HUT76369A - Novel soluble protein compounds - Google Patents

Description

The present invention relates to antagonists of human interleukin 4 (IL4) and / or human interleukin 13 (IL13), which are conditions resulting from undesired effects of IL4 and / or IL13, such as certain IgE-mediated allergic diseases, T-cell mediated autoimmunity. conditions and inadequate immune responses to infectious agents.

Interleukins are mediators of the immune response secretory peptide. Each of the known interleukins has several effects on the development, activation, proliferation and differentiation of cells of the immune system. IL4 plays a physiological role in these functions but can also contribute to the pathogenesis of the disease. IL4, associated with the development of B lymphocytes, results in the production of IgE antibodies that cause specific symptoms of certain allergic diseases such as extrinsic asthma, rhinitis, allergic conjunctivitis, atopic dermatitis and anaphylaxis. IL4 may also function as a general growth and differentiation factor in T lymphocytes, which may promote tissue damage in certain autoimmune conditions such as insulin dependent diabetes mellitus, multiple sclerosis and rheumatoid arthritis, and graft rejection. IL4 may also suppress the development of cell-mediated responses to combat infectious diseases. Antagonizing the effect on IL4 in T4 or B lymphocytes is therefore expected to have a beneficial effect on such diseases. IL13 has recently been identified and found to be a source of a large number of bio-bio · bio bio bio bio bio bio bio bio bio bio bio bio bio bio. ·

3 have logical characteristics similar to IL4 (Minty, A. et al., Natur. 362: 248-250 (1993)); these similar features include, for example, certain aspects of the receptor structure / function relationship [Aversa, G.

et al., J. Exp. Med. 178: 2213-2218 (1993).

Human IL4 consists of a single polypeptide chain of 129 amino acids with 2 possible N-glycosylation sites and 6 cysteines [Le, Η. V. et al., J. Biol. Chem. 263: 10817-10823 (1988)]. The amino acid sequence of IL4 and the positions of said hydrogen bridges are known (Carr, C. et al., 1991, Biochemistry 30, 1515-1523).

HIS-LYS-CYS-ASP-ILE-THR-LEU-GLN-GLU-ILE-LYS-THR-LEU-ASN20

SER-THR-LEU-GLU-GLN-LYS-THR-LEU-THR-CYS-THR-LEU-GLU-VAL-THR40

ASP-ILE-PHE-ALA-ALA-SER-LYS-ASN-THR-THR-GLU-LYS-GLU-THR-PHE50 60

CYS-ALA-ARG-ALA-VAL-THR-LEU-ARG-PHE-TYR-GLN-SER-HIS-HIS-GLU70

LYS-ASP-THR-ARG-CYS-LEU-GLY-ALA-THR-ALA-GLN-GLN-PHE-HIS-ARG80 90

HIS-LYS-ILE-GLN-LEU-ARG-PHE-LEU-LYS-ARG-ARG-LEU ASP ASN LEU100

TRP-GLY-LEU-ALA-GLY-LEU-ASN-SER-CYS-PRO-VAL-LYS-GLU-ALA-ASN110 120 GLN-SER-THR-LEU-GLU-ASN-PHE-LEU-GLU-ARG- LEU-LYS-THR-ILE-MET129

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

The disulfide bridges are 3 and 127, 24 and 65, and 46 and

They are among groups in the V position. The molecular weight of IL4 varies from 15 KDa (no glycosylation) to 60 KDa or more (hyperglycosylated IL4) depending on the degree of glycosylation.

The human IL4 DNA sequence has also been previously described (Yokota, T. et al., 1986, P.N.A.S. 83, 5894-5898).

WO 93/10235. International Patent Application Publication No. 4,647,125 describes IL4 mutants which are IL4 antagonists or partial antagonists.

European Patent Application 0 464 533 discloses fusion proteins comprising various portions of the constant region of an immunoglobulin molecule together with another human protein or a portion thereof.

The present invention relates to a soluble protein having IL4 and / or IL13 antagonist or partial antagonist activity comprising an IL4 mutant or variant linked to at least one human immunoglobulin constant region or fragment thereof.

The term mutant or variant includes any molecule, such as a truncated or other derivative of the IL4 protein, that retains its ability to antagonize IL4 and / or IL13 after internal administration to a human patient. Such other derivatives may be prepared by addition, deletion, substitution or rearrangement of amino acids, or chemical modification.

DNA polymers encoding mutants or variants of IL4 are identified by the · ·

- 19 1) 5 'CGA ACC ACT GAA TTC CGC ATT GCA GAG ATA 3' (includes EcoRI restriction site, GAATTC)

2) 5 'CAC AAA GAT CCT TAG GTA CCG CTC GAA CAC TIT GA 3' (includes a Kpnl restriction site, GGTACC)

The primers were used at a final concentration of 5 ng / μ, and dNTP was added to a total reaction volume of 100 μ 0,2 at a final concentration of 0.2 mM. Thirty-one PCR cycles were performed. The cycles consisted of the following steps: denaturation step at 94 ° C for 1 minute; A step of heating at 50 ° C for 1 minute 30 seconds; and an elongation step at 72 ° C for 1 minute 30 seconds. In the first cycle, the denaturation step duration was increased to 5 minutes, while in the last cycle, the elongation time was extended to 7 minutes. 2.5 units of Taq polymerase enzyme (Advanced Biotechnologies) were used in the PCR reaction. A 587bp PCR product was prepared. The product was purified using the Promega Magic PCR cleanup kit and the purified product was prepared as shown in FIG.

(restriction enzymes were obtained from Gibco BRL). The reaction mixture was heated at 37 ° C for 4 hours 30 minutes, then held at 70 ° C for 10 minutes and then precipitated with ethanol. Agarose gel electrophoretic analysis of the DNA thus obtained showed the presence of three bands of approximately 570bp, 463bp and 100bp. The

The 20 570bp fragment corresponds to the full-length IL4Y124D variant of IL4, which was the result of incomplete digestion. The two smaller fragments are contained in the IL4.Y124D cDNA

It was formed as a result of the EcoRI site. The 570bp band was purified by Geneclean ™. Subsequently, the product prepared by digestion with EcoRI and KpnI and purified by Genecleari ™ was coupled to Blescript KS ^+, ™ to generate a Bluescript KS + / IL4.Y124D recombinant. This recombinant DNA was prepared in large amounts using the Promega Magic Maxiprep procedure. The IL4.Y124D insert was excised from the Bluescript recombinant using Smal and KpnI. Recombinant DNA (20 µg) was incubated with 25 units of SmaI in reaction buffer 4 at 30 ° C overnight. Subsequently, 25 units of KpnI were added to the digest and incubated for 5 hours at 37 ° C. The resulting fragment of approximately 580bp was purified by Geneclean ™, resulting in an IL4.Y124D / SmaI / KpnI fragment.

(b) Structure of the IgG1 coding region

The COSFcLink vector (Table 1) shows corners 1-4 and 6-15, a

It contains the human IgG1 cDNA encoding CH2 1-110 and CH3 1-108 [Ellison J., Berson B. and Hood L. E., Nucleic Acids.

Research 110: 4071-4079 (1982)]. The 5 groups of the corner are changed from cysteine in the described IgG1 sequence to alanine by TGT? GCC modification in the nucleotide sequence. This is the human IgG plasma cell leukemia from ARH-77 • · · · • ·

- 21 (American Type Tissue Collection) was cloned by RT-PCR and confirmed by full sequencing with the known sequence (International Patent Application Publication No. WO 92/00985).

