AU1196402A - Novel compounds - Google Patents

Description

AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art:

C

C.

Name of Applicant: SmithKline Beecham PLC and SmithKline Beecham Corporation Actual Inventor(s): Michael Browne, Kay Elizabeth Murphy, Conrad Gerald Clinkenbeard, Peter Ronald Young, Allan Richard Shatzman Chapman, Helen Elizabeth Address for Service: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: NOVEL COMPOUNDS Our Ref: 660909 POF Code: 194607/194607, 85880 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): NOVEL COMPOUNDS This application is a divisional application of Australian Patent Application 21406/99 which in turn is a divisional of Australian Patent Application 33825/95, the entire contents of both earlier applications are herein incorporated by reference.

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.

c Interleukins are secreted peptide mediators of the immune response.

Each of the known interleukins has many effects on the development, 15 activation, proliferation and differentiation of cells of the immune system. IL4 has a physiological role in such functions, bu 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 extrinisic asthma, rhinitis, allergic i 20 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, S: 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).

W:\dskainkspdces\DIV 21406-99doc -3- Human lL-4 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 11L4 and the positions of these disulphide bridges are known (Carr, C. et al., (1991) Biochemistry 30, 1515-1523).

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

20

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

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

50 CYS-ARG-ALA-ALA-TH R-VAL-LEU-ARG-GLN-PHE-TYR-S ER-HIS-H IS-GLU-

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

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 09: GLN-SER-THR-LEU-GLU-ASN-PHE-LEU-GLU-ARG-LEU-LYS-THR-ILE-MET- 30129

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

C WINWOROUENNYM SPECNKM325DIV DOC 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 glycosylaton from (no glycosyladon) to 60KDa or more (hyperglycosylated IL4).

The DNA sequence for human IL4 has also been described by Yokota, T.

er. al., P.N.A.S. 1986 83 5894-5898.

WO 93/10235 describes certain mutants of IL4 which are 1L4 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 15 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 20 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 Mikaclian 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 er al Immunology 139, 1142 (1987)). IL4-stimulated T cell proliferation is inhibited in a dose-dependent manner.

Suitable [L4 mutants are disclosed in WO 93/10235, wherein at least one amino acid, naturally occuring in wild type 14 at any one of positions 120 to 128 4 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 IgG 1, 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 King Bodmer Turner Lawson Roberts 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 vo1173pp148 3 14 9 1 describe the alteration of 15 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 Fcy RI receptor, and substitution of glutamate for leucine in the latter increases the affinity. EP0307434 discloses various mutations including an L to E mutation at 20 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 IgG 1 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 Nature vo1216pp939- 9 4 1, 1967).

SPreferably 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 Berson B. and Hood L. Nucleic Acids Research vollO, pp4071- 4 0 7 9 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 Nature vo1216pp941-942, 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 Buxbaum J. and Hood L., DNA vollppl 1-18, 1981. In one example of a suitable mutation in IgG 4 residue of the hinge (residue 241, Kabat numbering) is altered from serine in the wild type to proline and residue 5 of CH2 (residue 248, Kabat numbering) is altered from leucine in the wild type to glutamate 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: 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.

15 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 m Glover 20 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 6 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 200-700C, generally in a volume of 50.l or less with 0.1-1Opg

DNA.

Enzymatic polymerisation of DNA may be carried out in virro 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°-370C, generally in a volume of 50pl 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 0 C to ambient, generally in a volume of 50pl or less.

The chemical synthesis of the DNA polymer or fragments may be carried out by conventional phosphotriester, phosphite or phosphoramidite chemistry, using solid 15 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 *i Chemie, Weinheim (1982),or in other scientific publications, for example MJ. 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; 20 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. Biernat, 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 reaction technology.

The precise structure of the DNA molecules and the way in which they are obtained depends upon the structure of the desired product. The design of a suitable strategy for the construction of the DNA molecule coding for the compound is a routine matter for the skilled worker in the art.

7 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.

20 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 e 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 :25 appropriate buffer at a temperature of 200-700C, generally in a volume of 50pd or less with 0.1-10pg 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 CaCl 2 (Cohen et al, Proc. Nat. Acad. Sci., 1973, 69, 2110) or with a solution comprising a mixture of RbC1, MnCI 2 potassium acetate and glycerol, and then with 3-[N-morpholino]proparie-sulphonic acid, RbCI and glycerol. Mammalian cells in culture may be transformed by calcium co-precipitation of the vector DNA onto the cells.

8 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 0

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. e al., Molecular and Cellular Biology 5, 1750-1759, 1985; Pavlakis G.N. and Hamer, Proceedings of the National Academy of Sciences (USA) 80, 397-401, 1983; Goeddel, D.V. et al., European Patent Application No. 0093619, 1983).

20 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 IL 3 such as IgE mediated allergic diseases and T cell mediated autoimmune conditions or chronic microbial infection.

The invention therefore further provides a pharmaceutical composition 25 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 1L13 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 Img to 300mg, but 9 typically will be in the region of 1 to 20mg per dose, in one or more doses, such as one to six doses per day, such that the daily dosage is in the range 0.02-40mg/kg.

Compositions suitable for injection may be in the form of solutions, suspensions or emulsions, or dry powders which are dissolved or suspended in a suitable vehicle prior to use.

Fluid unit dosage forms are prepared utilising the compound and a pyrogen-free sterile vehicle. The compound, depending on the vehicle and concentration used, can be either dissolved or suspended in the vehicle. Solutions may be used for all forms of parenteral administration, and are particularly used for intravenous infection. In preparing solutions the compound can be dissolved in the vehicle, the solution being made isotonic if necessary by addition of sodium chloride and sterilised by filtration through a sterile filter using aseptic techniques before filling into suitable sterile vials or ampoules and sealing. Alternatively, if solution rr 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 25 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 I1A4 and/or IL13 which comprises administering to the sufferer an effective amount of a compound of the invention.

10 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/IgGl fusion protein The construction of an IL4.Y124D/IgG1 chimeric cDNA, the expression of the corresponding protein in a mammalian expression system and its activity are 15 described.

1. Construction of DNA coding for fusion protein Construction of IL4.Y124D coding region A variant of the human IL4 gene, which has been described (Kruse, N, Tony, 20 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 I4A cDNA (purchased from British Biotechnology). The II4.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.

To amplify the IL4.Y124D molecule and add convenient restriction sites at each end for subcloning, a PCR reaction was performed using 20ng 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: 1) 5' CGA ACC ACT GAA TTC CGC ATT GCA GAG ATA 3' (includes an EcoRI restriction site, GAATTC) 2) 5' CAC AAA GAT CCT TAG GTA CCG CTC GAA CAC TTT GA 3' (includes a Kpnl restriction site, GGTACC) 11 Pnmers were used at a final concentration of 5ng/p.l, and dNTPs were added at a final concentration of 0.2mM in a total reaction volume of 100. 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 0 C, and an elongation step of 1 minute seconds at 72 0 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 587bp 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 370 C, the reaction was heated to 70 0 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 570bp, 463bp and 100bp The 570bp fragment represents the full-length IL4.Y124D variant of IL4 and was present because the 15 digest was incomplete. The two smaller fragments were produced due to the presence of an EcoRI site within the IL4.Y124D cDNA. The 570bp band was purified by the Geneclean TM procedure, and ligated into Bluescript KS TM which was prepared by digestion with EcoRI and KpnI followed by Geneclean TM. A Bluescript KS+/IL4.Y124D recombinant was thus generated. Large amounts of this 20 recombinant DNA were produced using the Promega "Magic Maxiprep" method.

oThe IL4.Y124D insert was excised from the Bluescript recombinant using SmaI and KpnI. 20pg recombinant DNA was incubated with 25 units SmaI in react buffer 4, at 30 0 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 580bp was purified by Geneclean T to generate an IL4.Y124D/SmaI/KpnI fragment.

