IE84574B1 - Fusion proteins with parts of immunoglobulins, their production and use - Google Patents

Description

PATENTS ACT, 1992 FUSION PROTEINS WITH PARTS OF IMMUNOGLOBULINS, THEIR PRODUCTION AND USE HOECHST AKTIENGESELLSCHAFT THE GENERAL HOSPITAL CORPORATION Description The invention relates to the field of engineered soluble fusion proteins consisting of human genetically proteins not belonging to the immunoglobulin family, or the The functional properties of the two fusion partners are, of parts thereof, and of various portions of constant region of immunoglobulin molecules. surprisingly, retained in the fusion protein.

EP—A O 325 262 and EP-A O 314 317 disclose corresponding fusion proteins consisting of various domains of the CD4 membrane protein. of human T cells and of human IgGl portions. some of these fusion proteins bind with the same affinity to the glycoprotein gpl2O of human immuno- deficiency virus as the cell—bound CD4 molecule. The CD4 the consequently, has a very similar tertiary structure to molecule belongs to immunoglobulin family and, that of immunoglobulin molecules. This also applies to the a chain of the T-cell antigen receptor, for which such fusions have also been described (Gascoigne et al., Proc. Natl. Acad. Sci. USA, Vol. 84 (1987), 2937-2940).

Hence, on the basis of the very similar domain structure, in this case retention of the biological activity of the two fusion partners in the fusion protein was to be expected.

The humarx proteins coupled. according to the invention preferably to the amino terminus of the constant region of immunoglobulixi do not belong to the immunoglobulin family and are to be assigned to the following classes: (i) membrane-associated proteins whose extracellular domain is wholly or partly introduced into the fusion.

These are in particular thromboplastin and cytokine receptors and growth factor receptors such as the cellular receptors for interleukin 4, interleukin 7, tumor necrosis factor, GM-CSF, G-CSF, (ii) non—membrane—associated soluble proteins which are erythropoietin; wholly or partly introduced into the fusion. These are w . 845 in particular proteins of therapeutic interest, such as, for example, erythropoietin and other cytokines and growth factors.

The fusion proteins can be prepared in known pro- and eukaryotic expression systems, but preferably in mammal- ian cells (for example CHO, COS and BHK cells).

EP—A O 269 455 describes protein in. which the sequence Ala—Pro—Thr—Ser—Ser—Thr~ a highly purified fusion Lys-Lys-Thr-Gln-Arg-Asn-Ser-Met-Leu is coupled to the N terminus of a human IgE Fc fragment, and a process for preparing and pmrifying this fusion protein, which is beneficial in the treatment of allergies.

The fusion proteins according to the invention are, by reason of their immunoglobulin portion, easy to purify by" affinity chromatography and have improved pharmacokinetic properties in vivo.

In many cases, the Fc part in the fusion protein is quite advantageous for use in therapy and diagnosis and thus results, for example, in improved pharmacokinetic (EP—A. 0 232 262). On the other hand, the possibility of removing the Fc part would be desirable properties for some applications, after the fusion protein has been expressed, detected and purified in the advantageous manner described. This is the case when the Fc portion proves an impediment for use in therapy and diagnosis, e.g. when the fusion protein is to serve as antigen for immunizations.

There are in existence various proteases whose use for this purpose appears conceivable. Papain or pepsin are employed, for example, to generate F(ab) immunoglobulins ed. Roitt, I.

Medical Publishing, (1989)), cleave in a particularly specific manner. Blood coagula- fragments from et al., but they do not (Immunology, Gower London tion factor Xa by contrast recognizes in a protein the relatively rare tetrapeptide sequence Ile—Glu—Gly—Arg and carries out a hydrolytic cleavage of the protein after the arginine residue. Cleavage sequences which contain the described tetrapeptide were introduced first by Nagai and Thogersen into a hybrid protein by genetic H.C., These authors were engineering means (Nagai, K. and Thogersen, 309 (1984), 8l0~8l2). able to show that the proteins expressed in E.

