HK1010216B - Fusion proteins with parts of immunoglobulins, their production and use - Google Patents

Description

The invention relates to genetically engineered soluble fusion proteins consisting of human proteins or parts thereof not belonging to the immunoglobulin family and various proportions of the constant region of immunoglobulin molecules.

EP-A 0325 262 and EP-A 0314 317 are known fusion proteins consisting of different domains of the human T cell CD4 membrane protein and human IgG1 components. Some of these fusion proteins bind with equal affinity to the human immunodeficiency virus gp120 glycoprotein as the cell-bound CD4 molecule. The CD4 molecule belongs to the immunoglobulin family and is therefore very similar in terms of its tertiary structure to immunoglobulin molecules. This also applies to the α-chain of the T cell antigen receptor, for which such fusion has also been described (Gascousion et al., Acad. Acad. Acad. Natd. USA, 2937 (1940), 84-29), 1987).

The human proteins of the invention, which are preferably coupled to the aminoterminus of the constant region of immunoglobulin, are not of the immunoglobulin family and are to be classified as follows: (i) membrane-bound proteins, the extracellular domain of which is wholly or partially incorporated into fusion. In particular, these are thromboplastin and cytokine and growth factor receptors, such as the cellular receptors for interleukin-4, interleukin-7, tumor necrosis factor, GM-CSF, G-CSF, erythropoietin; (ii) non-membrane-bound soluble proteins, wholly or partially incorporated into fusion. In particular, these are proteins of therapeutic interest, e.g. e.g. e.g. e.g. e.g. e.g. e.g. e.g. e.g. e.g. e.g. e.g. e.g. e.g. e.g. e.g. e.g. e.g. e.g. e.g. e.g. e.g. e.g. e.g. e.g. e.g. e.g. e.g. e.g. e.g. e.g. e.g. e.g. e.g. e. e.g. e. e.g. e.g. e. e.g. e. e. e.g. e. e. e. e.g. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e. e.

The fusion proteins can be produced in known pro- and eukaryotic expression systems, but preferably in mammalian cells (e.g. CHO, COS, BHK cells).

EP-A 0 269 455 describes a highly purified fusion protein in which the sequence Ala-Pro-Thr-Ser-Ser-Thr-Lys-Lys-Thr-Gln-Arg-Asn-Ser-Met-Leu is coupled to the N-terminus of a human IgE-Fc fragment and a method for the production and purification of this fusion protein useful in the treatment of allergies.

The fusion proteins of the invention are easily purified by affinity chromatography due to their immunoglobulin content and have improved pharmacokinetic properties in vivo.

In many cases the Fc component in the fusion protein is quite beneficial for therapeutic and diagnostic use, thus leading to, for example, improved pharmacokinetic properties (EP-A 0232 262). On the other hand, it would be desirable for some applications to be able to remove the Fc component after the fusion protein has been expressed, detected and purified in the beneficial manner described.

Papain or pepsin are used, for example, to produce F ((ab) fragments from immunoglobulins (Immunology, Roitt, I. et al., Gower Medical Publishing, London (1989)), but they do not divide very specifically. Blood clotting factor Xa, on the other hand, detects the relatively rare tetrapeptide sequence Ile-Glu-Gly-Arg in a protein and causes a hydrolytic breakdown of the protein after cleavage.

The invention thus concerns genetically engineered soluble fusion proteins consisting of human proteins or parts thereof not belonging to the immunoglobulin family and various proportions of the constant regions of heavy or light chains of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE). The immunoglobulin preferred is the constant part of the heavy chain of human IgG, particularly human IgG1, with fusion at the hinge region. In a particular embodiment, the Fc part is easily separated by a co-constructed splicing sequence via factor Xa.

The invention also relates to methods for the genetic production of these fusion proteins and their use in diagnostics and therapy.

The invention is finally explained in further examples.

Example 1: Thromboplastin fusion proteins

The clotting cascade is a process of central importance in the human body. The clotting cascade is finely regulated, in which a variety of cellular factors and plasma proteins interact. The totality of these proteins (and their cofactors) are called clotting factors. The end products of the clotting cascade are thrombin, which induces the aggregation of platelets (platelets), and fibrin, which stabilizes platelet thrombus. Thrombin catalyzes the formation of fibrin from fibrinogen and is itself formed by limited proteolysis of prothrombin.

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It is therefore conceivable that, apart from its use as a diagnostic agent (see below), thromboplastin may also be used as a component of therapeutic agents for the treatment of congenital or acquired blood clotting deficiencies, such as chronic haemophilia caused by a lack of factors VIII, IX or XI or acute blood clotting disorders due to, for example, liver or kidney disease.

