AU2140699A - Novel compounds - Google Patents
Novel compounds Download PDFInfo
- Publication number
- AU2140699A AU2140699A AU21406/99A AU2140699A AU2140699A AU 2140699 A AU2140699 A AU 2140699A AU 21406/99 A AU21406/99 A AU 21406/99A AU 2140699 A AU2140699 A AU 2140699A AU 2140699 A AU2140699 A AU 2140699A
- Authority
- AU
- Australia
- Prior art keywords
- dna
- compound according
- compound
- igg4
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Description
AUSTRALIA
Patents Act COMPLETE
SPECIFICATION
(ORIGINAL)
Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: Name of Applicant: SmithKline Beecham PLC, SmithKline Beecham Corporation Actual Inventor(s): Michael Joseph Browne Kay Elizabeth Murphy Conrad Gerald Chapman Helen Elizabeth Clinkenbeard Peter Ronald Young Allan Richard Shatzman Address for Service: PHILLIPS ORMONDE
FITZPATRICK
Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: NOVEL COMPOUNDS Our Ref 577166 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): -2- NOVEL COMPOUNDS This application is a divisional application of Australian Patent Application 33825/95 the entire contents of which 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.
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).
C %WW40ORDUENNYU\SPEGNK*\3392SMV
DOG
-3- Human 11L4 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. at., (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).
HI S-LYS-CYS-ASP-l LE-TH R-LEU-GLN-GLU-l LE-I LE-LYS-TH R-LEU-AS N- 20 SER-LEU-TH R-GLU-GLN-LYS-TH R-LEU-CYS-TH
R-GLU-LEU-THR-VAL-THR-
ASP-ILE-PHEAAAASER-LYS-.ASN-THR-.THRGLULYSGLU-THR-PHE-
CYS-ARG-ALAALAHRVALLEUARGGLNPHETYRSERHIS-HIS-GLU-
LYS-ASP-TH R-ARG-CYS-LEU-GLY-ALATHRALA-GLN-GLN-PHE-H
IS-ARG-
HIS-LYS-GLNLEULEARGPHELEU-LYS.ARGLEUASPARGASN-LEU-
100
TR-L-E-L-L-E-S-ERCSPOVLLSGUAAAN
110 120
GLN-SER-THR-LEU-GLU-ASN-PHELEU-GLU-ARG-.LEU-.LYSTHR-ILE-MET-
129
ARG-GLU-LYS-YR-SER-LYS-CYS-SER-SER
C WNODENk%~=~M 5 .O The disulphide bridges are between residues 3 and 127, 24 and 65, and 46 and 99. The molecular weight of ILA varies with the extent of glycosylation from (no glycosylation) 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 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 Immunology 139, 1142 (1987)), ILA-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 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 voll73pp 1483-1491, 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 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 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 vo1216pp939-941, 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 Berson B. and Hood L. Nucleic Acids Research vollO, pp 4 0 71-4079, 1982.
Residue 5 of the hinge is changed from cysteine in the published IgG I 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 IgG4, 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 ILA 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 1L4 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 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, I and III 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 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 200-70 0 C, generally in a volume of 50p.l or less with 0.1-10Opg
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 100-370C, 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 50.41 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 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; 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 etal., 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 transgcnic 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 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 appropriate buffer at a temperature of 200-700C, generally in a volume of 50p-l or less with 0.1-10 g DNA.
The recombinant host cell is prepared, in accordance with the invention, by transforming a host cell with a replicable expression vector of the invention under transforming conditions. Suitable transforming conditions are conventional and are described in, for example, Maniatis et al., cited above, or "DNA Cloning" Vol. 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 CaC12 (Cohen et al, Proc. Nat. Acad. Sci., 1973, 69, 2110) or with a solution comprising a mixture of RbCl, MnCl 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.11 D.M. Glover ed. IRL Press 1985; Kaufman, R.J. Z 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. e al., European Patent Application No. 0093619, 1983).
Compounds of the present invention have IL4 and/or 1L13 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 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 113 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 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.
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 L13.
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/IgG chimeric cDNA, the expression of the corresponding protein in a mammalian expression system and its activity are 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, 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.
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 KpnI restriction site, GGTACC) 11 Primers were used at a final concentration of 5ng/.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 0 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 1L4 and was present because the digest was incomplete. The two smaller fragments were produced due to the presence of an EcoRI site within the IL4.Y124D cDNA. The 570bp band was purified by the Geneclean TM procedure, and ligated into Bluescript KS TM which was prepared by digestion with EcoRI and 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. 20p.g recombinant DNA was incubated with 25 units Smai in react buffer 4, at 0 C overnight. 25 units of KpnI were then added to the digest, which was incubated at 37 0 C for 5 hours. The resulting fragment of approximately 580bp was purified by Geneclean T" to generate an IL4.Y124D/SmaI/KpnI fragment.
Construction of IgG 1 coding region The COSFcLink vector (Table 1) contains human IgG 1 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 L. Nucleic Acids Research vollO, pp4 0 7 1 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. 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 pUC 18 vector containing the human IgG 1 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 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 C 3' and GTA CTG CTC CTC CCG CGG CTT TGT CTT G 3' A DNA fragment containing the hinge-CH2 region was amplified from pUC 18-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 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.
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.
SFcR 1Cos4 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 Xbal 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' GCCAATGGACGTCTATAGTTCGATTAA 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: 5gg 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 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 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 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 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, 25tg 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 F- I 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 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.
The IL4.Y124D/IgG chimera inhibited 3 H thymidine incorporation by human peripheral blood T lymphocytes stimulated with 133pM IL4 in a dose dependent manner.
Example 2 IL4.Y124D/IgG4 fusion protein 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 XhoI 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 1:11-18, 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-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.