S" Construction of IgG1 coding region 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 Berson B. and Hood Nucleic Acids Research vollO, pp 4 0 7 1-4079, 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] The construction of COSFc began with a pUC18 vector containing the human IgG1 cDNA above (pUC18-Fc), which was digested with KpnI and SaclI, deleting the CH1, hinge and part of CH2. The deleted region was replaced with a PCR 12 amplified fragment containing the hinge-CH2 region as follows. Using the following PCR primers: TCG AGC TCG GTA CCG AGC CCA AAT CGG CCG ACA AAA CTC ACA C3' and 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 IgG 1 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 pUC 8Fcmod, and the junctions and PCR amplified region were sequenced for confirmation.

The entire hinge-CH2-CH3 insert in pUC18-Fcmod was removed in a single 20 DNA fragment with KpnI and XbaI, gel purified, and ligated into SFcRlCos4 cut with KpnI and XbaI to create COSFc.

SFcR Cos4 is a derivative of pST4DHFR (Deen, K McDougal, JS, Inacker, R, Folena-Wasserman, G, Arthos, J, Rosenberg, J, Maddon, PJ, Axel, R, and Sweet, RW. Nature 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 3-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.

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: AATTCGGTTACCTGCAGATATCAAGCT 3' 3' GCCAATGGACGTCTATAGTTCGAT'AA 13 The junction was sequenced to confirm orientation in the vector. The size of the final vector is 6.37 kb.

Construction of DNA coding for fusion protein.

To insert the IL4.Y124D cDNA, the COSFcLink vector was prepared by digesting with EcoRV and KpnI as follows: 5pg DNA was incubated with 15 units EcoRV in react 2 at 37 0 C for 5 hours, followed by ethanol precipitation. The resulting DNA was digested with KpnI in react 4 at 37 0 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 14.Y124D/IgG1 fusion protein. The ligation was achieved using an Amersham DNA ligation kit, product code RPN 1507, the reactions being incubated at 16 0

C

overnight. The ligation reaction products were transformed into Promega JM109 15 competent cells (high efficiency) and plated onto Luria Broth agar containing ampicillin at 50pg/ml. Transformants were cultured in Luria Broth (containing ampicillin at 50pg/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( 20 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.

2. Expression of the fusion protein HeLa cells were grown in MEMao medium (Gibco) with 10% foetal calf serum and 1% glutamine. For the assay, 1 x 106 HeLa cells were seeded in RPMI-1640 medium with 10% newborn calf serum, 1% glutamine ("seeding medium"), in a 75cm 2 flask, four days prior to transfection. On the day prior to transfection, a further 12.5mls seeding medium was added to each flask. On the day of transfection, the medium was changed to 15mls of "transfection medium" (MEM medium with Earle's salts containing 10% newborn calf serum and 1% non essential amino acids), at time zero. At time +3 hours, 251ig of the appropriate DNA in 0.125M CaCI 2 lx 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 12.5mls seeding medium containing 5mM sodium butyrate. The next day the cells were washed with PBS (Dulbecco's phosphate buffered saline) and 14 12.5mls "harvest medium" (RPMI-1640 with 2% of a 7.5% stock sodium bicarbonate solution) was added. After a further 24 hour incubation, the supernatants were removed, centrifuged at 1000rpm for 5 minutes to remove cell debris and stored at either 4°C or -20 0

C.

3. Biological Activity For assay of supernatant for IL4 antagonist activity: using the method described in Spits et al., J. Immunology J12, 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.

The IL4.Y124D/IgGI chimera inhibited 3 H thymidine incorporation by human peripheral blood T lymphocytes stimulated with 133pM IL4 in a dose 15 dependent manner.

Example 2 IL4.Y124D/IgG4 fusion protein 20 1. Construction of DNA coding for fusion protein PCR was performed to amplify the IL4.Y124D coding region and introduce a silent nucleotide substitution at the 3' end which creates a XhoI site. As substrate for the PCR reaction 20ng of linearised pDB951 plasmid (Example 1.1(c)) was used. The oligonucleotide primers used were as follows: 1) 5' CAC AAG TGC GAT ATC ACC TTA CAG GAG ATC 3' (includes an EcoRV restriction site, GATATC) 2) 5' CTC GGT ACC GCT CGA GCA CTT TGA GTC TTT 3' (includes a Xhol restriction site, CTCGAG).

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 Buxbaum J. and Hood DNA 111-18, 1981). Numerous silent substitutions were made to the published nucleotide sequence. The gene was assembled by combining two 0.5Kb synthetic DNA fragments. Each 0.5Kb fragment was made by annealing a series of 15 overlapping oligonucleotides and then filling in the gaps by PCR. The two fragments were joined at the SacII site and inserted into the pCR2 vector. A ApaI-BgIIl fragment containing the entire constant region was isolated and ligated into an expression vector, pCD, containing a humanized IL4 specific variable region.

This construct was used as the PCR substrate to amplify the hinge-CH2-CH3 region of IgG4.

The oligonucleotide primers used for amplification of the IgG4 hinge- CH2-CH3 region were as follows: 1) 5' GGT GGA CAA CTC GAG CGA GTC CAA ATA TGG 3' (includes a XhoI restriction site, CTCGAG) 2) 5' TTA CGT AGA TCT AGA CTA CAC TCA TIT ACC 3' (includes an XbaI site, TCTAGA).

The conditions for both PCR reactions were as described for the derivation of pDB951. Briefly, primers were used at 5ng/pl, and dNTPs at a final concentration of 0.2mM in a total reaction volume of 100l1. 2.5 Units of Taq polymerase enzyme from Advanced Biotechnologies were used and 31 cycles of PCR 20 performed. Cycles consisted of a denaturation step of 1 minute at 94C, an annealing step of 1 minute 30 seconds at 50C, and an elongation step of 1 minute 30 seconds at 72 0 C. On cycle 1 denaturation was extended to 5 minutes and on the final cycle elongation was extended to 7 minutes.

PCR products of approximately 700bp (hinge-CH2-CH3 of IgG4) and 25 400bp (IL4.Y124D) were obtained and purified using the Promega "Magic PCR cleanup" kit. The purified PCR reactions were then digested with the following enzymes to create "sticky ends": XhoI and XbaI for IgG4 and EcoRV and XhoI for ILA.Y124D. The digests were incubated at 37 0 C for 3 hours and then ethanol precipitated. The resulting DNAs were analysed by gel electrophoresis and gave sizes of approximately 690bp (hinge-CH2-CH3 of IgG4) and 370bp (IL4.Y124D).