Nature, vol. coli actually are specifically cleaved by factor Xa. However, there is as yet no published example of the possibility of such proteins also being expressed in eukaryotic and, animal cells after their especially, in and, purification, being cleaved by factor Xa. However, expression of the proteins according to the invention in animal cells is preferable because only in a cell system of this type is there expected to be secretion of, for example, normally membrane—associated. receptors as fusion partners with retention of their native structure and thus of their‘ biological activity; Secretion into the cell culture supernatant facilitates the subsequent straightforward purification of the fusion protein.

The soluble fusion proteins consisting of human proteins not the invention thus relates to genetically engineered belonging to immunoglobulin family, or parts thereof, and various portions of the constant regions of heavy or light chains of immunoglobulins of various (IgG, IgM, IgA, IgE). The immunoglobulin is the constant part of the heavy chain subclasses preferred of human IgG, particularly preferably of human IgGl, the fusion taking place at the hinge region. In a particular embodiment, the Fc part can be detached in a simple way by a cleavage sequence which is also incorporated and can be cleaved by factor Xa.

Furthermore, the invention relates to processes for the preparation of these fusion proteins by genetic engineering, and to the use thereof for diagnosis and therapy.

Finally, the invention is explained in further examples.

Example 1: Thromboplastin fusion proteins Blood coagulation is a process of central importance in the human body. There is appropriately delicate regula- tion of the coagulation cascade, in which a large number of cellular factors and plasma proteins cooperate. These proteins (and their cofactors) are The final products of the thrombin, which the aggregation of blood platelets, and fibrin which stabil- the platelet thrombus. Thrombin the formation of fibrin from fibrinogen and itself is formed in their entirety called coagulation factors. induces coagulation cascade are izes catalyzes by limited proteolysis of prothrombin. Activated factor X (factor Xa) is responsible for this step and, in the presence of factor Va and calcium ions, binds to platelet membranes and cleaves prothrombin.

Two ways exist for factor X to be activated, the extrin- sic and the intrinsic pathway. In the intrinsic pathway a series of factors is activated by proteolysis in order for each of them to form active proteases themselves. In the extrinsic pathway, there is increased synthesis of thromboplastin (tissue factor) by damaged cells, and it with VIIa calcium ions. It was formerly assumed that the activity activates factor X, together factor and of thromboplastin is confined to this reaction. However, the thromboplastin/VIIa activate the intrinsic pathway at the level of factor complex also intervenes to IX. Thus, a thromboplastin/VIIa complex is one of the most important physiological activators of blood coagulation.

It is therefore conceivable that thromboplastin, apart from its use as diagnostic aid (see below), can also be employed as constituent of therapeutic agents for treat- ing inborn or acquired blood coagulation deficiencies.

Examples of this are chronic hemophilias caused. by‘ a deficiency of factors VIII, IX or XI or else acute disturbances of blood coagulation as a consequence of, for example, liver or kidney disease. Use of such a therapeutic agent after surgicial intervention would also be conceivable.

Thromboplastin is an integral membrane protein which the Thromboplastin CDNA sequences have been published by a does not belong to immunoglobulin family. total of four groups (Fisher et al., Thromb. Res., vol. 48 (1987), 89-99; Morrisey et al., Cell, vol. 50 (1987), 129-135; Scarpati et al., Biochemistry, vol. 26 (1987), 5234-5238; Spicer et al., Proc. Natl. Acad. Sci. USA, vol. 84 (1987), 5148-5152). Thromboplastin CDNA contains an open reading frame which codes for a polypeptide of of which the 32 N—terminal amino acids act as signal peptide. Mature thromboplastin amino-acid residues, comprises 263 amino—acid residues and has a three-domain structure: i) (219 region (23 amino-terminal extracellular domain amino—acid residues); ii) transmembrane amino-acid residues); iii) cytoplasmic domain (carboxyl terminus; 21 amino—acid residues). In the extracellular domain there are three potential sites for N- glycosylation (Asn-X—Thr). Thromboplastin is normally glycosylated but glycosylation does not appear essential for the activity of the protein (Paborsky et al., Biochemistry, vol. 28 (1989), 8072-8077).