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 TTT ACC 3' (includes an Xbal 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 100pl. 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 0 C, an annealing step of 1 minute 30 seconds at 50°C, 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 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 Xbal for IgG4 and EcoRV and Xhol 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 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 IIA.Y124D PCR fragments by digesting pDB951 (IL4.Y124D in COSFcLink) with EcoRV and Xbal to remove most of the IL4.Y124D/IgG1 fusion molecule. The only part remaining is approximately 75bp at the 5' end of IL4, which is not present in the IL4.Y124D EcoRV/XhoI fragment produced by PCR amplification. 5.g of pDB951 DNA was digested in a total volume of 30|.l using react 2 buffer (GibcoBRL). The resulting 5.8Kb DNA fragment was purified using the Geneclean TM procedure.
16 The three fragments described (IL4.Y124D EcoRV/XhoI, hinge-CH2- CH3 of IgG4 Xhol/Xbal and the 5.8Kb fragment resulting from EcoRV/Xbal digestion of pDB951) were ligated together to form plasmid pDB952, which encodes the IL4.Y124D/IgG4 fusion protein. The ligation was carried out using a DNA ligation kit from Amersham (product code RPN 1507), incubating the reactions at 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 50pg/ml. Transformants were cultured in Luria Broth (containing ampicillin at 50p.g/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.
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, 25p.g of the appropriate DNA in 0.125M CaC1 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 1 2 .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 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 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 Xhol site as described in Example 2.
A second PCR reaction is performed to amplify the hinge-CH2-CH3 fragment of the human IgG4 heavy chain PE variant. 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 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.Y124D).
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 XhoI and Xbal for the hinge-CH2-CH3 of IgG4 PE fragment or EcoRV and XhoI for the IL4.Y124D fragment, followed by GenecleanTM. 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 690bp is purified by GenecleanTM 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 370bp is purified by GenecleanTM.
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- 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 50gg/ml. Transformants are cultured in Luria Broth (containing ampicillin at 50gg/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.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.
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, 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 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 L4.Y124D/IgG4 PE chimera inhibited 3 H thymidine incorporation by human peripheral blood T lymphocytes stimulated with 133pM IL4 in a dose dependent manner.
Example 4. Mammalian Expression vector containing DNA coding for IL4.Y124D/IgG4
PE.
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 phosphotransferase gene (NEO) inserted between the P-globin promoter and SV40 polyadenylation region for GeneticinTM selection, the DHFR selection cassette inserted between the 3-globin promoter and BGH polydenylation region for methotrexate (MTX) amplification, an ampicillih resistance gene for growth in bacteria, and a SV40 origin of replication.