A vector was prepared into which to ligate the hinge-CH2-CH3 of IgG4 and IL4.Y124D PCR fragments by digesting pDB951 (IL4.Y124D in COSFcLink) with EcoRV and Xbal to remove most of the IL4.Y124D/IgGl fusion molecule. The only part remaining is approximately 75bp at the 5' end of IL4, which is not present in the IL4.Y124D EcoRV/XhoI fragment produced by PCR amplification. 5g of pDB951 DNA was digested in a total volume of 30l using react 2 buffer (GibcoBRL). The resulting 5.8Kb DNA fragment was purified using the Geneclean TM procedure.

16 The three fragments described (IL4.Y124D EcoRV/XhoI, hinge-CH2- CH3 of IgG4 XhoI/Xbal and the 5.8Kb 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 160 C overnight. The ligation reaction products were transformed into Promega JM109 competent cells (high efficiency) and plated onto Luria Broth agar containing ampicillin at 50.g/ml. Transformants were cultured in Luria Broth (containing ampicillin at 50pg/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.

S. 2. Expression of the fusion protein HeLa cells were grown in MEMa medium (Gibco) with 10% foetal calf serum and 1% glutamine. For the assay, 1 x 106 HeLa cells were seeded in RPMI-1640 medium with 10% newborn calf serum, 1% glutamine ("seeding medium"), in a 75cm 2 flask, four days prior to transfection. On the day prior to transfection, a further 12.5mls seeding medium was added to each flask. On the day of transfection, the medium was changed to 15mls of "transfection medium" (MEM 25 medium with Earle's salts containing 10% newborn calf serum and 1% non essential amino acids), at time zero. At time +3 hours, 25p.g of the appropriate DNA in 0.125M CaCl 2 lx 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 12.5mls seeding medium containing 5mM sodium butyrate. The next day the cells were washed with PBS (Dulbecco's phosphate buffered saline) and 12.5mls "harvest medium" (RPMI-1640 with 2% of a 7.5% stock sodium bicarbonate solution) was added. After a further 24 hour incubation, the supernatants were removed, centrifuged at 1000rpm for 5 minutes to remove cell debris and stored at either 4 0 C or -20 0

C.

3. Biological Activity For assay of supernatant for IL4 antagonist activity: using the method described in Spits et al., J. Immunology 132, 1142 (1987), human peripheral blood lymphocytes were incubated for three days with phytohaemagluttinin, a T cell 17 mitogen, to upregulate the 1L4 receptor. The resultant blast cells were then stimulated for a further three days with IL4. Proliferation was measured by the incorporation of 3H thymidine.

The IL4.Y124D/IgG4 chimera inhibited 3 H thymidine incorporation by human peripheral blood T lymphocytes stimulated with 133pM IL4 in a dose dependent manner.

Example 3 IL4.Y124D/IgG4 PE fusion protein 1. Construction of DNA coding for fusion protein 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.

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 in the wild type to proline and residue 5 of CH2 (residue 248, Kabat numbering) is altered from leucine in the wild type to glutamate Angal King Bodmer M.W., Turner Lawson Roberts Pedley B. and Adair Molecular Immunology vol30ppl05-108, 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.

The IgG4 PE variant was created using PCR mutagenesis on the synthetic 25 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: referring to Table 8: residue 322 has been altered to in the PE variant from in the wild type; residue 333 has been altered to in the PE variant from in the wild type; and residues 343-344 have been altered to "GA" in the PE variant from "CT" in the wild type.

Oligonucleotide primers are used for amplification of the IgG4 PE variant hinge-CH2-CH3 region as described for the derivation of pDB952.

18 PCR products of approximately 700bp (hinge-CH2-CH3 of IgG4 PE mutant) and 400bp (IL4.Y124D) are obtained and purified using the Promega "Magic PCR cleanup" kit. The purified PCR reactions are then digested with the following enzymes to create "sticky ends": XhoI and XbaI for IgG4 PE and EcoRV and XhoI for IL4.Y124D. The digests are incubated at 37 0 C for 3 hours and then ethanol precipitated. The resulting DNAs are of sizes of approximately 690bp (hinge-CH2- CH3 of IgG4 PE) and 370bp (IL4.Y 124D).

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 TM which is prepared by digestion with either Xhol and Xbal for the hinge-CH2-CH3 of IgG4 PE fragment or EcoRV and XhoI for the IL4.Y124D fragment, followed by Geneclean T M 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 15 Maxiprep" method. The IgG4 PE hinge-CH2-CH3 fragment is excised from the Bluescript recombinant using Xhol and XbaI. The resulting fragment of approximately 690bp is purified by Geneclean T M 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 20 approximately 370bp is purified by Geneclean T M 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.

The three fragments described (IL4.Y124D EcoRV/XhoI, hinge-CH2- 25 CH3 of IgG4 PE variant Xhol/XbaI and the 5.8Kb fragment resulting from -i 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 0 C overnight. The ligation reaction products are transformed into Promega JM109 competent cells (high efficiency) and plated onto Luria Broth agar containing ampicillin at 50p.g/ml. Transformants are cultured in Luria Broth (containing ampicillin at 50p.g/ml) and DNA prepared using Promega "Magic Minipreps". Production of an IL4.Y124D/IgG4 PE variant recombinant DNA is verified by restriction digests, and the complete IL4.Y124D and hinge-CH2-CH3 IgG4 PE variant regions are verified by DNA sequencing. Table 9 describes the sequence of the coding region only of the IL4.Y 124D/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.

19 2. Expression of the fusion protein HeLa cells were grown in MEMa medium (Gibco) with 10% foetal calf serum and 1% glutamine. For the assay, 1 x 106 HeLa cells were seeded in RPMI-1640 medium with 10% newborn calf serum, 1% glutamine ("seeding medium"), in a 75cm 2 flask, four days prior to transfection. On the day prior to transfection, a further 12.5mls seeding medium was added to each flask. On the day of transfection, the medium was changed to 15mls of "transfection medium" (MEM medium with Earle's salts containing 10% newborn calf serum and 1% non essential amino acids), at time zero. At time +3 hours, 25g of the appropriate DNA in 0.125M CaCl2, lx 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 12.5mls seeding medium containing 5mM sodium butyrate. The next day the cells were washed with PBS (Dulbecco's phosphate buffered saline) and 12.5mls "harvest medium" (RPMI-1640 with 2% of a 7.5% stock sodium bicarbonate solution) was added. After a further 24 hour incubation, the supernatants were removed, centrifuged at 1000rpm for 5 minutes to remove cell debris and stored at 20 either 4 0 C or -20 0

C.

3. Biological Activity For assay of supernatant for IL4 antagonist activity: using the method described in Spits et al., J. Immunology 132, 1142 (1987), human peripheral blood lymphocytes were incubated for three days with phytohaemagluttinin, a T cell S. 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.

The IL4.Y124D/IgG4 PE chimera inhibited 3 H thymidine incorporation by human peripheral blood T lymphocytes stimulated with 133pM 114 in a dose dependent manner.

Example 4. Mammalian Expression vector containing DNA coding for IL4.Y124D/IgG4 PE.