The construction of COSFc started with a pUC18 vector (pUC18-Fc) containing the above human IgG1 cDNA. Digestion with KpnI and SacII broke the CH1, corner region and a portion of CH2. The deleted region was replaced by a PCR amplified fragment containing the corner CH2 region. The following PCR primers were used:

5 'TCG AGC TCG GTA CCG AGC CCA AAT CGG CCG ACA AAA CTC ACA C 3' and

5 'GTA CTG CTC CTC CCG CGG CTT TGT CTT G 3'

A DNA fragment containing the corner-CH2 region was amplified from pUC18-Fc, digested with KpnI and SacII, gel purified and then cloned into the KpnI / SacII digested pUC18-Fc vector. The Cys at 230 positions in the IgG1 heavy chain at Kabat numbering (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). t was changed to Ala by TGT → GCC substitution in the nucleotide sequence. The altered DNA sequence in one of the PCR primers introduced a unique KpnI site at the 5 'end of the heel. The resulting plasmid was designated pUC18Fcmod; switching points and PCR amplified number for proof ··· ·

- 22 caste sequenced.

The complete corner-CH2-CH3 insert in pUC18-Fcmod was removed with KpnI and XbaI in a single DNA fragment, gel purified, and excised with KpnI and XbaI to give SFcR1Cos4, resulting in COSFc.

SFcR1Cos4 is a pST4DHFR [Deen, K., McDougal, J. S., Inacker, R., Folena-Wasserman, G., Arthos, J., Rosenberg, J.,

Maddon, P.J., Axel, R., and Sweet, R.W., Naturre, 331, 82 (1988)], containing insertion of soluble I between the cytomegalovirus (CMV) promoter and the bovine growth hormone (BGH) polyadenylation sites. type Fc receptor (sFcR1), and further comprising: a dihydrofolate reductase (DHFR) cDNA inserted between the β-globin promoter and SV40 polyadenylation sites; an SV40 replication start; and an ampicillin-resistant gene for growth in bacteria. The vector is excised using KpnI and XbaI to remove the coding region for sFcR1 so that the COSFc vector contains the corner-CH2-CH3 region inserted between the CMV promoter and the BGH polyA region.

The COSFcLink vector was constructed from COSFc by inserting an oligonucleotide linker (linker) at the unique EcoRI site of the vector, which regenerates this EcoRI site and simultaneously introduces the BstEII, PstI and EcoRV cloning sites. The oligonucleotides used were:

· «· · 'AATTCGGTTACCTGCAGATATCAAGCT 3' 'GCCAATGGACGTCTATAGTTCGATTAA 5'

- 23 To confirm orientation within the vector, the interface was sequenced. The final vector was 6.27 kb.

(c) Structure of DNA encoding the fusion protein

For insertion of IL4.Y124D cDNA, the COSFcLink vector was prepared by digestion with EcoRV and KpnI as follows. 5 µg of DNA were incubated in reaction buffer # 2 at 37 ° C for 5 hours with 15 units of EcoRV followed by ethanol precipitation. The DNA thus obtained was digested with KpnI in reaction buffer 4 at 37 ° C for 3 hours, followed by ethanol precipitation. The IL4.Y124D / SmaI / KpnI and COSFcLink / EcoRV / KpnI fragments were linked together to create plasmid pDB951; plasmid pDB951 encodes the IL4.Y124D / IgG1 fusion protein. The linkage was performed using an Amersham DNA Link Kit RPN 1507; the reactions were incubated overnight at 16 ° C. The products of the coupling reactions were transformed into Promega JM109 competent cells with high efficiency and then placed on Luria Broth agar containing 50 pg / ml ampicillin. The transformants were grown in Luria Broth containing 50 pg / ml ampicillin and then DNA was prepared using Promega Magic Minipreps. Restriction of IL4.Y124D / COSFcLink Recombinant DNA Formation

- Confirmed by 24 digestions and DNA sequencing. The entire

The IL4.Y124D sequence and the connections with COSFcLink DNA were confirmed by DNA sequencing (Table 2). The coding sequence for recombinant IL4.Y124D / IgG1 DNA is shown in Table 3, while the amino acid sequence of the fusion protein is shown in Table 4. Recombinant IL4Y124D / COSFcLink DNA was prepared and purified using a cesium chloride gradient and used for transient transfection of HeLa cells.

2. Expression of the fusion protein

HeLa cells were grown in MEMa medium (Gibco) supplemented with 10% fetal calf serum and 1% glutamine. Four days before transfection, ⁶ HeLa cells were inoculated into 15 ml of RPMI-1640 medium (inoculum) supplemented with 10% newborn calf serum and 1% glutamine for four days prior to transfection. The day before transfection, an additional 12.5 ml of inoculum was added to each flask. At day 0 on the day of transfection, the medium was replaced with 15 ml of transfection medium (MEM medium with Earle salts containing 10% neonatal calf serum and 1% non-essential amino acids). The +3. hourly, 25 µg of the appropriate DNA in 0.125 M calcium chloride solution and 1xHBS (HEPES buffered saline) were added to the cells. A +7. At 1 hour, cells were treated with 15% glycerol and then incubated overnight in 12.5 ml of inoculum containing 5 mM sodium butyrate. The next day, the cells • w ···· ···· »* •» c · * • · · · · · ## · ·· *

- Washed with 25 PBS (Dulbecco's phosphate buffered saline) and 12.5 ml recovery medium (RPMI-1640 containing 2% 7.5% sodium bicarbonate) was added to the cells. . After an additional 24 hours incubation, the supernatants were removed, centrifuged at 1000 rpm for 5 minutes to remove cell debris, and stored at 4 ° C or -20 ° C.

3. Biological activity

Assay for supernatant IL4 antagonist activity

Spits et al., J. Immunology, 139, 1142 (1987). To upregulate the IL4 receptor, human peripheral blood lymphocytes were incubated for three days with phytohemagglutinin, a T-cell mitogen. The resulting blast cells were then stimulated with IL4 for a further three days. Proliferation was measured by incorporation of ³ H-thymidine.

The IL4.Y124D / IgG1 chimera inhibited the incorporation of ³ H-thymidine in human peripheral blood lymphocytes stimulated with 133 µM IL4.

Example 2

IL4.Y124D / IgG4 fusion protein

1. Construction of DNA encoding a fusion protein

PCR to amplify the IL4.Y124D coding region • ·

26 were completed, followed by a point mutation (silent) nucleotide substitution at the 3 'end of the molecule. As a substrate for the PCR reaction, 20 ng of linearized plasmid pDB951 [1. Example 1 (c)] was used. The oligonucleotide primers used were:

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 an Xhol restriction site, CTCGAG).

A second PCR reaction was performed to amplify the human IgG4 heavy chain corner-CH2-CH3 fragment. In this case, the substrate was a synthetic human IgG4 heavy chain cDNA having the sequence based on GB: HUMIGCD2 Genbank (Ellison J., Buxbaum J. and Hood LE, DNA, 1, 11-18 (1981)). is shown in Table. Several point mutations (silent) have been made relative to the known nucleotide sequence. The gene assembly was performed by combining two 0.5Kb synthetic DNA fragments. Both 0.5Kb fragments were prepared by heat treating a series of overlapping nucleotides and then inserting them into the spaces using PCR. The two 0.5Kb fragments were linked at the SacII site and inserted into the pCR2 vector. A 0.1 Kb Apal-BglII fragment containing the whole constant region was isolated and ligated to a pCD expression vector which contains a humanized IL4 specific variable region. This construct was used as a substrate for PCR to amplify the IgG4 corner-CH2-CH3 region.

