987), human peripheral blood lymphocytes were incubated for three days with phytohaemagluttinin, a T cell mitogen, to upregulate the IL4 receptor. The resultant blast cells were then stimulated for a further three days with IL4. Proliferation was measured by the incorporation of 3H thymidine. [0121]
The IL4.Y124D/IgG4 PE chimera inhibited [0122] ³H 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. [0123]
1. Construction of DNA [0124]
The pCDN vector (Aiyar, N., Baker, E., Wu, H-L., Nambi, P., Edwards, R. M., 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 β-globin promoter and SV40 polyadenylation region for Geneticin™ selection, the DHFR selection cassette inserted between the β-globin promoter and BGH polydenylation region for methotrexate (MTX) amplification, an ampicillin resistance gene for growth in bacteria, and a SV40 origin of replication. [0125]
To insert the IL4.Y124D/IgG4 PE cDNA, the pCDN vector was prepared by digesting with Nde1 and BstX1 as follows: 15 μg of DNA was incubated with 30 units of BstX1 in react 2 (Gibco-BRL) at 55° C. for 1 hour, and ethanol precipitated. The resulting DNA was digested with Nde1 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 BstX1 and Nde1 as follows: 15 μg of DNA was incubated with 30 units of BstX1 in react 2 at 55° C. for 1 hour, and ethanol precipitated. The resulting DNA was digested with Nde1 in react 2 at 37° C. for 1 hour, and ethanol precipitated. [0126]
The IL4.Y124D/IgG4 PE Nde1/BstX1 and pCDN Nde1/BstX1 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 μg/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 IL4.Y124D/IgG4 PE DNA was confirmed by sequencing. The pCDN-IL4.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. [0127]
2. Expression of the Fusion Protein [0128]
a) Transient Expression in COS [0129]
COS-1 cells were grown in DMEM medium with 10% fetal bovine serum. For the transfection, cells were seeded at 2×10[0130] ⁵cells into a 35 mm tissue culture dish 24 hours prior. A solution containing 1 μg of DNA in 100 μl of DMEM without serum is added to a solution containing 6 μl of LIPOFECTAMINE Reagent (Gibco-BRL) in 100 μl of DMEM without serum, gently swirled and incubated at room temperature for 45 minutes. The cells are washed once with serum free DMEM. 0.8 ml of serum free DMEM is added to the DNA-LIPOFECTAMINE SOLUTION, mixed gently and the diluted solution is overlayed on the cells. The cells are incubated at 37° C. for 5 hours, then 1 ml of DMEM containing 20% fetal bovine serum is added. The cells are assayed 48-72 hours later to determine expression levels.
b) Electroporation into CHO cells [0131]

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 μg of the pCDN-IL4.Y124D/IgG4 PE plasmid was digested using 30 units of Not1 at 37° C. for 3 hours to linearize the plasmid, and precipitated with ethanol. The resulting DNA was resuspended in 50 ul of 1×X TE (10 mM Tris, pH 8.0, 1 mM EDTA). The DNA was electroporated into 1×10 ⁷ACC-098 cells, using a Bio Rad Gene Pulser set at 380V and 25 μFd. The cells were resupended into growth medium at 2.5×10⁴cells/ml, and 200 μl 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 μg/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.