To insert the IL4.Y124D/IgG4 PE cDNA. the pCDN vector was prepared by digesting with Ndel and BstX1 as follows: 15.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: 15gg of DNA was incubated with 30 units of BstX1 in react 2 at 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 Ndel/BstX and pCDN Ndel/BstX1 fragments were ligated together to form the plasmid pCDN-IL4.Y124D/IgG4 PE. The ligation was achieved using 2 units ofT4 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 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 lgg of DNA inl00ul of DMEM without serum is added to a solution containing 6p. 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. 15itg 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 25gFd. The cells were resupended into growth medium at 2.5 X 104 cells/ml, and 200l of the cell suspension was plated into each well of a 96 well plate. 48 hours later the medium was switched to growth medium containing 400(g/ml G418 (Geneticin). Twenty one days post selection, conditioned medium from the colonies which arose were screened by Elisa assay. The highest expressing colonies were transferred to 24 well plates in order to be scaled up.
22 Table 1. DNA sequence of COSFcLink vector, 6367bp SEQID No:1I GACGTCGACGGATCGGGAGATCGGGGATCGATCCGTCGACGTACGACTAGTTATTATAG TAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTT 120 ACGGTAAATGGCCC GCCTGGCTGACCGCCAACGACCCCCGCCCATTGACGTCAATAATG 180 ACGTATGTITCCCATAGTAACGCCAATAGGGACT TTCCATTGACGTC-AATGGGTGGACTAT 240 TTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATcATATGCCAAGTACGCCCCCT 300 ATTGACGTCAATGACGGTAAATGGCCCGCCTGCCATTATGCCCAGTACATGACCTTATGG 360 GACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGG 420 TTTTGGCAGTACATCAATGGGCCTGG-ATAGCGGTTTGACTCACGGGGATTTCCAAGTCTC 480 CACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAA 540 TGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTC 600 TATATAAGCAGAGCTGGGTACGTGACCGTCAGATCGCCTGGAGACGCCATCGATTCGG 660 TTACCTGCAGATATCAAGCTAATTcGGTAcCGAGCCCAAATCGGCCGACAAAACTcACAC 720 ATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACcGTCAGTCTTCCTCTTCCCCCC 780 AAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGA 840 CGTGAGCCACGAAGACCCTGAGG-TCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCA 900 TAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGT 960 CCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGAGTACAGTGCAGGTCTCA 1020 CAAAGCCCTcccAGCCCCCATcGAGAAAACCATCTCCAAAGccAAAGGGCAGCCCCGAGA 1080 ACCACAGGTGTACACCCTGCCCCCATCCCG-GCATGAGCTGACCAAGAACCAGGTCAGCCT 1140 GACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCATG 1200 GCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTT 1260 CC-TCTACAGC-AAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG.-GGGACGTCTTCTCATG 1320 CTCCGTGATGCATGAGGC-TCTGCACAACCACTCACGCAGAAGAGCCTCTCCCT~GTCTCC 1380 GGGTAAATGAGTGTAGTCTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCA 1440 GCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGAGGTGCCACTCCCAC 1500 TGTCCTTTCCTAATAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTAT 1560 TCGGGTGGGGCGAACAGGGGATGAGCAACGC 1620 TGCTGGGGATGCGGTGGGCTCTATGGACCAGCTGGCTCGAGGATCTCCCGATC 1680 CCACTGTCCATCTTTCAATAAAAAGAATATT 1740 ACACCAATTCAGTAGTTGATTGAGCAAATGCGTTGCCAAAAAGGATGCTTTAGAGACAGT 1800 GTTCTCTGCACAGATALAGGACAAACATTATTCAGAGGGAGTACCCAGAGCTGAGACTCCT 1860 AAGCCAGTGAGTGGCACAGCATTCTAGGGAGATATGCTTGTCATCACCGAAGCCTGAT 1920 TCCGTAGAGCCACACCTTGGTAAGGGCCAATCTGCTCACACAGGATAGAGAGGGCAGGAG 1980 CCAGGGC-AGAGCATATAAGGTGAGGTAGGATCAGTTGCTCCTCACATTTGCTTCTGACAT 2040 AGTTGTGTTGGGAGCTTGGATAGCTTGGACAGCTCAGGGCTGCGATTTCGCGCCACTT 2100 GACGGCAATCCTAGCGTGAAGGCTGGTAGGATTTTATCCCCGCTGCCATCATGGTTCGAC 2160 CATTGAAcTGCATCGTCGCCGTGTcCCAA TATGGGGATTGGCAAGAACGGAGACCTAC 2220 CCTGGCCTCCGCTCAGGACGAGTTCAGTACTTCCAGATGACCACA.CCTCTTCAG 2280 TGGAAGGTAAACAGAATCTGGTGATTATGGGTAGGAAAACCTGGTTCTCCATTCCTGAGA 