1. Construction of DNA The pCDN vector (Aiyar, Baker, Wu, Nambi, Edwards, R.M., Trill, Ellis, Bergsma, D. Molecular and Cellular Biochemistry 131:75-86, 1994) contains the CMV promoter, a polylinker cloning region, and the BGH polyadenylation 20 region. This vector also contains a bacterial neomycin phosphoransferase gene (NEO) inserted between the P-globin promoter and SV40 polyadenylation region for Geneicin T M selection, the DHFR selection cassette inserted between the P-globin promoter and BGH polydenylation region for methotrexate (MTX) amplification, an ampicillin resistance gene for growth in bacteria, and a SV40 origin of replication.

To insert the IL4.Y 124D/IgG4 PE cDNA, the pCDN vector was prepared by digesting with Ndel and BstXl as follows: 15p.g of DNA was incubated with 30 units of BstX1 in react 2 (Gibco-BRL) at 55 0 C for 1 hour, and ethanol precipitated. The resulting DNA was digested with Ndel in react 2 at 37 0 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 Ndel as follows: 15g of DNA was incubated with 30 units of BstX1 in react 2 at 0 C for 1 hour, and ethanol precipitated. The resulting DNA was digested with Ndel in react 2 at 37 0 C for 1 hour, and ethanol precipitated.

i The IL4.Y124D/IgG4 PE Ndel/BstX1 and pCDN Ndel/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 0 C overnight. The ligation reaction products were transformed into Gibco-BRL DH5a competent cells (subcloning efficiency) and plated onto Luria Broth agar containing 75 ug/ml ampicillin. Transformants were cultured in Luria Broth (containing S 20 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.

2. Expression of the Fusion Protein a) Transient Expression in COS COS-1 cells were grown in DMEM medium with 10% fetal bovine serum. For the transfection, cells were seeded at 2 X 105 cells into a 35mm tissue culture dish 24 hours prior. A solution containing lpg of DNA inl00ul of DMEM without serum is added to a solution containing 61 of LIPOFECTAMINE Reagent (Gibco-BRL) in 100pl of DMEM without serum, gently swirled and incubated at room temperature for 45 minutes. The cells are washed once with serum free DMEM. 0.8ml 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 0 C for 5 hours, then Iml of DMEM containing 20% fetal bovine serum is added. The cells are assayed 48-72 hours later to determine expression levels.

21 b) Electroporation into CHO cells CHO cells, ACC-098 (a suspension cell line derived from CHO DG-44, Urlaub, G., Kas, Carothers, A.M. and Chasin, L.A. Cell, 33. 405-412, 1983) were grown in serum free growth medium WO 92/05246. 15.g of the pCDN-IL4.Y124D/IgG4

PE

plasmid was digested using 30 units of Notl at 37 0 C for 3 hours to linearize the plasmid, and precipitated with ethanol. The resulting DNA was resuspended in of 1 X TE (10mM Tris, pH 8.0, 1mM EDTA). The DNA was electroporated into 1 X 107 ACC-098 cells, using a Bio Rad Gene Pulser set at 380V and 25p.Fd. The cells were resupended into growth medium at 2.5 X 104 cells/ml, and 200pl 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 400lg/ml G418 (Genetcin). 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.