TABLE 1


DNA sequence of COSFcLink vector, 6367 bp

SEQ ID No:1
GACGTCGACGGATCGGGAGATCGGGGATCGATCCGTCGACGTACGACTAGTTATTAATAG	60

TAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTT	120

ACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATG	180

ACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTAT	240

TTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCT	300

ATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGG	360

GACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGG	420

TTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTC	480

CACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAA	540

TGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTC	600

TATATAAGCAGAGCTGGGTACGTGAACCGTCAGATCGCCTGGAGACGCCATCGAATTCGG	660

TTACCTGCAGATATCAAGCTAATTCGGTACCGAGCCCAAATCGGCCGACAAAACTCACAC	720

ATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCC	780

AAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGA	840

CGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCA	900

TAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGT	960

CCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAA	1020

CAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGA	1080

ACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCT	1140

GACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGG	1200

GCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTT	1260

CCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG	1320

CTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC	1380

GGGTAAATGAGTGTAGTCTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCA	1440

GCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCAC	1500

TGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTAT	1560

TCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCA	1620

TGCTGGGGATGCGGTGGGCTCTATGGAACCAGCTGGGGCTCGAGGGGGGATCTCCCGATC	1680

CCCAGCTTTGCTTCTCAATTTCTTATTTGCATAATGAGAAAAAAAGGAAAATTAATTTTA	1740

ACACCAATTCAGTAGTTGATTGAGCAAATGCGTTGCCAAAAAGGATGCTTTAGAGACAGT	1800

GTTCTCTGCACAGATAAGGACAAACATTATTCAGAGGGAGTACCCAGAGCTGAGACTCCT	1860

AAGCCAGTGAGTGGCACAGCATTCTAGGGAGAAATATGCTTGTCATCACCGAAGCCTGAT	1920

TCCGTAGAGCCACACCTTGGTAAGGGCCAATCTGCTCACACAGGATAGAGAGGGCAGGAG	1980

CCAGGGCAGAGCATATAAGGTGAGGTAGGATCAGTTGCTCCTCACATTTGCTTCTGACAT	2040

AGTTGTGTTGGGAGCTTGGATAGCTTGGACAGCTCAGGGCTGCGATTTCGCGCCAAACTT	2100

GACGGCAATCCTAGCGTGAAGGCTGGTAGGATTTTATCCCCGCTGCCATCATGGTTCGAC	2160

CATTGAACTGCATCGTCGCCGTGTCCCAAAATATGGGGATTGGCAAGAACGGAGACCTAC	2220

CCTGGCCTCCGCTCAGGAACGAGTTCAAGTACTTCCAAAGAATGACCACAACCTCTTCAG	2280

TGGAAGGTAAACAGAATCTGGTGATTATGGGTAGGAAAACCTGGTTCTCCATTCCTGAGA	2340

AGAATCGACCTTTAAAGGACAGAATTAATATAGTTCTCAGTAGAGAACTCAAAGAACCAC	2400

CACGAGGAGCTCATTTTCTTGCCAAAAGTTTGGATGATGCCTTAAGACTTATTGAACAAC	2460

CGGAATTGGCAAGTAAAGTAGACATGGTTTGGATAGTCGGAGGCAGTTCTGTTTACCAGG	2520

AAGCCATGAATCAACCAGGCCACCTTAGACTCTTTGTGACAAGGATCATGCAGGAATTTG	2580

AAAGTGACACGTTTTTCCCAGAAATTGATTTGGGGAAATATAAACTTCTCCCAGAATACC	2640

CAGGCGTCCTCTCTGAGGTCCAGGAGGAAAAAGGCATCAAGTATAAGTTTGAAGTCTACG	2700

AGAAGAAAGACTAACAGGAAGATGCTTTCAAGTTCTCTGCTCCCCTCCTAAAGCTATGCA	2760

TTTTTATAAGACCATGCTAGCTTGAACTTGTTTATTGCAGCTTATAATGGTTACAAATAA	2820

AGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGT	2880

TTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCAACGATAGCTTATCTGTGGGC	2940