2340 AGAATCGACCTTTAAAGGACAGATTATATAGTTCTCAGTAGAGCTCAGAACCAC 2400 CACGAGGAGCTCATTTTCTTGCCAAGTTTGGATGATGCCTTAGACTTATTGACAC 2460 CGGAATTGGCAAGTAAAGTAGACATGGTTTGGATAGTCGGAGGCAGTTCTGTTTACCAGG 2520 AGCCATGAATCAACCAGGCCACCTTAGACTCTTTGTGACAAGGATCATGCAGGAATTTG 2580 AAAGTGACACGTTTTTCCCAGAATTGATTTGGGGTATACTTCTCCCAGATACC 2640 CAGGCGTCCTCTCTGAGGTCCAGGAGGAAAAAGGCATCAAGTATAAGTTTGAAGTCTACG 2700 AGAAGAAAGACTAACAGGAAGATGCTTTCAGTTCTCTGCTCCCCTCCTAGCTATGCA 2760 TTTTTATAAGACCATGCTAGCTTGAACTTGTTTATTGCAGCTTATATGGTTACATAA 2820 AGCAATAGCATCACAAATTTCACAAATAAGCATTTTTTTCACTGCATTCTAGTTGTGGT 2880 TTGTCCAAACTCATCAATGTATCTTATCATGTCTGATCACCATAG;CTTATCTGTGG 2940 GATGCCAAGCACCTGGATGCTGTTGGTTTCCT6CTACTGATTTAGAGCCATTTGCCCCC 3000 23 TGAGGGGCTTGGGGCATAA-iTC~e-T-LCAAGGAGCATGCGAAGAAAGC 30 ATCAGAAACGCTTAATGAAGTAGTTTATAAT 3120 ACA-TAC-Tl TTAATTGAAACTAACACCTTATTCTTAATAATACACATTTCATA 3180 TGAAAGTATTTTACATAAGTAACTCAGATACATAGACAGCTATGATAGGTGTCC 3240 CTAAAAGTTCATTTATTAATTCTACATGATGAGCTGGCCATCAATTCCAGCTCAAT 3300 TCTACATAAAACAC-CAATACGATAAAACT= 3360 TAGCAAAAACTCTTCTCAAGGATAAAGACCTCTGGTGGAATCACCATGCCTGACCTAA 3420 AGCTGTACTACAGAGCAATTGTGATAAACTGCATGGTACTGATATAGAACGGACAAG 3480 TAACAGATGACAAA-CATGCCTACTACAAACA 3540 AACCATCCACTGGAAAAAGACAGCATTTTCACATGGTGCTGGCACACTGGTGGTT 3600 ATCATGGAGAAGAATGTGAATTGATCCATTCCTCTCCTTGTACTGGTCWTCTAA 3660 GTGTAGACCAAAACAAGCCGACTTGGAAATG 3720 GAAGCCAGTTGCCGGAAATCTATGAACAGCT 3780 TGCTGTAAGATCGAGAATTGACATGGGACCTCATGCTCCAGCTATCGGATCAA 3840 TTCCTCCAAAAAAGCCTCCTCACTACTTCTGGAATAGCTCAGAGGCCGAGGCGGCCTCGG 3900 CCTCTGCATAAATAAAAAAATTAGTCAGCCATGCATGGGGCGGAGATGGGCGACTG 3960 GGCGGAGTTAGGGGCGGGATGGGCGGAGTTAGGGGCGGGACTATGGTTGCTGACTATTG 4020 AGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGGGGACTTTCCACACCTGGTT 4080 GCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGGGGACTTT 4140 CCACACCCTAACTGACACACATTCCACAGAATTATTCCCGATCCCGTCGACCTCGAGAG 4200 CTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGATTGTTATCCGCTCACAATTCC 4260 ACACAACATACGAGCCGGAAGCATAAGTGTAAGCCTGGGGTGCCTAGAGTGAGCTA 4320 ACTCACATTAATTGCGTTGCGCCACTGCCCGCTTTCCAGTCGGGACCTGTCGTGCCA 4380 GCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTC 4440 CGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGC 4500 TCACTCAAGGCGGTAATACGGTTATCCACAGATCAGGGGATACGCAGGAGAACAT 4560 GTGAGC-AAAAGGCCAGCA;LGGCCAGGACCGTAAAAGGCCGCGTTGCTGGCGTTTTT 4620 CCATAGGCTCCGCCCCCTGACGAGCATCACAATCGACGCTCAGTCAGAGGTGGCG 4680 AACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAGCTCCCTCGTGCGCTC 4740 TCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGT 4800 GGCGCTTTCTCAATGCTCACGCTGTAGGTATCCAGTTCGGTGTAGGTCGTTCGCTCA 4860 GCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTA 4920 TCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAA 4980 CAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAGTGGTGGCCTAA 5040 CTACGGCTACACTAGAGGACAGTATTTGGTATCTGCGCTCTGCTGAGCCAGTTACCTT 5100 CGGAAAAAGAGTTGGTAGCTCTTGATCCGGCACAACCACCGCTGGTAGCGGTGGTTT 5160 TTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAGGATCTCA6GAGATCCTTTGAT 5220 CTTTTCTACGGGGTCTGACGCTCAGTGGACGAACTCACGTTAGGGATTTTGGTCAT 5280 GAGATTATCAAAAGGATCTTCACCTAGATCCTTTT T ATGAGTTT 2 .ATC 5340 AATCTAAAGTATATATGAGTWCTTGGTCTGACAGTTACCATGCTTATCAGTGAGGC 5400 ACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTA 5460 GATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGA 5520 CCCACGCTCACCGGCTCCAGATTTATCAGCATAACCAGCCAGCCGGAAGGGCCGAGCG 5580 CAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTATTGTTGCCGGGAAGC 5640 TAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCCGTTGTTGCCATTGCTACAGGCAT 5-700 CGTGG7GTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGTTCCCACGATCAAG 5760 GCGAGTTACATGATCCCCCATGTTGTGCAAAAGCGGTTAGCTCCTTCGGTCCTCCGAT 5820 CGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAA 5880 TTCTCTTACTGTCATGCCATCCGTAGATGCTTTTCTGTGACTGGTGAGTACTCACCAA 5940 GTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCATACGGGA 6000 TATCGGCCTGAACTAAATCCTATGAAGTTCG 6060 GCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTLCCCACTCGTGC 6120 ACCCAACTGATCTT CAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAACAGG 6180 AAGAAT%-GAAAGGAAGGCAAGAAGTATCCTC 6240 24 CTTCC-TTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACAT 6300 ATTTGAATGTATTTAGAAAAATAAACAAATAG-GG-TTCCGCGCACATTTCCCCGAAAAGT 6360 GCCACCT 6367 Table 2. DNA sequence of encoded Y 1 24D-lgG 1 fusion molecule in COSFcLink vector, 6926bp SEQ ID No:2 GACGTCGACGGATCGGGAGATCGGGGATCGATCCGTCGACGTACGACTAGTTATTAATAG TAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATA-ACTT 120 ACGGTAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCATATG 180 ACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTAT 240 TTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCT 300 ATTGAC:GTCAATGACGGThAATGGCCCGCCTGGCATTATGCC-CAGTACATGACCTTATGG 360 GACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGG 420 TTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAGTCTC 480 CACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAA 540 TGTCGTAACA.ACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTC 600 TATATAAGCAGAGCTGGGTACGTGAACCGTCAGATCGCCTGGAGACGCCATCGATTCGG 660 TTACCTGCAGATGGGCTGCAGGAATTCCGCATTGCAGAGATAATTGTATTTAAGTGCCTA 720 GCTCGATACAATAAACGCCATTTGACCATTCACCACATTGGTGTGCACCTCCAAGCTTAC 780 CTGCCATGGG'ICTCACCTCCCAACTGCTTCCCCCTCTGTTCTTCCTGCTAGCATGTGCCG 840 GCAACTTTGTCCACGGACACAAGTGCGATA'rCACCTTACAGGAGATCATCAAAACTTTGA 900 ACAGCCTCACAGAGCAGAGACTCTGTGCACCGAGTTGAcC-GTACAGAATCTTTGCTG 960 CCTCCAAGAACACAACTGAGAAGGAACCTTCTGCAGGGCTGCGACTGTGCTCCGCGT 1020 TCTACAGCCACCATGAGAAGGACACTCGCTGCCTGGGTGCGACTCACAGCAGTTCCACA 1080 GGAAGACGTCATCG;AGCCAAGACCGGCTGG 1140 GCTATCTTCGGAGACAACGGAGTGAATCTGA 1200 GGCTAAAGACGATCATGAGAGAGAAGACTCAAGTGTTCGAGCGGTACCGAGCCCAA 1260 CGGCCGACAAAACCACACATGCCCACCGTGCCCAGCACCTGACTCCTGGGACCGT 1320 cAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGG 1380 TCACATGCGTGGTGGGACGTGAGCCACGAAGACCCTGAGGTCGTCCTGTACG 1440 TGGACGGCGTGGAGGTGCATATGCCAGACAAGCCGCGGGAGGAGCAGTACACAC 1500 CGTACCGGGTGGTCAGCGTCCTCACCGCCTGCACCAGGACTGGCTGTGGCAGAGT 1560 ACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAG 1620 cCAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGA 1680 CCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCG 1740 TGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGG 1800 ACCGCGTCTTCTTCGAGTACTGCLGGAGGCG 1860 AGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACCGA 1920 AGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGTAGTCTAGAGCTCGCrGATCAGCCTCGA 1980 cTGTGCCTTCTAGTTGCCAGCCAcTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCC 2040 TGAGTCATCATTCTCT.TAAGGAATCTGATT 2100 TGGAGGCTCATTGGGGGGGGCGAACAGGAGT 2160 GGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGAACCAGCTGGGGCTC 2220 GAGGGGGGATCTCCCGATCCCCAGCTTTGCTTCTCTTTCTTATTTGCATATGAGA 2280 AAAAGGAAAATAATTTTAACACCAATTCAGTAGTTGATGAGCTGCGTTGCCAA 2340 AGGATGCTTTAGAGACAGTGTTCCTGCACAGATAGGACACA.TTATTCAGAGGGAGT 2400 ACCCAGAGCTGAGACCCTAAGCCAGTGAGTGACAGCATTCTAAGATATGCTT 2460 GTCATCACCGAAGCCGATTCCGTAGAGCCACACCTTGGTAGGGCCATCTGCTCACAC 2520 25 AGGATAGAGAGGGCAGGAGCCAGGGCAGAGCATATAAGGTGAGGTAGGATCAGTTGCTCC -2580 TCACATTTGCT-TCTGACATAGTTGTGTTGGGAGCTTGGATAGCTTGGACAGCTCAGGGCT 2640 GCGATTTCGCGCCAACTTGACGGCATCTAGCGTGAGGCTGGTAGGATTTTATCCCC 2700 GCTGCCATCATGGTTCGACCATTGAACTGCATCGTCGCCGTGTCCCAAATATGGGGATT 2760 GGCAAGAACGGAACCTACCCTGGCTCCGCTCAGGCGAGTTCAGTACTTCCAGA 2820 ATGACCACAACCCTTCAGTGGGGTACAGA-TCTGGTGATTATGGGTAGGAACC 2880 TGGTTCTCCATTCCGAGAAGATCGACCTTAGGACAGAATTATATAGTTCTCAGT 2940 