000**

C

22 Page(s) 3 -32 are claims pages they appear after the sequence listing Table 1. DNA sequence of COSFcLink vector, 6367bp SEQ ID No: 1 GACGTCGACGGATCGGGAGATCGGGGATCGATCCGTCGACGTACGACTAGTTATTAATAG TAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTT 120 ACGGTAAATGGCCCGCCTGGCTGACCGCCCACGACCCCCGCCCATTGACGTCATATG 3.80 ACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTAT 240 TTACGGTAAACTGCCCACTTGGCAGTACATCAGTGTATCATATGCCAAGTACGCCCCCT 300 ATTGACGTCAATGACGG.TAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGG 360 GACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGG 420 TTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTC 480 CACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCATCCGGGACTTTCCAA 540 TGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTC 600 TATATAGCAGAGCTGGGTACGTGACCGTCAGATCGCCTGGAGACGCCATCGATTCGG 660 TTACCTGCAGATATCAAGCTAATTCGGTACCGAGCCCATCGGCCGACA.ACTCACAC 720 ATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCC 780 -:oAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGA 840 :0%CTACAGAACCGGTAGTACGTAGGAGCTGGTC 900 a :0*TAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGT 960 CCTCACCGTCCTGCACCAGGACTGGCTGAATGGCGGAGTACGTGCAGGTCTCA 1020 so CAAAGCCCTCCCAGCCCCCATCGAGAAACCATCTCCAGCCAAGGGCAGCCCCGAGA 1080 ACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAGACCAGGTCAGCCT 11340 GACCTGCCTGGTCAGGCTTCTATCCCAGCGACATCGCCGTGAGTGGGAGAGCAATGG 1200 *GCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTT 1260 CC-TCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG 1320 CTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC 1380 :GGGTAAATGAGTGTAGTCTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCA 1440 *GCCATCTGTTGTTTGCCCCTCCCCCGGCCTTCCTTGACCCGAGGTGCCACTCCCAC 3.500 TGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGTGTCATTCTAT 1560 TCTGGGGGGTGGGGTGGGGCAGGACAGCAGGGGGAGGATTGGGGACATAGCAGCA 3.620 TGCTGGGGATGCGGTGGCTCTATGGACCAGCTGGGCTCGAGGGGATCTCCCGATC 1680 CCACTGTCCATCTTTCTATAAAAAGA-TATTA 1740 ACACCAATTCAGTAGTTGATTGAGCAATGCGTTGCCAAAGATGCTTTAGAGACAGT 1800 GTTCTCTGCACAGATAAGGACAAACATTATTCAGAGGGAGTACCCAGAGCTGAGACTCCT 1860 AAGCCAGTGAGTGGCACAGCATTCTAGGGAGAAATATGCTTGTCATCACCGAAGCCTGAT 1920 TCCGTAGAGCCACACCTTGGTAAGGGCCAATCTGCTCACACAGGATAGAGAGGGCAGGAG 1980 CCAGGGCAGAGCATATAAGGTGAGGTAGGATCAGTTGCTCCTCACATTTGCTTCTGACAT 2040 AGTTGTGTTGGGAGCTTGGATAGCTTGGACAGCTCAGGGCTGCGATTTCGCGCCACTT 2100 GACGGCAATCCTAGCGTGAAGGCTGGTAGGATTTTATCCCCGCTGCCATCATGGTTCGAC 23.60 CATTGAACTGCATCGTCGCCGTGTCCCAAAATATGGGATTGGCAAGALACGGAGACCTAC 2220 CCTGGCCTCCGCTCAGGACGAGTTCAAGTACTTCCAGATGACCACAACCTCTTCAG 2280 TGGAAGGTAAACAGAATCTGGTGATTATGGGTAGGAAACCTGGTTCTCCATTCCTGAGA 2340 AGAATCGACCTTTAAAGGACAGAATTAAATAGTTCTCAGTAGAGCTCAGAACCAC 2400 CACGAGGAGCTCATTTTCTTGCCAAAGTTTGGATGATGCCTAGACTTATTGAACAAC 2460 CGGAATTGGCAAGTAAAGTAGACATGGTTTGGATAGTCGGAGGCAGTTCTGTTTACCAG 2520 AACAGAC.CAGCCTAATTTTAAGACTCGATT 2580 AAGTGACACGTTTTTCCCAGAATTGATTGGGGAATATACTTCTCCCAGAATACC 2640 CAGGCGTCCTCTCTGAGGTCCAGGAGGAAAAAGGCATCAGTATAGTTTGAAGTCTACG 2700 AGAAGAAAGACTAACAGGAAGATGCTTTCAAGTTCTCTGCTCCCCTCCTAGCTATGCA 2760 TTTTTATAAGACCATGCTAGCTTGAACTTGTTTATTGCAGCTTATTGGTTACTAA 2820 AGCAATAGCATCACAAATTTCACAAATA.AAGCATTTTTTTCACTGCATTCTAGTTGTGGT 2880 TTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCAACGATAGCTTATCTGTGGC 2940 GATGCCAAGCACCTGGATGCTGTTGGTTTCCTGCTACTGATTTAGAAGCCATTTGCCCCC 3000 23 TGAGTGGGGCTTGGGAGCACTAACTTCTC::TTCAAAGGA.AGCAATGCAGAAAGAAAAGC 3060 ATACAAAGTATAAGCTGCCATGTAATAATGGAAGAAGATAAGGTTGTATGATTAGATTT 32.20 ACATACTTTGAATTGAACTAACACTTAATCTTATATATACACATTTCATA 3180 TGAAAGTATTTTACATAAGTAACTCAGATACATAGAAACAGCTATGATAGGTGTCC 3240 CTAAAAGTTCATTTATTAATTCTACAATGATGAGCTGGCCATCAATTCCAGCTCALT 3300 3360 TAGCAAAAkACTCTTCTCAAGGATAAAAGAACCTCTGGTGGAATCACCATGCCTGACCTA 3420 AGCTGTACTACAGAGCAATTGTGATAAAAACTGCATGGTACTGATATAGAAACGGACAAG 3480 TAACAGATGACAAACATGCCTACTACAAACA 3540 AACCATCCACTGGAAAAAAGACAGCATTTTCAACAAATGGTGCTGGCACAACTGGTGGTT 3600 ATCATGGAGAAGAATGTGAATTGATCCATTCCAATCTCCTTGTACTAAGGTCAAATCTAA 3660 GTGGATCAAGGAACTCCACATAAAACCAGAGACACTGAAACTTATAGAGGAGAAAGTGGG 3720 GAAAAGCCTCGAAGATATGGGCACAGGGGAAAAATTCCTGAATAGAACAGCAATGGCTTG 3780 TGCTGTAAGATCGAGAATTGACAAATGGGACCTCATGAAACTCCAAAGCTATCGGATCAA 3840 TTCCTCCAAAAAAGCCTCCTCACTACTTCTGGAATAGCTCAGAGGCCGAGGCGGCCTCGG 3900 CCTCTGCATAAATAAAAA.AAATTAGTCAGCCATGCATGGGGCGGAGAATGGGCGGAACTG 3960 GGCGGAGTTAGGGGCGGGATGGGCGGAGTTAGGGGCGGGACTATGGTTGCTGACTAATTG 4020 AGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGGGGACTTTCCACACCTGGTT 4080 GCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGGGGACTTT 4140 20 CCACACCCTAACTGACACACATTCCACAGAATTAATTCCCGATCCCGTCGACCTCGAGAG 4200 UCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCC 4260 *ACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTA 4320 ACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCA 4380 GCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTC 4440 CGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGC 4500 TCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACAT 4560 GTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTT 4620 CCATAGGCTCCGCICCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCG 4680 :AAACCCGACAGGACTATAAAGATACCAGGCGTT'TCCCCCTGGAAGCTCCCTCGTGCGCTC 4740 TCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGT 4800 GGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGFAGGTCGTTCGCTCCAA 4860 GCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTA 4920 *TCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAA 4980 *CAGGA2TAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAA- 5040 CTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTT 5100 CGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTT 5160 TTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGAT 5220 CTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCAT 5280 .*GAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATC 5340 ALATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGC 5400 ACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTA 5460 GATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGA 5520 CCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCG 5580 CAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTALATTGTTGCCGGGAAGC 5640 TAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCAT 5700 CGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAG 5760 GCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGAT 5820 CGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAA 5880 TTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAA 5940 GTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGA 6000 TAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGG 6060 GCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGC 6120 ACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGG 6180 AAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACT 6240 24