GATGCCAAGCACCTGGATGCTGTTGGTTTCCTGCTACTGATTTAGAAGCCATTTGCCCCC	3000

TGAGTGGGGCTTGGAGCACTAACTTTTCTCTTTCAAAGGAAGCAATGCAGAAAGAAAAGC	3060

ATACAAAGTATAAGCTGCCATGTAATAATGGAAGAAGATAAGGTTGTATGAATTAGATTT	3120

ACATACTTCTGAATTGAAACTAAACACCTTTAAATTCTTAAATATATAACACATTTCATA	3180

TGAAAGTATTTTACATAAGTAACTCAGATACATAGAAAACAAAGCTAATGATAGGTGTCC	3240

CTAAAAGTTCATTTATTAATTCTACAAATGATGAGCTGGCCATCAAAATTCCAGCTCAAT	3300

TCTTCAACGAATTAGAAAGAGCAATCTGCAAACTCATCTGGAATAACAAAAAACCTAGGA	3360

TAGCAAAAACTCTTCTCAAGGATAAAAGAACCTCTGGTGGAATCACCATGCCTGACCTAA	3420

AGCTGTACTACAGAGCAATTGTGATAAAAACTGCATGGTACTGATATAGAAACGGACAAG	3480

TAGACCAATGGAATAGAACCCACACACCTATGGTCACTTGATCTTCAACAAGAGAGCTAA	3540

AACCATCCACTGGAAAAAAGACAGCATTTTCAACAAATGGTGCTGGCACAACTGGTGGTT	3600

ATCATGGAGAAGAATGTGAATTGATCCATTCCAATCTCCTTGTACTAAGGTCAAATCTAA	3660

GTGGATCAAGGAACTCCACATAAAACCAGAGACACTGAAACTTATAGAGGAGAAAGTGGG	3720

GAAAAGCCTCGAAGATATGGGCACAGGGGAAAAATTCCTGAATAGAACAGCAATGGCTTG	3780

TGCTGTAAGATCGAGAATTGACAAATGGGACCTCATGAAACTCCAAAGCTATCGGATCAA	3840

TTCCTCCAAAAAAGCCTCCTCACTACTTCTGGAATAGCTCAGAGGCCGAGGCGGCCTCGG	3900

CCTCTGCATAAATAAAAAAAATTAGTCAGCCATGCATGGGGCGGAGAATGGGCGGAACTG	3960

GGCGGAGTTAGGGGCGGGATGGGCGGAGTTAGGGGCGGGACTATGGTTGCTGACTAATTG	4020

AGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGGGGACTTTCCACACCTGGTT	4080

GCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGGGGACTTT	4140

CCACACCCTAACTGACACACATTCCACAGAATTAATTCCCGATCCCGTCGACCTCGAGAG	4200

CTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCC	4260

ACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTA	4320

ACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCA	4380

GCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTC	4440

CGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGC	4500

TCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACAT	4560

GTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTT	4620

CCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCG	4680

AAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTC	4740

TCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGT	4800

GGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAA	4860

GCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTA	4920

TCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAA	4980

CAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAA	5040

CTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTT	5100

CGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTT	5160

TTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGAT	5220

CTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCAT	5280

GAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATC	5340

AATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGC	5400

ACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTA	5460

GATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGA	5520

CCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCG	5580

CAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGC	5640

TAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCAT	5700

CGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAG	5760

GCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGAT	5820

CGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAA	5880

TTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAA	5940

GTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGA	6000

TAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGG	6060

GCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGC	6120

ACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGG	6180

AAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACT	6240

CTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACAT	6300

ATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGT	6360

GCCACCT	6367

TABLE 2


DNA sequence of encoded Y124D-IgG1 fusion molecule in COSFcLink
vector, 6926bp

SEQ ID No:2
GACGTCGACGGATCGGGAGATCGGGGATCGATCCGTCGACGTACGACTAGTTATTAATAG	60

TAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTT	120

ACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATG	180

ACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTAT	240

TTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCT	300

ATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGG	360

GACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGG	420

TTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTC	480

CACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAA	540

TGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTC	600

TATATAAGCAGAGCTGGGTACGTGAACCGTCAGATCGCCTGGAGACGCCATCGAATTCGG	660

TTACCTGCAGATGGGCTGCAGGAATTCCGCATTGCAGAGATAATTGTATTTAAGTGCCTA	720

GCTCGATACAATAAACGCCATTTGACCATTCACCACATTGGTGTGCACCTCCAAGCTTAC	780

CTGCCATGGGTCTCACCTCCCAACTGCTTCCCCCTCTGTTCTTCTTGCTAGCATGTGCCG	840

GCAACTTTGTCCACGGACACAAGTGCGATATCACCTTACAGGAGATCATCAAAACTTTGA	900

ACAGCCTCACAGAGCAGAAGACTCTGTGCACCGAGTTGACCGTAACAGACATCTTTGCTG	960

CCTCCAAGAACACAACTGAGAAGGAAACCTTCTGCAGGGCTGCGACTGTGCTCCGGCAGT	1020

TCTACAGCCACCATGAGAAGGACACTCGCTGCCTGGGTGCGACTGCACAGCAGTTCCACA	1080

GGCACAAGCAGCTGATCCGATTCCTGAAACGGCTCGACAGGAACCTCTGGGGCCTGGCGG	1140

GCTTGAATTCCTGTCCTGTGAAGGAAGCCAACCAGAGTACGTTGGAAAACTTCTTGGAAA	1200

GGCTAAAGACGATCATGAGAGAGAAAGACTCAAAGTGTTCGAGCGGTACCGAGCCCAAAT	1260

CGGCCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGT	1320

CAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGG	1380

TCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACG	1440

TGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCA	1500

CGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGT	1560

ACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAG	1620

CCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGA	1680

CCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCG	1740

TGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGG	1800

ACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGC	1860

AGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGA	1920

AGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGTAGTCTAGAGCTCGCTGATCAGCCTCGA	1980

CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCC	2040

TGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTC	2100

TGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATT	2160

GGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGAACCAGCTGGGGCTC	2220

GAGGGGGGATCTCCCGATCCCCAGCTTTGCTTCTCAATTTCTTATTTGCATAATGAGAAA	2280

AAAAGGAAAATTAATTTTAACACCAATTCAGTAGTTGATTGAGCAAATGCGTTGCCAAAA	2340

AGGATGCTTTAGAGACAGTGTTCTCTGCACAGATAAGGACAAACATTATTCAGAGGGAGT	2400

ACCCAGAGCTGAGACTCCTAAGCCAGTGAGTGGCACAGCATTCTAGGGAGAAATATGCTT	2460

GTCATCACCGAAGCCTGATTCCGTAGAGCCACACCTTGGTAAGGGCCAATCTGCTCACAC	2520

AGGATAGAGAGGGCAGGAGCCAGGGCAGAGCATATAAGGTGAGGTAGGATCAGTTGCTCC	2580

TCACATTTGCTTCTGACATAGTTGTGTTGGGAGCTTGGATAGCTTGGACAGCTCAGGGCT	2640

GCGATTTCGCGCCAAACTTGACGGCAATCCTAGCGTGAAGGCTGGTAGGATTTTATCCCC	2700

GCTGCCATCATGGTTCGACCATTGAACTGCATCGTCGCCGTGTCCCAAAATATGGGGATT	2760

GGCAAGAACGGAGACCTACCCTGGCCTCCGCTCAGGAACGAGTTCAAGTACTTCCAAAGA	2820

ATGACCACAACCTCTTCAGTGGAAGGTAAACAGAATCTGGTGATTATGGGTAGGAAAACC	2880

TGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGACAGAATTAATATAGTTCTCAGT	2940