Thromboplastin is required as additive to plasma samples The status of the tested person can be found by the one- in diagnostic tests of coagulation. coagulation stage prothrombin clotting time determination (for example Quick's test). The thromboplastin required for human difficult to the yield is low and considerable amounts diagnostic tests is currently obtained from tissue, and the preparation process is standardize, of human starting material (placentae) must be supplied.

On the other hand, it is to be expected that preparation of native, membrane-bound thromboplastin by genetic engineering will also be difficult owing to complex These avoided by the fusion according to the invention to purification processes. difficulties can be immunoglobulin portions.

The thromboplastin fusion proteins according to the invention are secreted by mammalian cells CHO, BHK, cos cells) by affinity chromatography on protein .A-Sepharose and (for example into the culture medium, purified have surprisingly high activity in one~stage prothrombin clotting time determination.

Cloning of thromboplastin cDNA The sequence published by Scarpati et al., Biochemistry, vol. 26 (1987), 5234-5238, thromboplastin CDNA. Two oligonucleotide probe molecules was used for cloning the (see Fig. 1) were derived from this. These twc> probe molecules were used to screen a CDNA bank from human placenta Natl. Acad. Sci. USA, vol. 83 (Grundmann et al., (1986), 8024-8028).

Proc.

CDNA clones of various lengths were obtained. One clone, 2b-Apr5, which is used for the subsequent procedure, for the the CDNA 2 depicts the total sequence of the clone 2b-Apr5 with the thromboplastin codes same amino-acid sequence as described in Scarpati et al. Fig. amino—acid sequence deduced therefrom.

Construction of a hybrid plasmid pTF1Fc coding for thromboplastin fusion protein.

The plasmid pCD4E gamma 1 (EP 0 325 262 A2; deposited at the ATCC under the 67610) is expression. of a fusion. protein composed of human CD4 number No. used for receptor and human IgGl. The DNA sequence coding for the extracellular domain of CD4 is deleted from this plasmid using the restriction enzymes HindIII and BamHI. Only partial cleavage must be carried out with. the enzyme HindIII in this case, in order to cut at only one of the two HindIII sites contained in pCD4E gamma 1 (position 2198). The result is an opened vector in which a eukary- otic transcription regulation sequence (promoter) is followed by the open HindIII site. The open BamHI site is located. at the start of the coding regions for a pentapeptide linker, followed by the hinge and the CH2 and CH3 domains of human IgGl. The reading frame in the BamHI recognition sequence GGATCC is such that GAT is translated as aspartic acid. DNA amplification with thermostable DNA polymerase makes it possible to modify a given sequence in such a way that attached at both oligonucleotides able to hybridize with sequences in the any desired sequences are one or ends. Two '-untranslated region (A: 5' GATCGATTAAGCTTCGGAACCCGCTCGATCTCGCCGCC 3') or coding region (B: 5' GCATATCTGGATCCCCGTAGAATATTTCTCTGAATTCCCC 3') of thromboplastin cDNA were synthesized. Of these, nucleotide A is partially homologous with the sequence oligo- of the coding strand, and oligonucleotide B is partially homologous with the non-coding strand; cf. Fig. 3.

Thus, amplification results in a DNA fragment (827 bp) at the 5' end before the start of the coding sequence a HindIII the 3' three amino—acid residues of the transmembrane region a which contains (based on the coding strand) site, and at end after the codon for the first BamHI site. The reading frame in the BamHI cleavage site is such that ligation with the BamHI site in pCD4E gamma 1 results in a gene fusion with a frame the the thromboplastin CDNA to the stop codon of the heavy chain of IgGl. treatment with HindIII and BamHI, reading continuous from initiation codon of after into the vector pCD4E gamma 1, as described above, which had been cut with HindIII and BamHI. plasmid was called pTFlFc (Fig. 4).