CTTCC

7 TTTTTCAATATTATTGAAGCATTTATCAGGG.TTATTGTCTCATGAGCGGATACAT 6300 ATTTGAATGTATTTAGAAAATACAATAGGGGTTCCGCGCACATTTCCCCGAAGT 6360 GCCACCT 6367 Table 2. DNA sequence of encoded Y 124D- IgGl1 fusion molecule in COS FcLink vector, 6926bp SEQ ID No:2 GACGTCGACGGATCGGGAGATCGGGGATCGATCCGTCGACGTACGACTAGTTATTAATAG TAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTT 120 ACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCATATG 180 ACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCATGGGTGGACTAT 240 TTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCT 300 ATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGG 360 GACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGG 420 TTTTGGCAGTACATCATGGGCGTGGATAGCGGTTTGACTCACGGATTTCCAAGTCTC 480 *CACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCATCACGACTTTCCAA 540 *TGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTC 600 TATATAAGCAGAGCTGGGTACGTGAACCGTCAGATCGCCTGGAGACGCCATCGAATTCGG 660 TTACCTGCAGATGGGCTGCAGGATTCCGCATTGCAGAGATAATTGTATTTAAGTGCCTA 720 GCTCGATACAATAAACGCCATTTGACCATTCACCACATTGGTGTGCACCTCCAAGCTTAC 780 *CTGCCATGGGTCTCACCTCCCACTGCTTCCCCCTCTGTTCTTCCTGCTACATGTGCCG 840 GCAACTTTGTCCACGGACACAAGTGCGATATCACCTTACAGGAGATCATCAACTTTGA 900 ACAGCCTCACAGAGCAGAAGACTCTGTGCACCGAGTTGACCGTAACAGACATCTTTGCTG 960 *.:CCTCCAAGAACACAACTGAGAGGAACCTTCTGCAGGCTGCGACTGTGCTCCGAGT 1020 *TCTACAGCCACCATGAGAAGGACACTCGCTGCCTGGGTGCGACTGCACAGCAGTTCCACA 1080 GGCACAAGCAGCTGATCCGATTCCTGAACGGCTCGACAGGACCTCTGGGCCTGGCGG 1140 GCTTGA.ATTCCTGTCCTGTGAAGGAAGCCAACCAGAGTACGTTGGACTTCTTGGP. 1200 GGCTAAAGACGATCATGAGAGAGAGACTCAAGTGTTCGAGCGGTACCGAGCCAAAT 1260 CGGCCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGCTCCTGGGGACCGT 1320 CAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGG 1380 TCCTCTGGTGCTACAGAGCCGGTAGTACGTC 1440 TGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCA 1500 CGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGACTGGCTGATGGCAAGAGT 1560 :ACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAG.CCATCTCCAG 1620 CCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGA 1680 CCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAGGCTTCTATCCCAGCGACATCGCCG 1740 TGGAGTGGGAGAGCA.ATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGG 1800 ACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGC 1860 AGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGA 1920 AGAGCCTCTCCCTGTCTCCGGGTAA.ATGAGTGTAGTCTAGAGCTCGCTGATCAGCCTCGA 1980 CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCC 2040 TGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGATTGCATCGCATTGTC 2100 TGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCGGGGAGGATT 2160 GGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGAACCAGCTGGGGCTC 2220 GAGGGGGGATCTCCCGATCCCCAGCTTTGCTTCTCATTTCTTATTTGCATATGAGA 2280 AAAAGGAAAATTAATTTTAACACCAATTCAGTAGTTGATTGAGCTGCGTTGCCAA 2340 AGGATGCTTTAGAGACAGTGTTCTCTGCACAGATAAGGACAACATTATTCAGAGGGAGT 2400 ACCCAGAGCTGAGACTCCTAAGCCAGTGAGTGGCACAGCATTCTAGGAGATATGCTT 2460 GTCATCACCGAAGCCTGATTCCGTAGAGCCACACCTTGGTAAGGGCCAATCTGCTCACAC 2520 25 AGGATAGAGAGGGCAGGAGCCAGGGCAGAGCATATAAGGTGAGGTAGGATCAGTTGCTCC 2580 TCACATTTGCTTCTGACATAGTTGTGTTGGGAGCTTGGATAGCTTGGACAGCTCAGGGCT 2640 GCGATTTCGCGCCAAACTTGACGGCAATCCTAGCGTGAAGGCTGGTAGGATTTTATCCCC 2700 GCTGCCATCATGGTTCGACCATTGAACTGCATCGTCGCCGTGTCCCAAATATGGGGATT 2760 GGCAAGAACGGAGACCTACCCTGGCCTCCGCTCAGGCGAGTTCAGTACTTCCAGA 2820 ATGACCACAACCTCTTCAGTGGAAGGTAAACAGAATCTGGTGATTATGGGTAGGAAACC 2880 TGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAGGACAG.TTAATATAGTTCTCAGT 2940 AGAGAACTCAAAGAACCACCACGAGGAGCTCATTTTCTTGCCAAGTTTGGATGATGCC 3000 TTAAGACTTATTGAACAACCGGAATTGGCAAGTAAAGTAGACATGGTTTGGATAGTCGGA 3060 GGCAGTTCTGTTTACCAGGAAGCCATGAATCAACCAGGCCACCTTAGACTCTTTGTGACA 3120 AGGATCATGCAGGAATTTGAAAGTGACACGTTTTCCCAGATTGATTTGGGATAT 3180 AAACTTCTCCCAGAATACCCAGGCGTCCTCTCTGAGGTCCAGGAGGAAAAGGCATCAAG 3240 TATAAGTTTGAAGTCTACGAGAAGAAAGACTAACAGGAAGATTTTCGTTCTCTGCT 3300 CCCCTCCTAAAGCTATGCATTTTTATAAGACCATGCTAGCTTGMACTTGTTTATTGCAGC 3360 TTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACATAGCATTTTTTTC 3420 ACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCAA 3480 CGATAGCTTATCTGTGGGCGATGCCAAGCACCTGGATGCTGTTGG'rTTCCTGCTACTGAT 3540 TTAGAAGCCATTTGCCCCCTGAGTGGGGCTTGGGAGCACTCTTTCTCTTTCAGA 3600 GCAGAAAAAGAAAATTACGCTTAATG.GAAA 3660 GGTTGTATGAATTAGATTTACATACTTCTGAATTGAAACTACACCTTTAAATTCTTAA 3720 ***ATATATAACACATTTCATATGAAAGTATTTTACATAAGTAACTCAGATACATAGAACA 3780 AAGCTAATGATAGGTGTCCCTAAAAGTTCATTTATTAATTCTACATGATGAGCTGGCC 3840 ATCAAATTCCAGCTCAATTCTTCAACGAATTAGAAAGAGCATCTGCACTCATCTGG 3900 AATAACAAAAAACCTAGGATAGCAAAACTCTTCTCAAGGATAGAACCTCTGGTGGA 3960 ATCACCATGCCTGACCTAAAGCTGTACTACAGAGCAATTGTGATAAAAACTGCATGGTAC 4020 TGATATAGAAACGGACAAGTAGACCAATGGAATAGAACCCACACACCTATGGTCACTTGA 4080 TCTTCAACAAGAGAGCTAAACCATCCACTGGAAAAAAGACAGCATTTTCCTGT 4140 GCTGGCACAACTGGTGGTTATCATGGAGAAGAATGTGAATTGATCCATTCCAATCTCCTT 4200 GTACTAAGGTCAAATCTAAGTGGATCAAGGAACTCCACATAAACCAGAGACACTGAC 4260 TTATAGAGGAGAAAGTGGGGAAAAGCCTCGAAGATATGGGCACAGGAAATTCCTGA 4320 *ATAGAACAGCAATGGCTTGTGCTGTAAGATCGAGAATTGACAAATGGGACCTCATGWAC 4380 TCCAAAGCTATCGGATCAATTCCTCCAAAAAAGCCTCCTCACTACTTCTGGATAGCTCA 4440 GAGGCCGAGGCGGCCTCGGCCTCTGCATAAATAAAAAAAATTAGTCAGCCATGCATGG 4500 CGGAGAATGGGCGGAACTGGGCGGAGTTAGGGGCGGGATGGGCGGAGTTAGGGGCGGGAC 4560 TATGGTTGCTGACTAATTGAGATGC-ATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGG 4620 *GGACTTTCCACACCTGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGC 4680 TGGGGAGCCTGGGGACTTTCCACACCCTAACTGACACACATTCCACAGAATTAATTCCCG 4740 ATCCCGTCGACCTCGAGAGCTTGGCGTAATCATGGTCATAGCGTTTCCTGTGTGATT 4800 GTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTGCCTG 4860 GTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGT 4920 CGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTT 4980 TGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGC 5040 TGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATCGGTTATCCACAGAATCAGGGG 5100 ATAACGCAGGAAAGAACATGTGAGCAAAGGCCAGCAAAAGGCCAGGAACCGTAAGG 5160 CCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGAC 5220 GCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTG 5280 GAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCT 5340 TTCTCCCTTCGGGAAGCGTGGCGCTTTCTCALATGCTCACGCTGTAGGTATCTCAGTTCGG 5400 TGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCT 5460 GCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCAC 5520 TGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGT 5580 TCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTC 5640 TGCTGAAGCCAGTTACCTTCGGAAAAGAGTTGGAGCTCTTGATCCGGCCACCA 5700 CCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAGGAT 5760 26 CTCALAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGACGAAAACTCAC 5820 GTTAAGGGATTTTGGTCATGAGATTATCAGGATCTTCACCTAGATCCTTTTATT 5880 AAAAATGAAGTTTTAAATCA.ATCTAAkAGTATATATGAGTAAACTTGGTCTGACAGTTACC 5940 AATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTG 6000 CCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGT'G 6060 CTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCATACCAGC 62.20 CAGCCGGA.AGGGCCGAGCGCAGAAGTGGTCCTGCACTTTATCCGCCTCCATCCAGTCTA 6180 TTATTGCGAACAATATGTGCGTAATTCCAGT 6240 TTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCT 6300 CCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAGCGGTTA 6360 GCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGG 6420 TTATGGCAGCACTGCATAATTCTCTTACTGCATGCCATCCGAGATGCTTTTCTGTGA 6480 CTGGGATACAGCTCGGATGGAGGCACATGTT 6540 GCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGACTTTAAAAGTGCTCATCA 6600 TTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTT 6660 CGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTT 6720 CTGTACAACGAGCAAGCGAAAGGAAGGGCCG 6780 AATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATT 6840 GTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATACAATAGGGGTTCCGC 6900 20 GCACATTTCCCCGAAAAGTGCCACCT 6926 Table 3. DNA sequence of IL4.Y12L D/IgGl fusion molecule coding region, 1 164bp SEQ ID No:3 ATGGGTCTCACCTCCCAACTGCTTCCCCCTCTGTTCTTCCTGCTAGCATGTGCCGGCAAC TTTGTCCACGGACACAAGTGCGATATCACCTTACAGGAGATCATCACTTTGACAGC 120 CTCACAGAGCAGAGACTCTGTGCACCGAGTTGACCGTACAGACATCTTTGCTGCCTCC 180 AAGAACACAACTGAGAAGGAAACCTTCTGCAGGGCTGCGACTGTGCTCCGGCAGTTCTAC 240 .*30 AGCCACCATGAGAAGGACACTCGCTGCCTGGGTGCGACTGCAcAGCAGTTCCACAGGCAC 300 AAGCAGCTGATCCGATTCCTGAAACGGCTCGACAGGAACCTCTGGGGCCTGGCGGGCTTG 360 AATTCCTGTCCTGTGAAGGAAGCCAACCAGAGTACGTTGGAACTTCTTGGGGTA 420 AAGACGATCATGAGAGAGAAAGACTCAAAGTGTTCGAGCGGTACCGAGCCCAAATCGGCC 480 GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTC 540 TTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACA 600 TGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGAC 660 GGCGTGGAGGTGCATA.ATGCCAAGACAA.AGCCGCGGGAGGAGCAGTACACAGCACGTAC 720 **CGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGTGGCAGAGTACAAG 780 *.:TGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAACCATCTCCGCAA 840 GGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAG 900 AACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAG 960 TGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCC 2.020 GACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGG 1.080 AACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGC 11240 CTCTCCCTGTCTCCGGGTAAATGA 1164 Table 4. Sequence of encoded IL4.Y 124D/IgGlI fusion protein, 387aa SEQ ID No: 1 MGLTSQLLPP LFFLLACAGN FVHGHKCDIT LQEIIKTLNS LTEQKTLCTE 51 LTVTDIFAAS KNTTEKETFC RAATVLRQFY SHHEKDTRCL GATAQQFHR- 27 101 KOLJ RFLKP~L 011:1GLA L IILCPVY~F.A~:.'Q~~. 151 .SA DVTII7CPPCP APELLCP,' rLrPpY~r SP TiF"/.