AGAGAACTCAAAGAACCACCACGAGGAGCTCATTTTCTTGCCAAAAGTTTGGATGATGCC	3000

TTAAGACTTATTGAACAACCGGAATTGGCAAGTAAAGTAGACATGGTTTGGATAGTCGGA	3060

GGCAGTTCTGTTTACCAGGAAGCCATGAATCAACCAGGCCACCTTAGACTCTTTGTGACA	3120

AGGATCATGCAGGAATTTGAAAGTGACACGTTTTTCCCAGAAATTGATTTGGGGAAATAT	3180

AAACTTCTCCCAGAATACCCAGGCGTCCTCTCTGAGGTCCAGGAGGAAAAAGGCATCAAG	3240

TATAAGTTTGAAGTCTACGAGAAGAAAGACTAACAGGAAGATGCTTTCAAGTTCTCTGCT	3300

CCCCTCCTAAAGCTATGCATTTTTATAAGACCATGCTAGCTTGAACTTGTTTATTGCAGC	3360

TTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTC	3420

ACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCAA	3480

CGATAGCTTATCTGTGGGCGATGCCAAGCACCTGGATGCTGTTGGTTTCCTGCTACTGAT	3540

TTAGAAGCCATTTGCCCCCTGAGTGGGGCTTGGGAGCACTAACTTTCTCTTTCAAAGGAA	3600

GCAATGCAGAAAGAAAAGCATACAAAGTATAAGCTGCCATGTAATAATGGAAGAAGATAA	3660

GGTTGTATGAATTAGATTTACATACTTCTGAATTGAAACTAAACACCTTTAAATTCTTAA	3720

ATATATAACACATTTCATATGAAAGTATTTTACATAAGTAACTCAGATACATAGAAAACA	3780

AAGCTAATGATAGGTGTCCCTAAAAGTTCATTTATTAATTCTACAAATGATGAGCTGGCC	3840

ATCAAAATTCCAGCTCAATTCTTCAACGAATTAGAAAGAGCAATCTGCAAACTCATCTGG	3900

AATAACAAAAAACCTAGGATAGCAAAAACTCTTCTCAAGGATAAAAGAACCTCTGGTGGA	3960

ATCACCATGCCTGACCTAAAGCTGTACTACAGAGCAATTGTGATAAAAACTGCATGGTAC	4020

TGATATAGAAACGGACAAGTAGACCAATGGAATAGAACCCACACACCTATGGTCACTTGA	4080

TCTTCAACAAGAGAGCTAAAACCATCCACTGGAAAAAAGACAGCATTTTCAACAAATGGT	4140

GCTGGCACAACTGGTGGTTATCATGGAGAAGAATGTGAATTGATCCATTCCAATCTCCTT	4200

GTACTAAGGTCAAATCTAAGTGGATCAAGGAACTCCACATAAAACCAGAGACACTGAAAC	4260

TTATAGAGGAGAAAGTGGGGAAAAGCCTCGAAGATATGGGCACAGGGGAAAAATTCCTGA	4320

ATAGAACAGCAATGGCTTGTGCTGTAAGATCGAGAATTGACAAATGGGACCTCATGAAAC	4380

TCCAAAGCTATCGGATCAATTCCTCCAAAAAAGCCTCCTCACTACTTCTGGAATAGCTCA	4440

GAGGCCGAGGCGGCCTCGGCCTCTGCATAAATAAAAAAAATTAGTCAGCCATGCATGGGG	4500

CGGAGAATGGGCGGAACTGGGCGGAGTTAGGGGCGGGATGGGCGGAGTTAGGGGCGGGAC	4560

TATGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGG	4620

GGACTTTCCACACCTGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGC	4680

TGGGGAGCCTGGGGACTTTCCACACCCTAACTGACACACATTCCACAGAATTAATTCCCG	4740

ATCCCGTCGACCTCGAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATT	4800

GTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGG	4860

GTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGT	4920

CGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTT	4980

TGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGC	5040

TGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGG	5100

ATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGG	5160

CCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGAC	5220

GCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTG	5280

GAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCT	5340

TTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGG	5400

TGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCT	5460

GCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCAC	5520

TGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGT	5580

TCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTC	5640

TGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCA	5700

CCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGAT	5760

CTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCAC	5820

GTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATT	5880

AAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACC	5940

AATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTG	6000

CCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTG	6060

CTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGC	6120

CAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTA	6180

TTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTG	6240

TTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCT	6300

CCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTA	6360

GCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGG	6420

TTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGA	6480

CTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTT	6540

GCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCA	6600

TTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTT	6660

CGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTT	6720

CTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGA	6780

AATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATT	6840

GTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGC	6900

GCACATTTCCCCGAAAAGTGCCACCT	6926

TABLE 3


DNA sequence of IL4.Y124D/IgG1 fusion molecule coding region,
1164bp

SEQ ID No:3
ATGGGTCTCACCTCCCAACTGCTTCCCCCTCTGTTCTTCCTGCTAGCATGTGCCGGCAAC	60

TTTGTCCACGGACACAAGTGCGATATCACCTTACAGGAGATCATCAAAACTTTGAACAGC	120

CTCACAGAGCAGAAGACTCTGTGCACCGAGTTGACCGTAACAGACATCTTTGCTGCCTCC	180

AAGAACACAACTGAGAAGGAAACCTTCTGCAGGGCTGCGACTGTGCTCCGGCAGTTCTAC	240

AGCCACCATGAGAAGGACACTCGCTGCCTGGGTGCGACTGCACAGCAGTTCCACAGGCAC	300

AAGCAGCTGATCCGATTCCTGAAACGGCTCGACAGGAACCTCTGGGGCCTGGCGGGCTTG	360

AATTCCTGTCCTGTGAAGGAAGCCAACCAGAGTACGTTGGAAAACTTCTTGGAAAGGCTA	420

AAGACGATCATGAGAGAGAAAGACTCAAAGTGTTCGAGCGGTACCGAGCCCAAATCGGCC	480

GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTC	540

TTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACA	600

TGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGAC	660

GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTAC	720

CGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAG	780

TGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAA	840

GGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAG	900

AACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAG	960

TGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCC	1020

GACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGG	1080

AACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGC	1140

CTCTCCCTGTCTCCGGGTAAATGA	1164

TABLE 4


Sequence of encoded IL4.Y124D/IgG1 fusion protein, 387aa

SEQ ID No:4

1	MGLTSQLLPP LFFLLACAGN FVHGHKCDIT LQEIIKTLNS LTEQKTLCTE

51	LTVTDIFAAS KNTTEKETFC RAATVLRQFY SHHEKDTRCL GATAQQFHRH

101	KQLIRFLKRL DRNLWGLAGL NSCPVKEANQ STLENFLERL KTIMREKDSK

151	CSSGTEPKSA DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT

201	CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH

251	QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK

301	NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL

351	TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK*

TABLE 5


DNA sequence of synthetic IgG4 cDNA, 1006bp

SEQ ID No:5
GCTTCCACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAG	60

AGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCG	120

TGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA	180

GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACC	240

TACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCC	300

AAATATGGTCCCCCATGCCCATCATGCCCAGCACCTGAATTTCTGGGGGGACCATCAGTC	360

TTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACG	420

TGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGAT	480

GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTAC	540

CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAG	600

TGCAAGGTCTCCAACAAAGGCCTCCCGTCATCGATCGAGAAAACCATCTCCAAAGCCAAA	660

GGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAG	720

AACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAG	780

TGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCC	840

GACGGATCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGG	900

AATGTCTTCTCATGCTCCGTCATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGC	960