The desired fragment was obtained and, ligated (partially) The resulting Transfection of pTF1Fc into mammalian cells The fusion protein encoded. by the plasmid. pTF1Fc is called pTFlFc pTFlFc expressed in COS cells. For this purpose, COS cells were hereinafter. was transiently transfected with pTF1Fc with the aid of DEAE-dextran (EP A O 325 262). Indirect immunofluorescence investiga- tions revealed that the proportion of transfected cells was about 25%. the cells were This cell supernatant was harvested after a further three days. h after transfection, transferred into serum—free medium.

Purification of pTF1Fc fusion protein from cell culture supernatants ml of supernatant from transiently transfected COS cells were collected overnight in a batch process in a column containing 0.8 ml of protein A-Sepharose at 4°C, (50 mM tris and eluted in 0.5 ml frac- washed with 10 volumes of washing buffer buffer pH 8.6, 150 mM NaCl) tions with eluting buffer (93:7 100 mM citric acid: 100 mM The first 9 immediately neutralized with 0.1 ml of 2M tris buffer sodium citrate). fractions were pH 8.6 in each case and then combined, and the contained protein was transferred by three concentration/dilution cycles in an Amicon microconcentrator (Centricon 30) into TNE buffer (50 mM tris buffer pH 7.4, 50 mM Nacl, 1 mM EDTA). The pTFlFc obtained in this way is pure by SDS—PAGE electrophoresis (U.K. Lammli, Nature 227 (1970) 680-685). In the absence of reducing agents it behaves in the SDS-PAGE like a dimer (about 165 KDa).

Biological activity of purified TF1Fc in the prothrombin clotting time determination TFlFc fusion protein is active in low concentrations (> 50 ng/ml) determination in the one-stage prothrombin clotting time {Vinazzer, H. Gerinnungsphysiologie und Methoden im Blutgerinnungslabor (1979), Fisher Verlag Stuttgart). with the clotting times obtained with thromboplastin The clotting times achieved are comparable isolated from human placenta.

Example 2: Interleukin-4 receptor fusion proteins Interleukin-4 (IL-4) originally" called B—cell growth factor because it is is synthesized by T cells and was able to stimulate B-cell proliferation. It exerts a large number of effects on these cells. one in particular is the stimulation of synthesis of molecules of immunoglobulin subclasses IgGl and IgE in activated B 102 (1988) IL-4 also regulates the proliferation cells (Coffmann et al., Immunol. vol.

). In addition, ReV., and differentiation of T cells and other hemopoietic cells. It thus contributes to the regulation of allergic and other immunological reactions. IL-4 binds with high affinity to a specific receptor. The CDNA which codes for the human IL-4 receptor has been isolated (Idzerda et al., J. Exp. Med. vol. 171 (1990) 861-873. It is evident from analysis of the amino-acid sequence deduced from the CDNA sequence that the IL-4 receptor consists with the 25 N—terminal amino acids acting as signal peptide. Mature human IL-4 800 like has a three-domain structure: i) amino- (207 amino acids) of a total of 825 amino acids, receptor consists of amino acids and, thromboplastin, terminal extracellular domain amino acids); ii) transmembrane region (24 and iii) cytoplasmic domain (569 amino acids). In the extra- cellular domain there are six potential sites for (Asn-X-Thr/Ser). IL-4 homologies with human Il-6 receptor, with the B—subunit IL-2 with N~glycosylation receptor has of human receptor, mouse erythropoietin receptor and with rat prolactin receptor (Idzerda et al., cit.). Thus, like thromboplastin, member of the immunoglobulin family but is together with the homologous proteins mentioned to the Members of this loc. it is not a assigned new family of hematopoietin receptors. family have 4 cysteine residues and a conserved sequence (Trp-Ser-X-Trp-Ser) in the extracellular domain located near the transmembrane region in common.

On the basis of the described function of the IL-4/IL-4 receptor system, there is a possible therapeutic use of a recombinant form of the IL-4 receptor for suppressing ILmediated immune reactions (for example transplant rejection reaction, autoimmune diseases, allergic reac- tions).

The amount of substance required for therapy‘ make it necessary to prepare such molecules by genetic engineering. Because of the straightforward purification by affinity chromatography and improved pharmacokinetic properties, according to the invention the synthesis of soluble forms of the IL-4 receptor as immunoglobulin fusion protein is particularly advantageous.