201 (,,r/vDViI iF r) pT.,/rrWtiw, GVT.VI4IAr-7?Y PPrEQYLI.7Y P' V.V:r ~j rEI4. ij ei CPV'.VSJgALPA P ~I~K SYKAr 1YdPPEPOVY7 ''Pf:p Z CL- r 301 'J:7SVL-CL'1Y rrY P .DI AVE WE3fl0,PEIfJ Y Y 7 PPV S Fr. 7 Y: 351 T.VrY P 117/rZ'ZV?*L A L If 11I y LZ.:ZP'Y* Tablc 5. DNA sequecelC of svnhhctic IgG4 cDNA. Il(X)6hp SEQ ID GCTTCCACCMAGGGCCCATCCG'rCr CCCCC'TGGCGCCCTGCTCCAGGAGCACC'.CCGAG AGCACAGCCXCCCGGCTGCCTGTCAAGGACTAC1-rCCCCGAACCGGTGAC' ;TGTCG 120 TGGAACTCAGGCGCCCTGACCAGCGGCGrGCACACCTTCCCGG;CTGTCCThCAGTCC'rCA 18e0 CATCCCGACTG;GCGCCCTCWG CCCCAGC 240 7ACACCTGCAACGTAGATCACAJAGCCAGCAMCACAAGGTGGACAMGAGAGTTGAGTCC 3100 360 42 C *TGCGTGGTGG7GGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAAC7GG7ACGTGGAT 48 GOCGTGGAGGTGCATAATGCCAAGACA AAGCCGCGG'AGGAGCAG7TCAACAGCACGTAC 540 *CGrGTGCTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCT:MCGGCAAGGAGTACAAG 600 TGCA;,GTCTCCAACAAAGGCCTCCCGTCATCGATCGAGAA 'JACCATCTCCAAAGCCAAA 660 GGGCACCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAG 720 AA* AACCGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCACGACATCGCCGTGGAG 780 TGGGAGAGCMATGGGCAGCCGGAGAACAAC-.ACAAGACCACGCCTCCCGTGC-,CZACTCC 840 GACGGATCCTTCTTCCTCTACAGCAGGCTAACCGrGGACAAGAGCAGG7GGCAGGAG3GG 900 AATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAG;%GC 960 :CTCTCCCTGTCTCTGGGTAAATGAGTGTAGTC7AGATCTACG7AA7G 1006 Table 6. DNA sequence of 1L4.Y124D1IgG4 fusion molecule codin~g region, 1 149bp SEQ ID No:6 ATGGG7CTCACCTCCCAACTGCTTCC CC7CTGTTCTTCCTGCTAGCA7(,TGCCGGCAAC TTTGTCCACGG.ACACAAGTGCGATATCACCTTACAGGAGATCATCAAAACTTGAAbCAGC 12£ CTCACAGAGCAGAAGACTCTGTGCACCGAGTTGACCGTAACAGACANTCTTTGCTGCCTCC 180 AAGAACACAACTGAGAAGGAACCTTCTGCAGGGCTGCGACTGTGCTCC:;GCAGTTCTAC 240 *AGCCACCATGAGAAGGACACTCGTGCCGGGTGCGACTGCACAGCAGTTCCACAGGCAC 300 AAGCAGCTGATCCGATTCCTGAAACGGCTCGACAGGAAC:-TCGGOGCCTGGCGGGCT'rG 360 AATTCCTGTCCTGTGAAG.GAAGCCAACCAGAGTACG;TTGGAAAACT.TCT'IGGAAAGGCTA 420 AAGACGATCATGAGAGAGAAAGACTCAAAGTGC-.CGAGCGACGTCCAAATATGGTCCCCCA 480 :GCCCATCATGCCCAGCACCTGAATT'CTGGGGGGACCATCAGTCT'rCC-TGTTCCCCCCA 540 AAACCCAAGGACACTCTCAZGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGA.C 600 GTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCAT 660 AATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTG;TGGTCAGCGTC 720 CTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTAChAGTGCAAGGTCTCCALAC 780 AAAGGCCTCCCGTCA ZGATCGAGAAAACCATC CCAAAGCCAAAGGGCAGCCCCGAGAG 846O CCACAGGTGTACACCCIGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGG'1CAGCCTG 900 ACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCG GGAGTGGGAGAGCALATGGG 960 CAGCCGGQAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGATCCTTCTTC 1020 CTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGG'3GAATGTCTTV1'CATGC 1080 T CCGTGA7GCATGAGGCTCTGCACA.ACCACTACACACAGAAGAGO3CTC7CCCTGTCTCTG 1140 GGTAAATGA 1149 28 Trable 7. .Seq tc ncc of'encoded I L4.Y I 2141)/IgC04 I iji4 n prorein. 3X 2 amj SIEQ 11) No:7 I MC.LTz' L -P r J LACAA; LrEL .T LtI r F 7LC -F.