Thromboplastin is an integral membrane protein that does not belong to the immunoglobulin family. Thromboplastin cDNA sequences have been published by a 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-ended domain that encodes a polypeptidine domain of 295 amino acids, of which 32 amino acids function as signal terminals. Amino acids are normally found in the cytoplasmic membrane of Amino acids (Amino acids, Amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amine, amine, amine, amine, amine, amine, amine, amine, amine, amine, amine, amine, amine, amine, amine, amine, amine, amine, amine, amine, amine, amine, amine, amine, amine, amine, amine, amine, amine, amine, amine, amine, amine, amine, amine, amine, amine, amine, amine

Thromboplastin is needed as an additive to plasma samples in the diagnosis of clotting. The single-step prothrombin clotting time (e.g. Quick-test) allows the clotting status of the person to be determined. The thromboplastin required for diagnosis is currently obtained from human tissue, with the manufacturing process difficult to standardize, the yield is low and significant amounts of human starting material (placentas) must be provided.

The thromboplastin fusion proteins of the invention are excreted by mammalian cells (e.g. CHO, BHK, COS cells) into culture medium, purified to protein A-sepharose by affinity chromatography and have surprisingly high activity in the single-step prothrombin clotting time.

Cloning of thromboplastin cDNA

The cloning of the thromboplastin cDNA was carried out using the published sequence of Scarpati et al., Biochemistry, vol. 26 (1987), 5234-5238, from which two oligonucleotide probes (see Fig. 1) were derived and from which a cDNA bank from human placenta was sequenced (Grundmann et al., Proc. Natl. Acad. Sci. USA, vol. 83 (1986), 8024-8028).

A clone, 2b-Apr5, which is used for the further procedure, encodes for the same amino acid sequence as the cDNA described in Scarpati et al. The total sequence of the clone 2b-Apr5 with the derived thromboplastin amino acid sequence is shown in Fig. 2.

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

The plasmid pCD4E gamma 1 (EP 0 325 262 A2; deposited at ATCC under number 67610) is used to express a fusion protein from human CD4 receptor and human IgG1. The DNA sequence encoding the extracellular domain of CD4 is removed from this plasmid with the restriction enzymes HindIII and BamHI. Only a partial cleavage with the enzyme HindIII may be performed to cut at one of the two HindIII sites contained in pCD4E gamma 1 (position 2198).DNA amplification with thermostable DNA polymerase allows a given sequence to be altered by adding any sequence at one or both ends. Two oligonucleotides have been synthesised with sequences in the 5'-untranslated region (A: 5'GATCGATTAAGGAACCCTCGATCCGGG 3') or coding region (B: 5'GATCTATCCGATCCGG 3') respectively, which can be homologous to the OGAG-coding thromboplastin, which is homologous to the OGAG-coding thromboplastin.

Thus, after amplification, a DNA fragment (827 bp) is present which (in relation to the coding strand) contains a HindIII site at the 5' end before the start of the coding sequence, a BamHI site at the 3' end after the codons for the first three amino acid residues of the transmembrane region. The reading frame at the BamHI interface is such that after ligation with the BamHI site in pCD4E gamma 1 gene fusion is achieved with a continuous reading frame from the thromboplastin cDNA initiation ligand to the stop codon of the heavy ligand of IgG1. The desired fragment was obtained and after treatment with HindIII and the above described with the inhibited (BHI) gamma (III) and partially formed with the plasma pF4 (F4), the resulting ligand is called PTF1 (CD4F).

Transfection of pTF1Fc in mammalian cells

The fusion protein encoded by the plasmid pTF1Fc is hereinafter referred to as pTF1Fc. pTF1Fc was transiently expressed in COS cells by translocation of COS cells with pTF1Fc using DEAE dextran (EP A 0325 262).

Indirect immunofluorescence studies showed about 25% of the cells transfected, and 24 hours after transfection, the cells were transferred to a serum-free medium, and this cell surplus was harvested after another three days.

Purification of pTF1Fc fusion protein from cell culture residues

170 ml of residues of transiently transfected COS cells were collected overnight in a batch process at 4°C with 0.8 ml of protein A-sepharose in a column, washed with 10 volumes of washing buffer (50mM Tris buffer pH 8.6, 150mM NaCl) and eluted with elution buffer (100mM Citric acid: 100mM NaCitrate 93:7) into 0.5 ml fractions. The first 9 fractions were immediately neutralized, combined with 0.1 ml of 2M Tris buffer pH 8.6 each, and the protein contained was purified by three cycles of concentration/ dilution in the Microcontraconcentrator (Centricon 30) in Tris buffer (50mM Tris buffer pH 7.4, 50mM NaCl, 1mK) in an electrically conductive medium (DPAPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA) as described in Nature (DPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA, SPA,

Biological activity of purified TF1Fc in the prothrombin clotting time

TF1Fc fusion protein is active at low concentrations (> 50 ng/ml) in the single-step prothrombin clotting time (Vinazzer, H. Clotting physiology and methods in the blood clotting laboratory (1979), Fisher Verlag, Stuttgart).