The IL-4 receptor fusion proteins are secreted by (for example CHO, BHK, COS cells) the culture medium, purified by affinity chromatography mammalian cells into on protein A~Sepharose and have, surprisingly, identical functional properties to the extracellular domain of the intact membrane-bound IL-4 receptor molecule.

Construction of a hybrid plasmid pIL-4RFc coding for IL-4 receptor fusion protein.

Cutting of the plasmid pCD4EGamma1 with XhoI and BamHI results in an opened vector in which the open Xhol site The open BamHI site is located at the start of the coding is located downstream from the promoter sequence. regions for a pentapeptide linker, followed by the hinge and the CH2 and CH3 domains of human IgG1. The reading frame in the BamHI recognition sequence GGATCC is such that GAT is translated as aspartic acid. DNA amplifica- tion with thermostable DNA polymerase makes it possible to modify a given sequence in such a way that any desired sequences are attached at one or both ends. Two oligonucleotides able to hybridize with sequences in the '-untranslated region (A: 5' GATCCAGTACTCGAGAGAGAAGCCGGGCGTGGTGGCTCATGC 3') or coding region (B: 5‘ CTATGACATGGATCCTGCTCGAAGGGCTCCCTGTAGGAGTTGTG 3') of the IL-4 receptor CDNA which is cloned in the vector pDC302/T22-8 (Idzerda et al. cit.) were , loc. synthesized. Of these, oligonucleotide A is partially homologous with the sequence of the coding strand, and oligonucleotide B is partially homologous with the non- coding strand; cf. Fig. 5. Amplification using thermo- stable DNA polymerase results in a DNA fragment (836 bp) which, based on the coding strand, contains at the 5‘ end before the start of the coding sequence an Xhol site, and at the 3' end before the last codon of the extracellular domain a BamHI site. The reading frame in the BamHI cleavage site is such that ligation with the BamHI site :u1 pCD4E gamma 1. results lJ1 a gene fusion with a reading frame continuous from the initiation codon of the IL-4 receptor cDNA to the stop codon of the heavy chain of IgG1. The desired fragment was obtained and, after treatment with XhoI and BamHI, ligated into the vector pCD4E gamma 1, described above, which. had been cut with XhoI/BamHI. The resulting plasmid was called pIL4RFc (Fig. 6).

Transfection of pIL4RFc into mammalian cells The fusion protein encoded by the plasmid pIL4RFc is called pIL4RFc pIL4RFc was expressed in COS cells. For this purpose, COS cells were transfected with pIL4RFc with the aid of DEAE-dextran (EP A O 325 262). Indirect immunofluorescence investiga- hereinafter. transiently tions revealed that the proportion of transfected cells was about 25%. 24 h after transfection, the cells were medium. This cell transferred into serum—free supernatant was harvested after a further three days.

Purification of IL4RFc fusion protein from cell culture supernatants ml of supernatant from transiently transfected COS cells were collected overnight in a batch process in a column containing 1.6 ml of protein A-Sepharose at 4°C, buffer (50 mM tris and eluted in 0.5 ml frac- (93:7 100 mM citric acid: The first 9 immediately neutralized with 0.1 ml of 2M tris buffer washed with 10 volumes of washing buffer pH 8.6, 150 mM Nacl) tions with eluting buffer 100 mM sodium citrate). fractions were pH 8.6 in each case and then combined, and the contained protein was transferred by three concentration/dilution cycles in an Amicon microconcentrator (Centricon 30) into TNE buffer (50 mM tris buffer pH 7.4, 50 mM Nacl, 1 mM EDTA). The IL4RFc obtained in this way is pure by SDS—PAGE electrophoresis (U.K. Lammli, Nature 227 (1970) 680-685). In the absence of reducing agents it behaves in the SDS-PAGE like a dimer (about 150 KDa).