The following oligonucleotide printers were used to amplify the IgG4 corner-CH2-CH3 region:

1) 5 'GGT GGA CAA CTC GAG CGA GTC CAA ATA TGG 3' (Includes Xhol Restriction Site, CTCGAG)

2) 5 'TTA CGT AGA TCT AGA CTA CAC TCA TTT ACC 3' (includes Xbal space, TCTAGA).

The reaction conditions for both PCRs are the same as for pDB951. Primers were used at a concentration of 5 ng / μΐ and dNTP at a final concentration of 0.2 mM in a total reaction volume of 100 μΐ. The PCR reaction used 2.5 units of Taq polymerase enzyme (Advanced Biotechnologies) and performed 31 PCR cycles. Each cycle consisted of the following steps: denaturation step at 94 ^e C. for 1 min; ^This tempering step carried out at 50 C for 1 min 30 sec; and an elongation step at 72 ° C for 1 minute 30 seconds. In the first cycle, the denaturation step duration was increased to 5 minutes, while in the last cycle, the elongation time was extended to 7 minutes.

One is approximately 700bp (IgG4 corner-CH2-CH3) and one is

A 400bp (IL4.Y124D) PCR product was prepared. The products were purified using the Promega Magic PCR cleanup kit. Cleaned space to create adhesive ends • ·

- 28 mice were digested with Xhol and XbaI for IgG4 and EcoRV and XhoI for IL4.Y124D. The reaction mixture was incubated for 3 hours at 37 ^e C, followed by ethanol precipitation. Agarose gel electrophoretic analysis of the resulting DNAs revealed the presence of two bands of approximately 690bp (IgG4 corner-CH2-CH3) and 370bp (IL4.Y124D).

A vector was constructed to which the IgG4 corner-CH2-CH3 and IL4.Y124D PCR fragments were ligated. The vector was constructed by digesting pDB951 (IL4.Y124D in COSFcLink) with EcoRV and XbaI to remove most of the IL4.Y124D / IgG1 fusion molecule. The only residue is approximately 75bp at the 5 'end of IL4, which is not present in the EcoRV / XhoI fragment generated by PCR amplification. Reagent # 2 (GibcoBRL) was digested with 5 µg of pDB951 DNA in a total volume of 30 μΐ. The resulting 5.8Kb DNA fragment was purified using Geneclean ™.

The three fragments (i.e., the IL4.Y124D EcoRV / XhoI, the IgG4 XhoI / XbaI angle-CH2-CH3 fragment and the 5.8Kb fragment obtained by digestion of pDB951 EcoRV / XbaI) were ligated together to form plasmid pDB952; plasmid pDB952 encodes the IL4.Y124D / IgG4 fusion protein. The linkage was performed using an Amersham DNA Link Kit RPN 1507; the reactions were incubated overnight at 16 ° C. The products of the coupling reactions were transformed into Promega JM109 competent cells with high efficiency and then transferred to Luria Broth agar containing 50 μg / ml ampicillin. The transformants were cultured in Luria Broth containing 50 μg / ml ampicillin and DNA was prepared using Promega Magic Minipreps. The formation of IL4.Y124D / IgG4 recombinant DNA was confirmed by restriction digestion. The entire IL4.Y124D and IgG4 corner-CH2-CH3 sections were determined by DNA sequencing. The sequence of the coding region of the IL4.Y124D / IgG4 fusion molecule is shown in Table 6, while the amino acid sequence of the fusion protein is shown in Table 7. Recombinant IL4Y124D / IgG4 DNA was prepared and purified using a cesium chloride gradient and used for transient transfection of HeLa cells.

2. Expression of the fusion protein

HeLa cells were grown in MEMa medium (Gibco) supplemented with 10% fetal calf serum and 1% glutamine. Four days before transfection, ⁶ HeLa cells were inoculated into 15 ml of RPMI-1640 medium (inoculum) supplemented with 10% neonate calf serum and 1% glutamine for four days prior to transfection. The day before transfection, an additional 12.5 ml of inoculum was added to each flask. At day 0 on the day of transfection, the medium was replaced with 15 ml of transfection medium (MEM medium containing Earle salts containing 10% neonatal calf serum and 1% non-essential amino acids). The +3. 1 hour in 0.125 M calcium chloride solution

25 μg of DNA and 1xHBS (HEPES buffered saline) were added to the cells. A +7. at hour

30 cells were treated with 15% glycerol and then incubated overnight in 12.5 ml of inoculum containing 5 mM sodium butyrate. The next day, the cells were washed with PBS (Dulbecco's phosphate buffered saline) and then in 12.5 mL of recovery medium (RPMI-1640 containing 2% 7.5% sodium bicarbonate). added to the cells. After an additional 24 hours incubation, the supernatants were removed, centrifuged at 1000 rpm for 5 minutes to remove cellular debris, and stored at 4 ° C or -20 ° C.

3. Biological activity

Supernatant IL4 antagonist activity was assayed using the method of Spits (Spits et al., 1987, J. Immunology, 139, 1142). To upregulate the IL4 receptor, human peripheral blood lymphocytes were incubated for three days with phytohemagglutinin, a T-cell mitogen. The resulting blast cells were then stimulated with IL4 for a further three days. Proliferation was measured by incorporation of H-thymidine.

The chimeric IL4.Y124D / IgG4 inhibited the incorporation of ³ H-thymidine in human peripheral blood T lymphocytes stimulated with 133 µM IL4.

- 31 Example 3

IL4.Y124D / IgG4 PE fusion protein

1. Construction of DNA encoding a fusion protein

To amplify the coding region of IL4.Y124D, a PCR was performed followed by a Xhol site point mutation (silent) nucleotide substitution at the 3 'end, as described in Example 2.

A second PCR reaction was performed to amplify the human IgG4 heavy chain PE variant corner-CH2-CH3 fragment. In IgG4 PE, the 10 groups of the heel (241 Kabat numbered groups) are replaced by proline (P) instead of the wild-type serine (S), while the 5 groups of CH2 (the

Kabat pity group 248) has been changed from wild-type leucine (L) to glutamate (E). Angal et al., Described an IgG4 molecule in which Kabat numbered group 241 is replaced by proline (Angal, S., King, D.J., Bodmer, M.W., Turner, A., Lawson, ADG, Róbert, G., Pedley, B., and Adair, R., Molecular Immunology, 30, 105-108 (1993). This exchange increases the serum half-life of the IgG4 molecule.

The IgG4 PE variant was generated by PCR mutagenesis with the synthetic human IgG4 heavy chain cDNA described in Table 5, and linked to the pCD expression vector. This plasmid was used as a substrate in the PCR reaction used to amplify the PE-CH2-CH3 fragment of IgG4 PE. The sequence of the PE variant IgG4 is shown in Figure 8.

• ·

- Table 32. The groups of the IgG4 nucleotide sequence that have been altered to produce the PE variant are as follows in Table 8:

group 322 was changed from wild-type T to C in the PE variant;

group 333 was changed from wild-type A to G in the PE variant; and groups 343-344 from CT in the wild type

Replaced with GA in PE variant.

The same oligonucleotide primers as described for pDB952 were used to amplify the IgG4 PE variant corner-CH2-CH3 region.

A PCR product of approximately 700bp (IgG4 PE mutant corner-CH2-CH3) and 400bp (IL4.Y124D) was prepared. The products were purified using the Promega Magic PCR cleanup kit. The purified products were digested with Xhol and XbaI for IgG4 PE and EcoRV and XhoI for IL4.Y124D to form the adhesive ends. The reaction mixture was incubated for 3 hours at 37 ° C and then precipitated with ethanol. Agarose gel electrophoretic analysis of the DNAs thus obtained showed the presence of two bands of approximately 690bp (IgG4 PE corner-CH2-CH3) and 370bp (IL4.Y124D).