CTCTCCCTGTCTCTGGGTAAATGAGTGTAGTCTAGATCTACGTATG	1006

TABLE 6


DNA sequence of IL4.Y124D/IgG4 fusion molecule coding region,
1149bp

SEQ ID No:6
ATGGGTCTCACCTCCCAACTGCTTCCCCCTCTGTTCTTCCTGCTAGCATGTGCCCGCAAC	60

TTTGTCCACGGACACAAGTGCGATATCACCTTACAGGAGATCATCAAAACTTTGAACAGC	120

CTCACAGAGCAGAAGACTCTGTGCACCGAGTTGACCGTAACAGACATCTTTGCTGCCTCC	180

AAGAACACAACTGAGAAGGAAACCTTCTGCAGGGCTGCGACTGTGCTCCGGCAGTTCTAC	240

AGCCACCATGAGAAGGACACTCGCTGCCTGGGTGCGACTGCACAGCAGTTCCACAGGCAC	300

AAGCAGCTGATCCGATTCCTGAAACGGCTCGACAGGAACCTCTGGGGCCTGGCGGGCTTG	360

AATTCCTGTCCTGTGAAGGAAGCCAACCAGAGTACGTTGGAAAACTTCTTGGAAAGGCTA	420

AAGACGATCATGAGAGAGAAAGACTCAAAGTGCTCGAGCGAGTCCAAATATGGTCCCCCA	480

TGCCCATCATGCCCAGCACCTGAATTTCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCA	540

AAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGAC	600

GTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCAT	660

AATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTC	720

CTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAAC	780

AAAGGCCTCCCGTCATCGATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAG	840

CCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTG	900

ACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGG	960

CAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGATCCTTCTTC	1020

CTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGC	1080

TCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTG	1140

TABLE 7


Sequence of encoded IL4.Y124D/IgG4 fusion protein, 382aa

SEQ ID No:7

1	MGLTSQLLPP LFFLLACAGN FVHGHKCDIT LQEIIKTLNS LTEQKTLCTE

51	LTVTDIFAAS KNTTEKETFC RAATVLRQFY SHHEKDTRCL GATAQQFHRH

101	KQLIRFLKRL DRNLWGLAGL NSCPVKEANQ STLENFLERL KTIMREKDSK

151	CSSESKYGPP CPSCPAPEFL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD

201	VSQEDPEVQF NWYVDGVEVH NAKTKPREEQ FNSTYRVVSV LTVLHQDWLN

251	GKEYKCKVSN KGLPSSIEKT ISKAKGQPRE PQVYTLPPSQ EEMTKNQVSL

301	TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSRLTVDKS

351	RWQEGNVFSC SVMHEALHNH YTQKSLSLSL GK*

TABLE 8


DNA sequence of IgG4 PE variant, 984 bp

GCTAGTACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAG	60
GAGCACCTCCGAG

AGCACgGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACC	120
GGTGACGGTGTCG

TGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGT	180
CCTACAGTCCTCA

GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTT	240
GGGCACGAAGACC

TACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAA	300
GAGAGTTGAGTCC

AAATATGGTCCCCCATGCCCAcCATGCCCAGCgCCTGAaTTtgaGGG	360
GGGACCATCAGTC

TTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGAC	420
CCCTGAGGTCACG

TGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAA	480
CTGGTACGTGGAT

GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTT	540
CAACAGCACGTAC

CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGG	600
CAAGGAGTACAAG

TGCAAGGTCTCCAACAAAGGCCTCCCGTCaTCgATCGAGAAAACCAT	660
CTCCAAAGCCAAA

GGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGA	720
GGAGATGACCAAG

AACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGA	780
CATCGCCGTGGAG

TGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCC	840
CGTGCTGGACTCC
960
GACGGaTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAG	900
GTGGCAGGAGGGG

AATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTA	960
CACACAGAAGAGC

CTCTCCCTGTCTCTGGGTAAATGA	984

TABLE 9


DNA sequence of IL4.Y124D/IgG4 PE fusion molecule coding region,
1149 bp

ATGGGTCTCACCTCCCAACTGCTTCCCCCTCTGTTCTTCCTGCTAGCATGTGCCGGCAAC	60	SEQ ID No:9

TTTGTCCACGGACACAAGTGCGATATCACCTTACAGGAGATCATCAAAACTTTGAACAGC	120

CTCACAGAGCAGAAGACTCTGTGCACCGAGTTGACCGTAACAGACATCTTTGCTGCCTCC	180

AAGAACACAACTGAGAAGGAAACCTTCTGCAGGGCTGCGACTGTGCTCCGGCAGTTCTAC	240

AGCCACCATGAGAAGGACACTCGCTGCCTGGGTGCGACTGCACAGCAGTTCCACAGGCAC	300

AAGCAGCTGATCCGATTCCTGAAACGGCTCGACAGGAACCTCTGGGGCCTGGCGGGCTTG	360

AATTCCTGTCCTGTGAAGGAAGCCAACCAGAGTACGTTGGAAAACTTCTTGGAAAGGCTA	420

AAGACGATCATGAGAGAGAAAGACTCAAAGTGCTCGAGCGAGTCCAAATATGGTCCCCCA	480

TGCCCACCATGCCCAGCgCCTGAATTTGAGGGGGGACCATCAGTCTTCCTGTTCCCCCCA	540

AAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGAC	600

GTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCAT	660

AATGCCAAGACAAAGCCGCGGGAGCAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTC	720

CTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAAC	780

AAAGGCCTCCCGTCaTCgATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAG	840

CCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTG	900

ACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGG	960

CAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGaTCCTTCTTC	1020

CTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGC	1080

TCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTG	1140

GGTAAATGA	1149

TABLE 10


Sequence of encoded IL4.Y124D/IgG4 PE variant
fusion protein, 382aa

1	MGLTSQLLPP LFFLLACAGN FVHGHKCDIT	SEQ ID No:10
	LQEIIKTLNS LTEQKTLCTE

51	LTVTDIFAAS KNTTEKETFC RAATVLRQFY
	SHHEKDTRCL GATAQQFHRH

101	KQLIRFLKRL DRNLWGLAGL NSCPVKEANQ
	STLENFLERL KTIMREKDSK

151	CSSESKYGPP CPPCPAPEFE GGPSVFLFPP
	KPKDTLMISR TPEVTCVVVD

201	VSQEDPEVQF NWYVDGVEVH NAKTKPREEQ
	FNSTYRVVSV LTVLHQDWLN

251	GKEYKCKVSN KGLPSSIEKT ISKAKGQPRE
	PQVYTLPPSQ EEMTKNQVSL

301	TCLVKGFYPS DIAVEWESNG QPENNYKTTP
	PVLDSDGSFF LYSRLTVDKS

351	RWQEGNVFSC SVMHEALHNH YTQKSLSLSL
	GK*

Claims

1. 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.

2. A compound according to claim 1, 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.

3. A compound according to claim 2, wherein the tyrosine naturally occurring at position 124 is replaced by a different natural amino acid.

4. A compound according to claim 3, wherein the tyrosine naturally occurring at position 124 is replaced by aspartic acid.

5. A compound according to claim 1, wherein the immunoglobulin is of the IgG subclass

6. A compound according to claim 5, wherein the constant domain(s) or fragment thereof is the whole or a substantial part of the constant region of the heavy chain of human IgG.

7. A compound according to claim 5, wherein the constant domain(s) or fragment thereof is the whole or a substantial part of the constant region of the heavy chain of human IgG4.

8. A compound according to claim 1, having the amino acid sequence represented by SEQ ID No:4, SEQ ID No:7 or SEQ ID No: 10.

9. A process for preparing a compound according to claim 1, which process comprises expressing DNA encoding said compound in a recombinant host cell and recovering the product.

10. A process according to claim 9, which comprises:

ii) transforming a host cell with said vector;

iv) recovering said compound.

11. A DNA polymer comprising a nucleotide sequence that encodes a compound according to claim 1.

12. A DNA polymer according to claim 11, which comprises or consists of the sequence of SEQ ID No:3, SEQ ID No:6 or SEQ ID No:9.

13. A replicable expression vector comprising a DNA polymer according to claim 11.

14. A host cell transformed with a replicable expression vector according to claim 13.

15. A pharmaceutical composition comprising a compound according to claim 1 and a pharmaceutically acceptable carrier.

16. 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 according to claim 1.