Biological activity of purified IL4RFc I—radiolabeled IL-4 with the (KD=O.5 nM) IL4RFc proteins binds same affinity as membrane-bound intact IL-4 receptor. It inhibits the proliferation. of the IL cell line CTLLHuIL-4RI clone D (Idzerda et cit.) in concentrations of 10-1000 ng/ml. In dependent al., addition, it loc. is outstandingly suitable for developing IL-4 binding assays because it can be bound via its Fc part to uucrotiter plates previously coated with, for rabbit IgG, this likewise binds its ligand with high affinity. example, anti—human and in form Example 3: Erythropoietin fusion proteins (EPO) is consists of 166 amino acids and is Mature erythropoietin a glycoprotein which essential for the stimulates the development of erythrocytes. It maturation and the terminal differentiation of erythroid _ 13 _ precursor cells. The CDNA for human EPO has been cloned (EP—A-O 267 678) mature EPO and a signal peptide of 22 amino acids which The cDNA can be used to EPO in modified. mammalian cells and the EPO can be and codes for the 166 amino acids of is essential for secretion. prepare recombinant functional genetically employed clinically for the therapy of anemic manifestations of various etiologies (for example associated. with. acute renal failure). of the improved pharmacokinetic properties, Because straightforward purification and the according to the invention the synthesis of EPO as immunoglobulin fusion protein is particularly advantageous.

Construction of a hybrid plasmid pEPOFc erythropoietin fusion protein. coding for This construction. was carried. out in analogy to that described in Example 2 (section: "Construction of a hybrid plasmid pIL—4RFc coding for IL-4 receptor fusion protein"). Two oligonucleotides able to hybridize with sequences in the vicinity of the initiation codon (A: 5'GATCGATCTCGAGATGGGGGTGCACGAATGTCCTGCCTGGCTGTGG 3') and of the stop codon (B: 5' CTGGAATCGGATCCCCTGTCCTGCAGGCCTCCCCTGTGTACAGC 3') of the EPO CDNA (EP—A O 267 678) were synthesized. Of these, oligonucleotide A cloned in the vector pCES is partially homologous with the sequence of the coding strand, and oligonucleotide B is partially homologous Fig. 7. results with thermostable DNA polymerase in a DNA frag- (598 bp) which, the contains at the 5' end in front of the initiation codon an XhoI site and in which at the 3' with the non-coding strand, cf. Amplification ment based on coding strand, end the codon for the penultimate C-terminal amino—acid residue of EPO (Asp) The reading frame in the BamHI cleavage site is such that is present iii a BamHI recognition sequence. ligation with the BamHI site in pCD4E gamma 1 results in a gene fusion with a reading frame continuous from the initiation codon of EPO CDNA to the stop codon of the heavy chain of IgG1. The desired fragment was obtained and, after treatment with XhoI and BamHI, ligated into the vector pCD4E gamma 1, described above, which had been cut with XhoI/BamHI. The resulting plasmid was called pEPOFC (Fig. 8).

Claims (7)

Claims

1. A soluble fusion protein consisting of the extracellular portion of human IL-4 receptor or a functional part thereof and of_ an Fc part of an immunoglobulin molecule selected from one of the immunoglobulin classes IgG, IgM, IgA and IgE.

2. A fusion protein as claimed in claim 1, wherein the portion of the immunoglobulin molecule is connected via its hinge region to the extracellular part of the IL-4 receptor.

3. A fusion protein as claimed in claim 1, wherein the portion of the immunoglobulin molecule consists of the constant region of the heavy chain of human IgG.

4. A fusion protein as claimed in claim 3, wherein the portion of the immunoglobulin molecule consists of the constant region of the heavy chain of human IgGl or a protein A-binding fragment thereof.

5. A process for preparing fusion proteins as claimed in any of claims 1 to 4, which comprises intro- ducing the DNA coding for these constructs into a mam- malian cell expression system and, after expression, purifying the fusion protein which has been formed by affinity chromatography via the immunoglobulin portion.

6. The use of the fusion proteins as claimed in any of claims 1 to 4 for in vitro diagnosis.

7. A fusion protein as claimed in any of claims 1 to 4 as pharmaceutical. F. R. KELLY & CO., AGENTS FOR THE APPLICANTS