The IgG4 PE variant is a corner-CH2-CH3 fragment and

To generate larger amounts of the IL4.Y124D fragment, the purified and digested PCR products were coupled to Bluescript KS ⁺ ™, which was prepared by using XhoI and XbaI for the IgG4 PE corner-CH2-33 -CH3 fragment and

The IL4.Y124D fragment is digested with EcoRV and Xhol and purified by Geneclean ™. Accordingly, a Bluescript KS ⁺ / IgG4 PE corner-CH2-CH3 recombinant and a Bluescript KS ⁺ /IL4.Y124D recombinant are generated. These DNAs are prepared in large quantities using the Promega Magic Maxiprep procedure. The IgG4 PE corner-CH2-CH3 fragment was excised using XhoI and XbaI

Bluescript from recombinant. The resulting approximately 690bp fragment was purified by Geneclean® ^{4 to} obtain a large amount of the IgG4 PE corner-CH2-CH3 XhoI / XbaI fragment. The IL4.Y124D fragment was excised from the Bluescript recombinant using EciRV and XhoI. The resulting fragment of approximately 370bp was purified by Geneclean ™.

A vector was constructed to which the IgG4 PE corner-CH2-CH3 and IL4.Y124D fragments were ligated. The vector was constructed by digesting pDB951 with EcoRV and XbaI as described for pDB952.

The three fragments (i.e., the IL4.Y124D EcoRV / XhoI, the XhoI / XbaI corner-CH2-CH3 fragment of the IgG4 PE variant, and the 5.8Kb fragment obtained by digestion of pDB951 EcoRV / XbaI) were ligated together to form plasmid pDB953. The linkage was performed using an Amersham DNA Link Kit RPN 1507; the reactions were incubated overnight at 16 ° C. The products of the coupling reactions with high efficiency into Promega JM109 competent cells • ·

34 transformed and placed on Luria Broth agar containing 50 µg / ml ampicillin. Transformants were grown in Luria Broth containing 50 µg / ml ampicillin and DNA was prepared using Promega Magic Minipreps. The formation of recombinant DNA for the PE variant IL4.Y124D / IgG4 was confirmed by restriction digestion. The complete IL4.Y124D and IgG4 PE variant variant CH2-CH3 region was determined by DNA sequencing. The sequence of the coding region of the PE fusion molecule IL4.Y124D / IgG4 is shown in Table 9, while the amino acid sequence of the fusion protein is shown in Table 10. The

Recombinant IL4Y124D / IgG4 PE was prepared and purified using a cesium chloride gradient and used for transient transfection of HeLa cells.

2. Expression of the fusion protein

HeLa cells were grown in MEMa medium (Gibco) supplemented with 10% fetal calf serum and 1% glutamine. A g

four days prior to transfection, 1 x 10 HeLa 2 cells were inoculated into 15 ml of RPMI-1640 medium (inoculum) supplemented with 10% neonate calf serum and 1% glutamine in a 75 cm flask. The day before the transfection, additional

12.5 ml of inoculum was added to each flask. At day 0 on the day of transfection, the medium was replaced with 15 ml of transfection medium (MEM medium with Earle salts containing 10% neonatal calf serum and 1% non-essential amino acids). The +3. 1 hour in 0.125 M calcium chloride solution

25 μg of DNA plus lxHBS (HEPES buffered physiopharm)

- 35 ··· · · β β β β β β «« «« «« «« «« l gi l l l l l l l l β β l β β β β β β β β β β β β β β β β β β) β)) β))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))) A +7. At 1 hour, cells were treated with 15% glycerol and then incubated overnight in 12.5 ml of inoculum containing 5 mM sodium butyrate. The next day, the cells were washed with PBS (Dulbecco's phosphate-buffered saline) and then in 12.5 mL of recovery medium (RPMI-1640 containing 2% 7.5% sodium bicarbonate). added to the cells. After a further 24 hours incubation, the supernatants were removed, centrifuged at 1000 rpm / min to remove cell fragments 5 minutes and then stored at 4 ° C or -20 ^e C.

3. Biological activity

Supernatant IL4 antagonist activity was assayed using the method of Spits (Spits et al., 1987, J. Immunology, 139, 1142). To upregulate the IL4 receptor, human peripheral blood lymphocytes were incubated for three days with phytohemagglutinin, a T-cell mitogen. The resulting blast cells were then stimulated with IL4 for a further three days. Proliferation was measured by incorporation of ³ H-thymidine.

The IL4.Y124D / IgG4 PE chimera inhibited the incorporation of ³ H-thymidine in human peripheral blood T lymphocytes stimulated with 133 µM IL4.

ί · · »

- 36 Example 4

Mammalian Expression Vector Containing DNA Encoding IL4.Y124D / IgG4 PE Fusion Protein

1. The structure of DNA

The pCDN vector [Aiyar, N., Baker, E., Wu, H.-L., Nambi, P., Edwards, R.M., Trill, J.J., Ellis, C., Bergsma, D., Molecular and Cellular Biochemistry, 131: 75-86 (1994)] includes the CMV promoter, a polylinker cloning site, and the BGH polyadenylation site. The vector further includes: a bacterial neomycin phosphotransferase gene (NEO) inserted between the β-globin promoter and the SV40 polyadenylation region for Geneticin ™ selection; a DHFR selection cassette inserted between the β-globin promoter and the BGH polyadenylation region for methotrexate (MTX) amplification; an ampicillin-resistant gene for bacterial culture; and an SV40 replication start.

For insertion of the IL4.Y124D / IgG4 PE cDNA, the pCDN vector was prepared by digestion with NdeI and BstX1 as follows. 15 μg of DNA was incubated with 30 units of BstX1 in Reaction Buffer 2 (Gibco-BRL) at 55 ° C for 1 hour and then ethanol precipitated. The resulting DNA was digested with reaction with NdeI in reaction buffer # 2 for one hour at 37 ° C, followed by ethanol precipitation. The IL4.Y124D / IgG4 PE fragment was prepared from pDB953 (Example 3 1.) by digestion with BstX1 and NdeI as follows. 15 μς ·· * »·« · · »* · ······ · ·· ·» * ·

37 DNA were incubated in reaction buffer # 2 with 30 units of BstXl for 1 hour at 55 ° C and then ethanol precipitated. The resulting DNA was digested with reaction with NdeI in reaction buffer # 2 for one hour at 37 ° C, followed by ethanol precipitation.

The IL4.Y124D / IgG4 PE Ndel / BstX1 and pCDN Ndel / BstX1 fragments were linked together to form the plasmid pCDN-IL4.Y124D / IgG4 PE. The coupling was performed using 2 units of T4 DNA Ligation (Gibco BRL) and T4 DNA Ligation buffer; the reactions were incubated overnight at 16 ° C. The products of the coupling reactions were transformed into GibcoBRL DH5a competent cells with subcloning efficiency and placed on Luria Broth agar containing 75 pg / ml ampicillin. The transformants were grown in Luria Broth containing 50 pg / ml ampicillin and then subjected to alkaline lysis to generate DNA. The formation of IL4.Y124D / IgG4 PE DNA was confirmed by restriction digestion. The complete sequence of the recombinant IL4.Y124D / IgG4 PE DNA was confirmed by sequencing. Recombinant DNA from pCDN-IL4.Y124D / IgG4 PE was prepared and purified using Qiagen columns, then used for transient infection of COS cells and electroporated into CHO cells to form stable clones.