51 *LVTO I FAS V!?;-EYE7TC PAAT VLP' Fy SHHEY.DTRCL CATAQQFMH r.LI PLYP~L DP.-ILWGLAGI. IISCPVKEAZI-d STLElIVLER. K-a IMPEKeD'W CZS EZKYGPP CPSCPAPErL GGPSVVFLFPP KPKDTUL1I'P 7PEVTCVVV I)201 I/SOEDPEVOF ?JWYDG*,/VH NAKTKPREkQ; FNSTYRWZV LTVLHODWLIU :51 GKEYKCKVSN V.GLPSSIEKT ISKAKGQP&E POVYTLPiSQ EE-MTKNOVSL 301 TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLOSDGSFF LYSRLTVDIKS 351 PWQECNVFSC SVNHEALHIIH YTQXSLSLSL rK Table 8. DNA sequence of lgC4 PE varian~t. 984bp SEQ ID No:8 AGCACgGCCGCCCTGGGCTGCCGGTCAAGG.ACG TCCCG.ACCGOGAG;CGG7UTCG 120 TG*CCGGGCTACACGG*CAAC CCGCCCTAATCC 180 GGCCATCTACGGGTACGGCTCGACTGCCAGC 240 TAACGACTGTAC GCACAACAGTGCAAAGGC 300 AAkATATGGTCCCCCATGC 'ACTCCGgCG&~t&Gvf'ACTAT 360 TTCGTCCCAACAGAATTCTACCCGCCTAGCV- 420 TGGGTGGAGGGCG~GCCCAGCATCATGAGGA 480 -CGAATCCAAAAG~CGAGA#ACACGAGA 540 CGGGTACTCCCGCTCCCGATGTACGAGATCA 600 TGAGTTCAAAGCCC-~T~ACAAACACCAACA- 660 GGCGCCAACAAGCTC CTCCCTCAGWGT.ACA 720 AACGTACTACGCGTAAGCTTCCACAACCGGA 780 GATGGACGAAA-ACAAGACC;CCCGCTGCC 840 GAGaCTCTCCAACGCAACTGCAAAGGCG~YG 900 c'TCTCCCTGTCTCTGGGTAAATCA 984 Table 9. DNA sequence of fL.A.Y1I24D/1gG4 PE fusion molecule coding region, 1 I149bp SEQ ID No:9 ATGTTACCCATCTCC CT-TTCTCACTTCGCA TTGCAGAAAGGGTTACT=WGTACAATTACG 120 C7AAACGAATTTCCGGTGCGACGCTTTCGCC 180 AAACC.CGGAGACTC'CAGCGGCGGTCGATCA 240 AGCCAGGLGAATGTCTGTCATCCGACAAGA 300 AACGTACGTCTAAGCCAAGACCiGGCGCGCT 36~0 ;LTCTTCGGAGAGCACGGACTGAACTTGGAAAGGCTA 420 AAAGTAGGGGAGCCAGGC.CACATCATTGCCC 480 TGCACTCCGgCGATGGGGGCACGCTCCT%-CC 540 AACCAGCCCCTACCCGACCGGTAGGGGTGG 600 GTACA~%GCCGGTCGTACGTCTGTGCGAGGA 660 AATGCCAAACAAGCCGCGGGAGGAGCAG'CAACACACGTACCGTGTGTCAGCGTC 720 29 CTCACGTCTGACCGGATGGTGACGGCLAGAGTCAATGCAGGCTCAAC780 AAGCTCG~TgTGGAACACCAACAAGCGCCAA 840 CCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTG 900 ACCTGCCTGGTCAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGAGAATG 960 CAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGa TCCTTCTTC 1020 CTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGATGTCTTCTCAtGC 1080 TCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTG 1140 GGTAAATGA 1149 .0 Table 10. Sequence of encoded 1L4X1I24D/IgG4 PE variant fusion protein, 382aa SEQ ID No: 1 MGLTSQLLPP 51 LTVTDIFAAS 101 KQLIRFLKRL 151 CSSESKYGPP 201 VSQEDPEVQF 251 GKEYKCKVSN 301 TCLVKGFYPS 351 RWQEGNVFSC

LFFLLACAGN

KNTTEKETFC

DRNLWGLAGL

CPPCPAPEFE

NWYVDGVEVH

I(GLPSSIEKT

D IAVEWESNG

SVMHEALHNH

FVHGHKCD IT

RAATVLRQFY

NSCPVKEANQ

GGP SVFLFPP NAKTKP REEQ

ISKAKGQPRE

QP ENNYKTTP

YTQKSLSLSL

LQEI IKTLNS

SHHEKDTRCL

STLENFLERL

KPKDTLMISR

FNSTYRVVSV

PQVYTLPPSQ

PVLDSDGSFF

GK*

LTEQKTLCTE

GATAQQFHRH

KT IMREKDSK TP EVTCVVVD

LTVLHQDWLN

EFMTKaNQVS L

LYSRLTVDKS

30

Claims (15)

1. A soluble protein having IL4 and/or IL 13 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. S. 15 4. A compound according to claim 3, wherein the tyrosine naturally occurring at position 124 is replaced by aspartic acid. a A compound according to any one of the preceding claims, 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 Shuman 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

9. A process for preparing a compound according to any one of the preceding claims, which process comprises expressing DNA encoding said compound in a recombinant host cell and recovering the product. 31 32 A process according to claim 9, which comprises: 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.

11. A DNA polymer comprising a nucleotide sequence that encodes a compound according to any one of claims 1 to 8.

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. S 15 14. A host cell transformed with a replicable expression vector according to claim 13.

15. A pharmaceutical composition comprising a compound according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.

16. A method of treating conditions resulting from undesirable actions of IL4 S 20 and/or IL13 which comprises administering to the sufferer an effective amount of a compound according to claim 1.

17. A compound according to any one of claims 1 to 8 for use in therapy.

18. A compound according to any one of claims 1 to 8 for use in the treatment of conditions resulting from undesirable actions of IL4 and/or IL3.

19. Use of a compound according to any one of claims 1 to 8 in the manufacture of a medicament for use in the treatment of conditions resulting from undesirable actions of IL4 and/or IL13. DATED: 17 January, 2002 PHILLIPS ORMONDE FITZPATRICK Attorneys for: 4 d SMITHKLINE BEECHAM PC and SMITHKLINE BEECHAM CORPORATION W:ciskaJink\species\DIV 2140-99.doc