AGAGAACTCAAAGAACCACCACGAGGAGCTCATTTCTTGCCAAGTTTGGATGATGCC 3000 TTAATATACACGATGAATAGAAAGTTGTGCG 3060 GGCAGTTCTGTTTACCAGGAGCCATGAATCACCAGGCCACCTTAGACTCTTTGTGACA 3120 AGGATCATGCAGGAATTTGAAGTGACACGTTTTCCCAGATTGATTTGGAATAT 3180 AACTTC.GAACAGGCTTTAGCAGGAAAGACA 3240 TAAGTGATTCAAGAGCTAAGAAGTTAGTTTC 3300 CCCCTAGTTCTTTTAACAGTGTGATGTATCG 3360 TTTAGTAAAAACAACTCCATTAAAAACTTTT 3420 ACTGCATTCTAGTTGTGGTTTGTCCACTCATCAATGTATCTTATCATGTCTGGATCAA 3480 CGATAGCTTATCTGTGGGCGATGCCAAGCACCTGGATGCTGTTGGTTTCCTGCTACTGAT 3540 TTAGAAGCCATTTGCCCCCTGAGTGGGGCTTGGGAGCACTCTTTCTCTTTCAGA 3600 GCAGAAAAAGAAAATTAGTCAGATAGAGAAA 3660 GGTTTATAATAAATCGATGACAAACTAATTA 3720 ATATATAACACATTTCATATGWGTATTTTACATAGTACTCAGATACATAGAAAACA 3780 AAGCTAATGATAGGTGTCCCTAAAGTTCATTTATTATTCTACAAATGATGAGCTGGCC 3840 ATAATCACCATTCAGATAAAACACGAATACG 3900 AAACAAACAGTGAAATCTTAGAAAGACCGTG 3960 ATCACCATGCCTGACCTAAGCTGTACACAGAGCTTGTGATAACTGCATGGTAC 4020 4080 TCTACAAACAACACATGAAAGCGATTACATG 4140 GCGCCATGGTACTGGAGAGGATACATCACCT 4200 GTACTAAGGTCATCTAAGTGGATCAGGACTCCACATAACCAGAGACACTGAC 4260 TTTGGAAATGGAACTGAGTTGCCGGAAATCG 4320 ATGAACAGCTTCGAGTCAATGCATGACCTAA 4380 TCCAAAGCTATCGGATCAATTCCTCCAAGCCTCCTCACTACTTCT.GGLAAGCTCA 4440 GAGGCCGAGGCGGCCTCGGCCTCTGCATATAAAAAATTAGTCAGCCATGCATGGGG 4500 CGGAGAATGGGCGGAACTGGGCGGAGTTAGGGGCGGGATGGCGAGTTAGGGGCGAC 4560 TATGGTTGCTGACTAATTGAGATGCATGCTTTGCAACTTCTGCCTGCTGGGGAGCCTG 4620 GGACTTTCCACACCTGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGC 4680 TGGGGAGCCTGGGGACTTTCCACACCCTACTGACACACATTCCACAGATTAATTCCCG 4740 ATCCCGTCGACCTCGAGAGTTGGCGTATCATGGTCATAGCTGTTTCCTGTGTGATT 4800 GTACGTAATCAAAAAAGGCGACTAGGAACTG 4860 GTGCCTAATGAGTGAGCTAACTCACATTA.TTGCGTTGCGCTCACTGCCCGCTT~TCCAGT 4920 CGGGAAACCTGTCGTGCCAGCTGCATTAATGATCGGCCAACGCGCGGGGAGAGGCGGTT 4980 TGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGC 5040 TGCGGCGAGCGGTATCAGCTCACTCAGGCGGTAATACGGTTATCCACAGATCAGGGG 5100 ATAGAGAGAAGGGAAGGCGAAGCAGACTAAG 5160 CCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCTGACGAGCATCACAAATCGAC 5220 GCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAGATACCAGGCGTTTCCCCCTG 5280 GAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCT 5340 TTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATGCTCACGCTGTAGGTATCTCAGTTCGG 5400 TGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCT 5460 GCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAGACACGACTTATCGCCAC 5520 TGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGT 5580 TCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTC 5640 TGTAGCGTCTCGAAGGTGTGTTGTCGAAAAC 5700 CCCGTGGTGTTTGTGAACGAATCCCGAAAGA 5760 26 CTCAAGAAGATCCTTGATCTTTTCTACGGGGCTGACGCTCAGTGGACGAACTCAC 5820 GTTAAGGGATTTTGGTCATGAGATTATCAAAGGATCTTCACCTAGATCCTTTTAAATT 5880 AAAAATGAAGTTTTAAATCAATCTAAGTATATATGAGTACTTGGTCTGACAGTTACC 5940 AATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTG 6000 CCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTG 6060 CTGCAATGATACCGCGAGACCCACGCTCACCGCTCCAGATTTATCAGCATACCAGC 6120 CAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCACTTTATCCGCCTCCATCCAGTCTA 6180 -LATGTCGGACLGGAGAGTGCGTAA7TCCAGT 6240 TTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCT"TCATTCAGCT 6300 CCGGTTCCCAACGATCAGGCGAGTTACATGATCCCCCATGTTGTGCAAGCGGTTA 6360 GCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAGTTGGCCGCAGTGTTATCACTCATGG 6420 TTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGA 6480 CTGGGATACAGCTCGGATGGAGGCACATGTT 6540 GCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGACTTTAAAAGTGCTCATCA 6600 TTGAAGTTCGGGAATTCAGTTACCGTAACAT 6660 CGATGTAACCCACTCGTGCACCCACTGATCTTCAGCATCTTTTACTTTCCCAGCGTTT 6720 CTGTACAACGAGCAAGCGAAAGGAAGGGCCG 6780 ATGTTGAATACTCATACTCTTCCTTTTTCATATTATTGAGCATTTATCAGGGTTATT 6840 GTTAGGGAAAATGAGATTGAATACATGGTCG 6900 GCACATTTCCCCGAAGTGCCACCT 6926 