2. Expression of the fusion protein

a) Transient expression in COS cells

COS-1 cells were grown in DMEM medium supplemented with 10% fetal calf serum. 24 hours before transfection • ·

- Ban 2χ10 inoculated 38 ⁵ cells per 35 mm tissue culture dishes. To a solution of 6 μΐ LIPOFECTAMINE Reagent (Gibco-BRL) in 100 μΐ serum-free DMEM was added 1 μg of DNA solution in 100 μΐ serum-free DMEM, and the mixture was gently mixed and incubated for 45 minutes at room temperature. The cells were washed once with serum-free DMEM. 0.8 ml of serum-free DMEM was added to the DNA-LIPOFECTAMINE solution, the mixture was gently mixed, and the diluted solution was layered over the cells. Cells were incubated for 5 hours at 37 ° C and then 1 ml of DMEM containing 20% fetal calf serum was added. Cells were assayed 48-72 hours later to determine expression.

b) Electroporation into CHO cells

ACC-098 CHO cells {Suspension cell line from DG-44 CHO (Urlaub, G., Kas, E., Carothers, AM, and Chasin, LA, Cell, 33, 405-412 (1983))} WO 92/05246. No. 4,600,198, grown in serum-free culture medium as described in International Patent Application Publication No. 3,620,123. To linearize the plasmid, 15 μg of pCDN-IL4.Y124D / IgG4 PE plasmid was digested with 30 units of NotI at 37 ° C for 3 hours and then ethanol precipitated. The DNA thus obtained was suspended once in 50 μΐ TE (10 mM Tris, pH 8.0, 1 mM EDTA). DNA was electroporated into 1x10 ACC-098 cells using a Bio Rad Gene Pulser (380 V and 25 μFd). Cells were suspended at ^{2.5 to} 10 ⁴ cells / ml in culture medium.

- In 39 wells, 200 μΐ cell suspension was added to each well of a 96-well plate. 48 hours later, the medium was changed to culture medium containing 400 μg / ml G418 (Geneticin). 21 days after selection, conditioned media from colonies were screened by Elisa assay. For amplification purposes, the largest expression colonies were transferred to 24-well plates.

• ·

Table 1: DNA sequence of the COSFeLink vector, 6367bp

SEQ ID NO: 1

GACGTCGACGGATCGGGAGATCGGGGATCGATCCGTCGACGT ACGACT AGTT ATT AATAG AATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTT T 6 0 0 120 18 ACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATG ACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTAT TTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCT 240 300 360 ATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGG GACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGG TTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTC 420 4 80 540 CACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAA TGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTC TATATAAGCAGAGCTGGGTACGTGAACCGTCAGATCGCCTGGAGACGCCATCGAATTCGG 600 660 720 TTACCTGCAGATATCAAGCTAATTCGGTACCGAGCCCAAATCGGCCGACAAAACTCACAC ATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCC AAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGA 780 840 900 CGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCA TAATGCCAAGACAAAGCC GCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGC.GT 960 CCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAA CAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGA 1020 1080 1140 ACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCT GACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGG GCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTT 1200 1260 1320 CCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG CTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC GGGTAAATGAGTGTAGTCTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCA 1380 14 40 1500 GCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCAC

TGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTAT TCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCA 1560 1620 1680 TGCTGGGGATGCGGTGGGCTCTATGGAACCAGCTGGGGCTCGAGGGGGGATCTCCCGATC CCCAGCTTTGCTTCTCAATTTCTTATTTGCATAATGAGAAAAAAAGGAAAATTAATTTTA 1740

ACACCAATTCAGTAGTTGATTGAGCAAATGCGTTGCCAAAAAGGATGCTTTAGAGACAGT 18 00 GTTCTCTGCACAGATAAGGACAAACATTATTCAGAGGGAGTACCCAGAGCTGAGACTCCT 1860 AAGCCAGTGAGTGGCACAGCATTCTAGGGAGAAATGGAGGAT

TCCGTAGAGCCACACCTTGGTAAGGGCCAATCTGCTCACACAGGATAGAGAGGGCAGGAG 1980

CCAGGGCAGAGCATATAAGGTGAGGTAGGATCAGTTGCTCCTCACATTTGCTTCTGACAT 2040

- 41 AGTTGTGTTGGGAGCTTGGATAGCTTGGACAGCTCAGGGCTGCGATTTCGCGCCAAACTT

GACGGCAATCCTAGCGTGAAGGCTGGTAGGATTTTATCCCCGCTGCCATCATGGTTCGAC

CATTGAACTGCATCGTCGCCGTGTCCCAAAATATGGGGATTGGCAAGAACGGAGACCTAC

CCTGGCCTCCGCTCAGGAACGAGTTCAAGTACTTCCAAAGAATGACCACAACCTCTTCAG

TGGAAGGTAAACAGAATCTGGTGATTATGGGTAGGAAAACCTGGTTCTCCATTCCTGAGA

AGAATCGACCTTTAAAGGACAGAATTAATATAGTTCTCAGTAGAGAACTCAAAGAACCAC

CACGAGGAGCTCATTTTCTTGCCAAAAGTTTGGATGATGCCTTAAGACTTATTGAACAAC

CGGAATTGGCAAGTAAAGTAGACATGGTTTGGATAGTCGGAGGCAGTTCTGTTTACCAGG

AAGCCATGAATCAACCAGGCCACCTTAGACTCTTTGTGACAAGGATCATGCAGGAATTTG

AAAGTGACACGTTTTTCCCAGAAATTGATTTGGGGAAATATAAACTTCTCCCAGAATACC

CAGGCGTCCTCTCTGAGGTCCAGGAGGAAAAAGGCATCAAGTATAAGTTTGAAGTCTACG

AGAAGAAAGACTAACAGGAAGATGCTTTCAAGTTCTCTGCTCCCCTCCTAAAGCTATGCA

TTTTTATAAGACCATGCTAGCTTGAACTTGTTTATTGCAGCTTATAATGGTTACAAATAA

AGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGT

TTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCAACGATAGCTTATCTGTGGGC

GATGCCAAGCACCTGGATGCTGTTGGTTTCCTGCTACTGATTTAGAAGCCATTTGCCCCC

TGAGTGGGGCTTGGGAGCACTAACTTTCTCTTTCAAAGGAAGCAATGCAGAAAGAAAAGC

ATACAAAGTATAAGCTGCCATGTAATAATGGAAGAAGATAAGGTTGTATGAATTAGATTT

ACATACTTCTGAATTGAAACTAAACACCTTTAAATTCTTAAATATATAACACATTTCATA

TGAAAGTATTTTACATAAGTAACTCAGATACATAGAAAACAAAGCTAATGATAGGTGTCC

CTAAAAGTTCATTTATTAATTCTACAAATGATGAGCTGGCCATCAAAATTCCAGCTCAAT

TCTTCAACGAATTAGAAAGAGCAATCTGCAAACTCATCTGGAATAACAAAAAACCTAGGA

TAGCAAAAACTCTTCTCAAGGATAAAAGAACCTCTGGTGGAATCACCATGCCTGACCTAA

AGCTGTACTACAGAGCAATTGTGATAAAAACTGCATGGTACTGATATAGAAACGGACAAG

TAGACCAATGGAATAGAACCCACACACCTATGGTCACTTGATCTTCAACAAGAGAGCTAA

AACCATCCACTGGAAAAAAGACAGCATTTTCAACAAATGGTGCTGGCACAACTGGTGGTT

ATCATGGAGAAGAATGTGAATTGATCCATTCCAATCTCCTTGTACTAAGGTCAAATCTAA

GTGGATCAAGGAACTCCACATAAAACCAGAGACACTGAAACTTATAGAGGAGAAAGTGGG

GAAAAGCCTCGAAGATATGGGCACAGGGGAAAAATTCCTGAATAGAACAGCAATGGCTTG

TGCTGTAAGATCGAGAATTGACAAATGGGACCTCATGAAACTCCAAAGCTATCGGATCAA

TTCCTCCAAAAAAGCCTCCTCACTACTTCTGGAATAGCTCAGAGGCCGAGGCGGCCTCGG

CCTCTGCATAAATAAAAAAAATTAGTCAGCCATGCATGGGGCGGAGAATGGGCGGAACTG

GGCGGAGTTAGGGGCGGGATGGGCGGAGTTAGGGGCGGGACTATGGTTGCTGACTAATTG

AGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGGGGACTTTCCACACCTGGTT

GCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGGGGACTTT

CCACACCCTAACTGACACACATTCCACAGAATTAATTCCCGATCCCGTCGACCTCGAGAG

CTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCC

ACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTA

ACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCA

2100

2160

2220

2280

2340

2400

2460

2520

2580

2640

2700

2760

2820

2880

2940

3000

3060

3120

3180

3240

3300

3360

3420

3480

3540

3600

3660

3720

3780

3840

3900

3960

4020

4080

4140

4200

4260

4320

4380 • · ··

- 42 GCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTC CGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGC 4440 4500 45 60 TCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACAT GTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTT CCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCG 4620 4680 4740 AAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTC TCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGT GGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAA 4800 4860 4920 GCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTA TCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAA CAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAA 4980 5040 5100 CTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTT CGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTT TTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGAT 5160 5220 5280 GAG CTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCAT ATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATC AATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGC 5340 5400 5460 ACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTA GATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGA CCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCG 5520 5580 5640 CAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGC TAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCAT CGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAG 5700 5760 5820 GCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGAT CGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAA TTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAA 5880 5940 6000 GTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGA TAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGG GCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGC 6060 6120 6180 ACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGG AAGGCAAAATGCC GCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACT 6240 CTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACAT 6300 ATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCGGGACACTA

- 43 - Table 2: DNA sequence encoding the Y124D-IgG1 fusion molecule in the COSFcLink vector, 6926bp

SEQ ID NO: 2

GACGTCGACGGATCGGGAGATCGGGGATCGATCCGTCGACGTACGACTAGTTATTAATAG TAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTT 6 0 120 180 ACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATG ACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTAT TTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCT 240 300 360 ATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGG GACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGG TTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTC 420 480 540 CACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAA TGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTC TATATAAGCAGAGCTGGGTACGTGAACCGTCAGATCGCCTGGAGACGCCATCGAATTCGG 600 660 720 TTACCTGCAGATGGGCTGCAGGAATTCCGCATTGCAGAGATAATTGTATTTAAGTGCCTA GCTCGATACAATAAACGCCATTTGACCATTCACCACATTGGTGTGCACCTCCAAGCTTAC CTGCCATGGGTCTCACCTCCCAACTGCTTCCCCCTCTGTTCTTCCTGCTAGCATGTGCCG 780 840 900 GCAACTTTGTCCACGGACACAAGTGCGATATCACCTTACAGGAGATCATCAAAACTTTGA ACAGCCTCACAGAGCAGAAGACTCT GTGCACCGAGTTGACCGTAACAGACATCTTTGCTG CCTCCAAGAACACAACTGAGAAGGAAACCTTCTGCAGGGCTGCGACTGTGCTCCGGCAGT 960 1020 1080 TCTACAGCCACCATGAGAAGGACACTCGCTGCCTGGGTGCGACTGCACAGCAGTTCCACA GGCACAAGCAGCTGATCCGATTCCTGAAACGGCTCGACAGGAACCTCTGGGGCCTGGCGG GCTTGAATTCCTGTCCTGTGAAGGAAGCCAACCAGAGTACGTTGGAAAACTTCTTGGAAA 1140 1200 1260 GGCTAAAGACGATCATGAGAGAGAAAGACTCAAAGTGTTCGAGCGGTACCGAGCCCAAAT CGGCCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGT CAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGG 1320 1380 1440 TCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACG TGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCA CGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGT 1500 1560 1620 ACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAG CCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGA CCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCG 1680 1740 1800 TGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGG ACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCA CCGTGGACAAGAGCAGGTGGCAGC 1860 AGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGA 1920 AGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGTAGTCTAGAGCTCGCGAGCAG

- 44 CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCC TGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTC 2040 2100 2160 TGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATT GGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGAACCAGCTGGGGCTC GAGGGGGGATCTCCCGATCCCCAGCTTTGCTTCTCAATTTCTTATTTGCATAATGAGAAA 2220 2280 2340 AAAAGGAAAATTAATTTTAACACCAATTCAGTAGTTGATTGAGCAAATGCGTTGCCAAAA AGGATGCTTTAGAGACAGTGTTCTCTGCACAGATAAGGACAAACATTATTCAGAGGGAGT ACCCAGAGCTGAGACTCCTAAGCCAGTGAGTGGCACAGCATTCTAGGGAGAAATATGCTT 2400 2460 2520 GTCATCACCGAAGCCTGATTCCGTAGAGCCACACCTTGGTAAGGGCCAATCTGCTCACAC AGGATAGAGAGGGCAGGAGCCAGGGCAGAGCATATAAGGTGAGGTAGGATCAGTTGCTCC TCACATTTGCTTCTGACATAGTTGTGTTGGGAGCTTGGATAGCTTGGACAGCTCAGGGCT 2580 2640 2700 GCGATTTCGCGCCAAACTTGACGGCAATCCTAGCGTGAAGGCTGGTAGGATTTTATCCCC GCTGCCATCATGGTTCGACCATTGAACTGCATCGTCGCCGTGTCCCAAAATATGGGGATT GGCAAGAACGGAGACCTACCCTGGCCTCCGCTCAGGAACGAGTTCAAGTACTTCCAAAGA 2760 2820 2880 ATGACCACAACCTCTTCAGTGGAAGGTAAACAGAATCTGGTGATTATGGGTAGGAAAACC TGGTT CTCCATTCCTGAGAAGAATCGACCTTTAAAGGACAGAATTAATATAGTTCTCAGT AGAGAACTCAAAGAACCACCACGAGGAGCTCATTTTCTTGCCAAAAGTTTGGATGATGCC 2940 3000 3060 TTAAGACTTATTGAACAACCGGAATTGGCAAGTAAAGTAGACATGGTTTGGATAGTCGGA GGCAGTTCTGTTTACCAGGAAGCCATGAATCAACCAGGCCACCTTAGACTCTTTGTGACA AGGATCATGCAGGAATTTGAAAGTGACACGTTTTTCCCAGAAATTGATTTGGGGAAATAT 3120 3180 3240 AAACTTCTCCCAGAATACCCAGGCGTCCTCTCTGAGGTCCAGGAGGAAAAAGGCATCAAG TATAAGTTTGAAGTCTACGAGAAGAAAGACTAACAGGAAGATGCTTTCAAGTTCTCTGCT CCCCTCCTAAAGCTATGCATTTTTATAAGACCATGCTAGCTTGAACTTGTTTATTGCAGC 3300 3360 3420 TTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTC ACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCAA CGATAGCTTATCTGTGGGCGATGCCAAGCACCTGGATGCTGTTGGTTTCCTGCTACTGAT 3480 3540 3600 TTAGAAGCCATTTGCCCCCTGAGTGGGGCTTGGGAGCACTAACTTTCTCTTTCAAAGGAA GCAATGCAGAAAGAAAAGCATACAAAGTATAAGCTGCCATGTAATAATGGAAGAAGATAA GGTTGTATGAATTAGATTTACATACTTCTGAATTGAAACTAAACACCTTTAAATTCTTAA 3660 3720 3780 ATATATAACACATTTCATATGAAAGTATTTTACATAAGTAACTCAGATACATAGAAAACA AAGCTAATGATAGGT GTCCCTAAAAGTTCATTTATTAATTCTACAAATGATGAGCTGGCC 3840 ATCAAAATTCCAGCTCAATTCTTCAACGAATTAGAAAGAGCAATCTGCAAACTCATCTGG AATAACAAAAAACCTAGGATAGCAAAAACTCTTCTCAAGGATAAAAGAACCTCTGGTGGA 3900 3960 4020 ATCACCATGCCTGACCTAAAGCTGTACTACAGAGCAATTGTGATAAAAACTGCATGGTAC TGATATAGAAACGGACAAGTAGACCAATGGAATAGAACCCACACACCTATGGTCACTTGA TCTTCAACAAGAGAGCTAAAACCATCCACTGGAAAAAAGACAGCATTTTCAACAAATGGT 4080 4140 4200 GCTGGCACAACTGGTGGTTATCATGGAGAAGAATGTGAATTGATCCATTCCAATCTCCTT GTACTAAGGTCAAATCTAAGTGGATCAAGGAACTCCACATAAAACCAGAGACACTGAAAC 4260