Table 3. DNA sequence of IL4.Yl2tD/IgGI fusion molecule coding region, 1164bp SEQ ID No:3 ATGGGTCTCACCTCCCAACTGCTTCCCCCTCTGTr.TCTTCCTGCTAGCATGTGCCGGCAC TTTGTCCACGGACACAGTGCGATATCACCTTACAGGAGATCATCACTTTGA.ACAC 120 CTCACAGAGCAGAAGACTCTGTGCACCGAGTTGACCGTACAGAATCT.T.TCTGCCTCC 180 AAGAACACAACTGAGAAGGAAACCTTCTGCAGGGCTGCGACT.GTGCTCCGGCAGTTCTAC 240 AGCCAGGAGCCCCGCGGTCATCCGATCAAGA 300 AAGCAGCTGATCCGATTCCTGAAACGGCTCGACAGGAACCTCTGGGGCCTGGCGGGCTTG 360 AATCGCTTAGAGCACGATCTGAACTTGAAGT 420 AAGACGATCATGAGAGAGAAGACTCAGTGTTCGAGCGGTACCGAGCCCATCGGCC 480 GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTC 540 TTCCTCTTCCCCCCAACCCAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACA 600 TGCGTGGTGGTGGACGTGAGCCACGAGACCCTGAGGTCAGTTCAACTGGTACGTGGAC 660 GGCGTGGAGGTGCATATGCCAAGACAAGCCGCGGGAGGAGCAGTACAACAGCACGTAC 720 CGGGTACTCCCGCTCCCGATGTATGAGATCA 780 TGAGTTCAAACCCCGCCCTGGAACTTCAGCA 840 GGGCAGCCCCGAGA~cC-ACAGGTGTAr-CCCTGCCCCCATCCCGGGATGAGCTGACAG 900 AACCAGGTCAGCCTGACCTGCCTGTCAAGGCTTCTATCCCAGCGACATCGCCGTGGAG 960 TGGGGATGCGCGGAACTCAACCCTCGGTGCC 1020 GACGGCTCCTTCTTCCTCTACAGCAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGG 1080 ACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGC 1140 CTCTCCCTGTCTCCGGGTAAATGA 1164 Table 4. Sequence of encoded IL4.Y124DJIgGlI fusion protein, 387aa SEQ lID No:4 1 MGLTSQLLPP LFFLLACAGN FVHGHKCDIT LQEIIKTLNS
LTEQKTLCTE
51 LTVTDIFAAS KNTTEKETFC RAATVLRQFY SHHEKDTRCL
GATAQQFHRR
27 101 NOLJ RFLISAL OP:,Z1MGfLA 1 c.L :ZP VI -Q u L KT lmik srf 151 S:z7I~ A ri'te- 17CPPCP APELr FLPPypIc7 201 rD 1rD PEf'PtFWYvr GVF.VHtlAr7.) PPrEry!~ Ytl-, p,,r 251 A2tG~~ Cy','S'J'ALPA P ~IEY.71 SKA Y~P E Pe),'y7 P P, E 5301 :117- I AVE 't r7 -pPVL": z r r 351 m Ys pwsrr, Avzzx ALHIM 1Y-4 P Y Tahlc 5. DINA scqucnec of synthctic lgG4 cDNA. I (X)6hp SEQ ID GCTCCAGGCACCCrCCC~,CCTCCAGGAC-CGr 6G ACAAC:CC-CGTCTGTAGGCATCCCACGGS;GC 120 TGACCGCCCCCACGCGACCTCCCCGCTCCC.C 180 S ATTCCCTACGGGGGCGGCTC ACTGCAcGAAGAcc 240 TAACCACTGTAACCACACCAGTGCAAATGCC 300 AATTGCCCTCCTAGCACACTAT-,TCGGACTAT 363 TTCGTC CAACCAGCCCCTACCCGC-TAGCC 42 C TCGCTGGAGGGCGACCCCAGCATCA-G-ACAG, 48r GGCGAGCAATCkGCArCC~AGCA-TACGAGA 540 CG GGTACTCCCCCTCCAGAGCPAGCAGGACA 600 TCAXCCACA GCTCtCACACAA'ACCATCTCCAAAGCCAAA 660 GGCCCCAACAAGGAACTCCCTCAG GGTACA 720 AkACC. .,TACTACGCGTAAGCTTCCACAACCGGA 780 TGGGCATGCGCGGAACACAACCCTCCGAC-C 8340 CAGACTCTCCAACGCACCTGCAACG-GCGAZG 900 AAGCTTAGTCTAGACGGTTCCACCAAAAAGG 960 CTTCTTTTGTATATTCCAACACAT 1006 Table 6. DNA sequence of ILA.Y124D/IgG4 fusion molecule codirig region. I 149bp SEQ ID No:6 ATGTTACCCATCTC-CCGTC'CTCLArsGCCA6 TTCCA~4CCATCAACCTACGAAC-CAATTACG 120 CTAAACGAATTTCCGGTACTAAA.TTTCGCC 180 AAACCATAAGAACTTCACGTCATT=CGATCA 240 AGCCAGGAGCCCCGCCTCATCCGATCAAGA 300 AACGTACGTCTAAGCCGCGACTTGGCGCGCT 360 A.ATTCCTGTCCTGTGAAGGAAGCAACCAGAGTACrjrrQ AC.-CTTGGAAAGGCTA 420 AAAGTAGGGGAGCI-AATC.GGCGLCATTGCCC 480 TGCACTCCGACGAT.TGGGACTATTC-GTCCC 540 AACCAGCCCCTACCCGACCGGTAGGGGTGGA 600 GTACAGLGCCCGTCGTACGTCTGTGGGAGGA 660 AAcc~.CAGCCGAGrATCLCGACACT;GTACT 720 CTACTCGACGATCCGAGGAGAT~,GGAAGCCAL 780 AAGCTCGCTGTGGAACTTCAGCACGCGCCAA 840 CCCGTTCCCGCCACCGGGAACCAGACGTACT 900 ACTCTGCAGCTTCCACGCTGCTGGGGGGATG 960 CACGAACATCAACCCTCCTCCATCAGACTCT 1020 CTTCGAGTACTGCAACAGGCGAGGAGCTTAG 1080 -CCGTGA7GCATGAGGCCGCAC?.ACCACTACACACAGAGAGCTCCCTGTCCTCG 1140 GGTAArATGA 1149 28 Taible 7. Sequjcncec ~ericoded 1L4. Y 1 24 1)/IgCG4 I tmon prmein, IX2aj SI:Q 11) No:7 I IIGLT7ZL-PP LFF:2..ACAw,', LQ E: :Y.TLIO LT. c1 v 51 L-7-TD I AAS Y::77EETC PAA7VLPCFY SHHEKDTRCL CATAQ OFHPH PFLrPL DP!ILWGLAGI. ISCPV)KEMPQ STI UFURLL KeTIMP.ErD-' 151 CSSESKYGPP CPSCPAPErL GGPS'I'VLFPP KPK'DTLMISP T.P E V- CVVj 201 150EDPEVOF 14WYI.DGVEVH NAKTKPRE: Fl4STYRWZ5V LTVLMQDWLUi GKEYKCKVSN ?KCLPSSIEKT ISKAKCGQPRE POVYTLPk'SQ EEMTIQJQVSI.