TTATAGAGGAGAAAGTGGGGAAAAGCCTCGAAGATATGGGCACAGGGGAAAAATTCCTGA 4320

- 45 ATAGAACAGCAATGGCTTGTGCTGTAAGATCGAGAATTGACAAATGGGACCTCATGAAAC 4380 TCCAAAGCTATCGGATCAATTCCTCCAAAAAAGCCTCCTCACTACTTCTGGAATAGCTCA 4440

GAGGCCGAGGCGGCCTCGGCCTCTGCATAAATAAAAAAAATTAGTCAGCCATGCATGGGG 4500 CGGAGAATGGGCGGAACTGGGCGGAGTTAGGGGCGGGATGGGCGGAGTTAGGGGCGGGAC 4560

TATGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGG 4620

GGACTTTCCACACCTGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGC 4680

TGGGGAGCCTGGGGACTTTCCACACCCTAACTGACACACATTCCACAGAATTAATTCCCG 4740 ATCCCGTCGACCTCGAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATT 4800 GTTATCCGCTCACAATTCCACACAGTAGAA

GTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGT CGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTT 4920 4980 5040 TGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGC TGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGG 5100

ATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGG CCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGAC 5160 5220 5280 GCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTG GAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCT TTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGG 5340 5400 5460 TGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCT GCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCAC TGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGT 5520 5580 5640 TCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTC TGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCA CCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGAT 5700 5760 5820 CTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCAC GTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATT 5880

AAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACC 5940 AATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTG 6000

CCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTG CTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGC 6060 6120 6180 CAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTA TTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTG 6240

TTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCT 6300

CCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTA 6360 GCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGG 6420

TTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGA 6480

CTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTT 6540

GCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCA 6600

TTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTT 6660 • · · • ·

- 46 CGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTT 6720

CTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGA 67 80

AATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATT 6840

GTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGC 6900

GCACATTTCCCCGAAAAGTGCCACCT 6926

Table 3: Coding region for IL4.Y124D / IgG1 fusion molecule

DNA sequence, 1164bp

SEQ ID NO: 3

ATGGGTCTCACCTCCCAACTGCTTCCCCCTCTGTTCTTCCTGCTAGCATGTGCCGGCAAC 60

TTTGTCCACGGACACAAGTGCGATATCACCTTACAGGAGATCATCAAAACTTTGAACAGC 120

CTCACAGAGCAGAAGACTCTGTGCACCGAGTTGACCGTAACAGACATCTTTGCTGCCTCC 180

AAGAACACAACTGAGAAGGAAACCTTCTGCAGGGCTGCGACTGTGCTCCGGCAGTTCTAC 240

AGCCACCATGAGAAGGACACTCGCTGCCTGGGTGCGACTGCACAGCAGTTCCACAGGCAC 300

AAGCAGCTGATCCGATTCCTGAAACGGCTCGACAGGAACCTCTGGGGCCTGGCGGGCTTG 360

AATTCCTGTCCTGTGAAGGAAGCCAACCAGAGTACGTTGGAAAACTTCTTGGAAAGGCTA 420

AAGACGATCATGAGAGAGAAAGACTCAAAGTGTTCGAGCGGTACCGAGCCCAAATCGGCC 480

GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTC 540

TTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACA 600

TGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGAC 660

GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTAC 720

CGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAG 780

TGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAA 840

GGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAG 900

AACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAG 960

TGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCC 1020

GACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGG 1080

AACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGC 1140

CTCTCCCTGTCTCCGGGTAAATGA 1164

- 47 Table 4: Sequence of the encoded IL4.Y124D / IgG1 fusion protein, 387aa

SEQ ID NO: 4

MGLTSQLLPP LFFLLACAGN FVHGHKCDIT LQEIIKTLNS

LTVTDIFAAS KNTTEKETFC RAATVLRQFY SHHEKDTRCL

101 KQLIRFLKRL DRNLWGLAGL NSCPVKEANQ STLENFLERL

151 CSSGTEPKSA DKTHTCPPCP APELLGGPSV FLFPPKPKDT

201 CVWDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY

251 QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT

301 NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS

351 TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK *

LTEQKTLCTE

GATAQQFHRH

KTIMREKDSK

LMISRTPEVT

RVVSVLTVLH

LPPSRDELTK

DGSFFLYSKL

Table 5: DNA sequence of synthetic IgG4 cDNA, 1006bp

SEQ ID NO: 5

GCTTCCACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAG 6 0

AGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCG 120

TGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA 180

GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACC 240

TACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCC 300

AAATATGGTCCCCCATGCCCATCATGCCCAGCACCTGAATTTCTGGGGGGACCATCAGTC 360

TTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACG 420

TGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGAT 480

GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTAC 540

CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAG 600

TGCAAGGTCTCCAACAAAGGCCTCCCGTCATCGATCGAGAAAACCATCTCCAAAGCCAAA 660

GGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAG 720

AACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAG 780

TGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCC 840

GACGGATCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGG 900

AATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGC 960

CTCTCCCTGTCTCTGGGTAAATGAGTGTAGTCTAGATCTACGTATG

1006 ····································································································································································································· ···

Table 6: The coding region for the IL4.Y124D / IgG4 fusion protein

DNA sequence, 1149bp

SEQ ID NO: 6

ATGGGTCTCACCTCCCAACTGCTTCCCCCTCTGTTCTTCCTGCTAGCATGTGCCGGCAAC 60

TTTGTCCACGGACACAAGTGCGATATCACCTTACAGGAGATCATCAAAACTTTGAACAGC 120

CTCACAGAGCAGAAGACTCTGTGCACCGAGTTGACCGTAACAGACATCTTTGCTGCCTCC 180

AAGAACACAACTGAGAAGGAAACCTTCTGCAGGGCTGCGACTGTGCTCCGGCAGTTCTAC 240

AGCCACCATGAGAAGGACACTCGCTGCCTGGGTGCGACTGCACAGCAGTTCCACAGGCAC 300

AAGCAGCTGATCCGATTCCTGAAACGGCTCGACAGGAACCTCTGGGGCCTGGCGGGCTTG 360

AATTCCTGTCCTGTGAAGGAAGCCAACCAGAGTACGTTGGAAAACTTCTTGGAAAGGCTA 420

AAGACGATCATGAGAGAGAAAGACTCAAAGTGCTCGAGCGAGTCCAAATATGGTCCCCCA 480

TGCCCATCATGCCCAGCACCTGAATTTCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCA 540

AAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGAC 600

GTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCAT 660

AATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTC 720

CTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAAC 780

AAAGGCCTCCCGTCATCGATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAG 840

CCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTG 900

ACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGG 960

CAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGATCCTTCTTC 1020

CTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGC 1080

TCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTG 1140

GGTAAATGA

1149 · ♦ · ·

- 49 Table 7

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

SEQ ID NO: 7

MGLTSQLLPP LFFLLACAGN FVHGHKCDIT LQEIIKTLNS LTEQKTLCTE 51 LTVTDIFAAS KNTTEKETFC RAATVLRQFY SHHEKDTRCL GATAQQFHRH 101 KQLIRFLKRL DRNLWGLAGL NSCPVKEANQ STLENFLERL KTIMREKDSK 151 GGPSVFLFPP KPKDTLMISR CSSESKYGPP CPSCPAPEFL TPEVTCVWD NWYVDGVEVH NAKTKPREEQ 201 VSQEDPEVQF FNSTYRWSV LTVLHQDWLN 251 GKEYKCKVSN KGLPSSIEKT ISKAKGQPRE PQVYTLPPSQ EEMTKNQVSL 301 TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSRLTVDKS

351 RWQEGNVFSC SVMHEALHNH YTQKSLSLSL GK *

Table 8: DNA sequence of the PE variant of IgG4, 984bp

SEQ ID NO: 8

GCTAGTACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAG

AGCACgGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCG

TGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA

GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACC

TACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCC

AAATATGGTCCCCCATGCCCAcCATGCCCAGCgCCTGAaTTtgaGGGGGGACCATCAGTC

TTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACG

TGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGAT

GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTAC

CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAG

TGCAAGGTCTCCAACAAAGGCCTCCCGTCATCGATCGAGAAAACCATCTCCAAAGCCAAA

GGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAG

AACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAG

TGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCC

GACGGaTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGG

AATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGC

CTCTCCCTGTCTCTGGGTAAATGA

120

180

240

300

360

420

480

540

600

660

720

780

840

900

960

984 · * «·

Table 9: DNA sequence of the IL4.Y124D / IgG4 PE fusion molecule, 1149bp

SEQ ID NO: 9

ATGGGTCT.CACCTCCCAACTGCTTCCCCCTCTGTTCTTCCTGCTAGCATGTGCCGGCAAC TTTGTCCACGGACACAAGTGCGATATCACCTTACAGGAGATCATCAAAACTTTGAACAGC 60 120 180 CTCACAGAGCAGAAGACTCTGTGCACCGAGTTGACCGTAACAGACATCTTTGCTGCCTCC AAGAACACAACTGAGAAGGAAACCTTCTGCAGGGCTGCGACTGTGCTCCGGCAGTTCTAC AGCCACCATGAGAAGGACACTCGCTGCCTGGGTGCGACTGCACAGCAGTTCCACAGGCAC 240 300 360 AAGCAGCTGATCCGATTCCTGAAACGGCTCGACAGGAACCTCTGGGGCCTGGCGGGCTTG AATTCCTGTCCTGTGAAGGAAGCCAACCAGAGTACGTTGGAAAACTTCTTGGAAAGGCTA AAGACGATCATGAGAGAGAAAGACTCAAAGTGCTCGAGCGAGTCCAAATATGGTCCCCCA 420 480 540 TGCCCACCATGCCCAGCgCCTGAATTTGAGGGGGGACCATCAGTCTTCCTGTTCCCCCCA AAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGAC GTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCAT 600 660 720 AATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTC CTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAAC AAAGGCCTCCCGTCATCGATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAG 780 840 900 CCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTG ACCTGCCTGGTCAAAGGCTTCTACC CCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGG 960 CAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGaTCCTTCTTC CTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGC 1020 1080 1140 TCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTG GGTAAATGA 1199

Table 10: Sequence of the encoded IL4.Y124D / IgG4 PE variant fusion protein, 382aa

SEQ ID NO: 10

MGLTSQLLPP LFFLLACAGN FVHGHKCDIT LQEIIKTLNS LTEQKTLCTE 51 LTVTDIFAAS KNTTEKETFC RAATVLRQFY SHHEKDTRCL GATAQQFHRH 101 KQLIRFLKRL DRNLWGLAGL NSCPVKEANQ STLENFLERL KTIMREKDSK 151 GGPSVFLFPP KPKDTLMISR CSSESKYGPP CPPCPAPEFE TPEVTCVWD NWYVDGVEVH NAKTKPREEQ 201 VSQEDPEVQF FNSTYRWSV LTVLHQDWLN 251 GKEYKCKVSN KGLPSSIEKT ISKAKGQPRE PQVYTLPPSQ EEMTKNQVSL 301 TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSRLTVDKS

351 RWQEGNVFSC SVMHEALHNH YTQKSLSLSL GK *

Claims (16)

PATENT CLAIMS A soluble protein having IL4 and / or IL13 antagonist or partial antagonist activity, comprising at least one human immunoglobulin constant region or fragment thereof comprising an IL4 mutant or variant. A compound according to claim 1, wherein at least one naturally occurring amino acid at position 120-128 of wild-type IL4 is replaced by a different natural amino acid. A compound according to claim 2, wherein the naturally occurring tyrosine at position 124 is replaced by a different natural amino acid. A compound according to claim 3, wherein the naturally occurring tyrosine at position 124 is replaced by aspartic acid. 5. A compound according to any one of claims 1 to 5, wherein the immunoglobulin is of the IgG subclass. The compound of claim 5, wherein the constant region or fragment thereof is all or a substantial portion of the human IgG heavy chain constant region. The compound of claim 5, wherein the constant region or fragment thereof is all or a substantial portion of the human IgG4 heavy chain constant region. A compound according to claim 1 having the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10. »·· ··» ** · i · * · * • · * · * • »· · · *« · · * - 52 9. A method of any of claims 1-8. For the preparation of a compound according to any one of claims 1 to 3, characterized in that the DNA encoding the compound is expressed in a recombinant host cell and the product is recovered. The process of claim 9, wherein i) providing a replicable expression vector capable of expressing in a host cell a DNA polymer comprising a nucleotide sequence encoding the compound; ii) transforming a host cell with the vector; iii) culturing the transformed host cell under conditions which allow expression of the DNA polymer to produce the compound; and iv) recovering the compound. 11. A DNA polymer which is a DNA polymer of any one of claims 1-8. A nucleotide sequence encoding a compound according to any one of claims 1 to 6. 12. A DNA polymer according to claim 11, comprising SEQ ID NO: 3, SEQ ID NO: 6 or SEQ ID NO: 9, or SEQ ID NO: 3, SEQ ID NO: 6 or SEQ ID NO: 9. A replicable expression vector comprising a DNA polymer according to claim 11. 14. A host cell transformed with a replicable expression vector according to claim 13. 15. A pharmaceutical composition comprising a compound according to any one of claims 1-8. A compound according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier. r »·· - 53 »··" ·· · 5Λ t »ti · e * · · · · · · · · · · · · · · 16. One of claims 1-8. Use of a compound according to any one of claims 1 to 6 for the manufacture of a medicament for the treatment of conditions resulting from the undesirable effects of IL4 and / or IL13.