301 TCLVKCrYPS DIAVEWESNO OPENNYKTTF PVLODtGSFF LYSRLTVDK'S 351 PWQEGNVFSC SVHHEALHIIH YTOKSLSLSL ,K- Table 8. DNA scquerncc of IgG4 PE varia-d. 984bp SEQ ID No:8 GCTAGTArCAAGGCCCATCCC77TTCCCC::GGCTCTC7:7Z ACC:.CCGAG ACAgCGCTGCGCG-CAr.C.CTCC:ACGGCGGC 120 TGGAACTCAGGCGCCCTGACCAGCGGCG-GCACACC. 7CCCGCcTCTCCTACG-7CCTCA 180 GGACTCTACTCCCCAGCAGCGTGGTGACCGGCCTCCAGCAGcGGGCACGAC 240 TACACCGCAACTAGACACAAGCCCAGCACACCA QQGACAAGGAGACC 300 AAATATGCTCCCCCATGC :ACCATGCCCAGCqCCTGAsTTtqaGGGGGI.GACCArCAGTC 360 TTCGTCCCAACAWCCCTAIACCCGCCTAGCC 420 TGGGTGGAGGrCAGAACCGG-CA-.CATGAGGA 480 GGGGA-TCTAGCAAAACrCGAGGATCAACC;A 540 CGGGTACTCCCGCTCCCGATGTACGAGATCA 600 TGAG'CCACAGCTCG&aTgTGr.AACTTCAGCA 660 GGCGCCA~CAAGG7CCCTCCCTCAGWGTACA 720 A;CAGCGCGCTC-GTAAGCTTCCAC~-TGC'GA 780 TGAAC.TGCCC;AA'-ACAAGCAGCCCTCGA~- 840 GAGaCTCTCCAACGCACGGAAGGAGGCG~,G 900 AA7GTCT7CTCATGCTCCGTGATGCA7GAGG--CTGCACAACCACTACACCAGAAGAGC 960 CTCTCCCTGTCTCTGGGTAAATGA 984 Table 9. DNA sequence of IL4.Y I24DIIgG4 PE fusion moleculc coding region. I 149bp SEQ ID No:9 ATGTTACCCATCTCC CGTTCTCACTTCGCA TTGCAGAAAGGGTTACTCWGTACAATTACG 120 C:AAACGAATTTCCCGTGCGACGCTTTCGCC 180 AAACCATAAGAACTT--GG%.GGCGGTC-CGTCA 240 AGCCAGGAGCCCCGCCGTCATCCCATCAAGA 300 AACGTACGTCTAAGCCAAGACCLG/'CGCGCT 31S0 AATCGCTCCAGACA CGGAGTGAACT7GGAAAGGCTA 420 AAAGTAGGGGAGCCAGG-CACATCATTGCCC 480 TGCACTCCGgCGATGGGGGCACGCTCGTCCC 540 AACCAGCCCCTACCCGCC-rGTAGGG.GTc-rA 600 GTACAGAACCAGCATCACGTCTGTGGGATC- 660 AATGCCAACACAAGCCGCGGGAGGAGCA::CAACAGCACCTCCGTGGGTCAGC'TC 720 29
CCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAGACCAGGTCAGCCTG
CAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGaTCCTTCTTC CTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGATGTCTTCTCAt1GC
TCCGTGATGCATGAGGCTCTCACACCACTACACACAGAAGAGCCTCTCCCTGTCTCTG
GGTAAATGA
780 840 900 960 1020 1080 1140 1149 Table 10. Sequence of encoded IL4.Y124D/1gG4 PE variant fusion protein, 382aa SEQ DD No: 1 MGLTSQLLPP 51 LTVTDIFAAS 101 KQLIRFLKRL 151 CSSESKYGPP 201 VSQEDPEVQF 251 GKEYKCKVSN 301 TCLVKGFYPS 351 RWQEGNVFSC
LFFLLACAGN
KUTTEKETFC
DRNLWGLAGL
CPPCPAPEFE
NWYVDGVEVH
KGLPSSIEKT
D IAVEWESNG SVMHiEALHNH
FVHGHKCDIT
P.AATVLRQFY
NSCPVKEAZJQ
GGPSVFLFPP
NAKTKPREEQ
ISKAKGQPRE-
QPENNYKTTP
YTQKSLSLSL
LQEI IKTLNS
SHHEKDTRCL
STLENFLERL
KPKDTLMISP.
FNSTYRVVSV
POVYTLPPSQ
PVLDSDGSFF
GK*
LTEQKTLCTE
GATAQQFHRH
KTINP.E1DSK
TPEVTCVVVD
LTVLHQDWUJ
EEMTKNQVSL
LYSRLTVDKS
30
Claims (16)
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.
4. A compound according to claim 3, wherein the tyrosine naturally occurring at position 124 is replaced by aspartic acid. 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 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
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 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) transformning 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 I 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.
14. A host cell transformed with a replicable expression vector according to claim 13. A pharmaceutical composition comprising a compound according to any one of claims 1 to 8 and a pharmaceutical-ly acceptable carrier.
16. A method of treating conditions resulting from undesirable actions of 1L4 and/or EL 13 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 IL 13.
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 hA4 and/or IL 13. DATED: 24 March 1999 PHILLIPS ORMONDE FITZPATRICK Attorneys for: SMITHKRJINE BEECHAM PLC and SMITHKLINE BEECHAM CORPORATION 32
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB9415379 | 1994-07-29 | ||
GB9415379A GB9415379D0 (en) | 1994-07-29 | 1994-07-29 | Novel compounds |
US46829795A | 1995-06-06 | 1995-06-06 | |
US468297 | 1995-06-06 | ||
AU33825/95A AU3382595A (en) | 1994-07-29 | 1995-07-28 | Novel compounds |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU33825/95A Division AU3382595A (en) | 1994-07-29 | 1995-07-28 | Novel compounds |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU11964/02A Division AU1196402A (en) | 1994-07-29 | 2002-01-21 | Novel compounds |
Publications (1)
Publication Number | Publication Date |
---|---|
AU2140699A true AU2140699A (en) | 1999-06-03 |
Family
ID=27153550
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU21406/99A Abandoned AU2140699A (en) | 1994-07-29 | 1999-03-25 | Novel compounds |
Country Status (1)
Country | Link |
---|---|
AU (1) | AU2140699A (en) |
-
1999
- 1999-03-25 AU AU21406/99A patent/AU2140699A/en not_active Abandoned
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US5783181A (en) | Therapeutic uses of fusion proteins between mutant IL 4/IL13 antagonists and immunoglobulins | |
EP0770135A1 (en) | Novel compounds | |
US10829535B2 (en) | Interleukin-2 muteins for the expansion of T-regulatory cells | |
EP0832219A2 (en) | Chimeric leptin fused to immunoglobulin domain and use | |
US5457038A (en) | Natural killer stimulatory factor | |
EP0441900B1 (en) | Natural killer stimulatory factor | |
US20070104680A1 (en) | Antibodies to natural killer stimulatory factor | |
US6433142B1 (en) | Megakaryocyte stimulating factors | |
AU2140699A (en) | Novel compounds | |
AU1196402A (en) | Novel compounds | |
WO2000006596A2 (en) | Interferon alpha hybrids |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
MK5 | Application lapsed section 142(2)(e) - patent request and compl. specification not accepted |