Example 2: Interleukin-4 receptor fusion proteins

Interleukin-4 (IL-4) is synthesized by T cells and was originally called B cell growth factor because it can stimulate B cell proliferation. It exerts a variety of effects on these cells. In particular, this is the stimulation of the synthesis of molecules of the immunoglobulin subclasses IgG1 and IgE in activated B cells (Coffmann et al., Immunol. Rev., vol. 102 (1988) 5). In addition, IL-4 also regulates the proliferation and differentiation of T cells and other haulipotent cells. It thus contributes to the regulation of allergic and immunological reactions in other cells. IL-4 binds with high affinity to a specific human receptor cDNA, for which IL-4 has been coded for the receptor et al. (Ildera, et al. 1988).The analysis of the amino acid sequence derived from the cDNA sequence shows that the IL-4 receptor consists of a total of 825 amino acids, with the N-terminals of 25 amino acids acting as a signaling peptide. The mature human IL-4 receptor consists of 800 amino acids and has a three-dominated structure like thromboplastin: (i) amino-terminal extracellular domain (207 amino acids); (ii) transmembrane region (24 amino acids) and (iii) cytoplasmic domain (569 amino acids). In the extracellular domain, there are six potential sites for N-glycosylation (T-X-Toietin/serine). IL-4 is a member of the human receptor family, and is not a member of the human receptor family (I-II, IL-2, and IL-2, and is not a member of the human receptor family, but of the human receptor family β-E.The new family of haematopoietin receptors, which includes 4 cysteine residues and a conserved sequence (Trp-Ser-X-Trp-Ser) in the extracellular domain, is located near the transmembrane region.

Due to the described function of the IL-4/ IL-4 receptor system, a therapeutic use of a recombinant form of the IL-4 receptor to suppress IL-4-mediated immune responses (e. g. transplant rejection, autoimmune diseases, allergic reactions) is possible.

The quantities of substances required for a therapy make it necessary to produce such molecules by genetic engineering. According to the invention, the synthesis of soluble forms of the IL-4 receptor as an immunoglobulin infusion protein is particularly advantageous because of the ease of purification by affinity chromatography and the improved pharmacokinetic properties.

The IL-4 receptor fusion proteins are excreted by mammalian cells (e.g. CHO, BHK, COS cells) into the culture medium, purified to protein A-sepharosis via affinity chromatography and have surprisingly identical functional properties to the extracellular domain of the intact membrane bound IL-4 receptor molecule.

Design of a hybrid plasmid encoding IL-4 receptor fusion protein pIL-4RFc.

Err1:Expecting ',' delimiter: line 1 column 168 (char 167)The test chemical is a DNA fragment (836 bp) which is located on the coding strand at the 5' end before the start of the coding sequence, contains an XhoI site, at the 3' end before the last codon of the extracellular domain contains a gamma-BAM-C. The reading at the IgHI interface is that the read-only reading of the LHI-C is achieved by initiating a stop codon in the ILG-41 receptor by reading the LHI-C from the KCD4 receptor in the LHI-C to the LHI-C receptor in the LCD4 receptor.The desired fragment was obtained and ligated after treatment with XhoI and BamHI into the XhoI/BamHI-intersected vector pCD4E gamma 1 described above.

Transfection of pIL4RFc in mammalian cells

The fusion protein encoded by the plasmid pIL4RFc is hereinafter referred to as pIL4RFc. pIL4RFc was transiently expressed in COS cells. COS cells were translocated with pIL4RFc using DEAE dextran (EP A 0325 262). Indirect immunofluorescence studies showed approximately 25% of translocated cells. 24 h after transfection, the cells were transplanted into serum-free medium. This cell transfer was harvested after another three days.

Cleaning of IL4RFc fusion protein from cell culture residues

500 ml of residues of transiently transfected COS cells were collected overnight in a batch process at 4°C with 1.6 ml of protein A-sepharose in a column, washed with 10 volumes of washing buffer (50mM Tris buffer pH 8.6, 150mM NaCl) and eluted with elution buffer (100mM citric acid: 100mM NaCitrate 93:7) into 0.5 ml fractions. The first 9 fractions were immediately neutralized, combined with 0.1 ml of 2M Tris buffer pH 8.6 each, and the protein contained was purified by three cycles of concentration/ dilution in the Amicon microconcentrator (Centricon 30) in T-buffer (50mM Tris buffer pH 7.5, 50mM NaCl, 1m KDS) and dissolved in the L-DPAPAPA (L-DPAPAPAPA) (PPA 2280-740) in an electrical solution containing about 150 KDS.

Biological activity of purified IL4RFc

IL4RFc binds 125I radiolabelled IL-4 with the same affinity (KD=0.5nM) as membrane bound, intact IL-4 receptor. It inhibits proliferation of IL-4 dependent cell line CTLLHuIL-4RI Clone D (Idzerda et al., a.a.O.) at concentrations of 10-1000 ng/ml. It is also excellent for the development of IL-4 binding tests, as it can bind via its Fc part to e.g. rabbit anti-human IgG coated microplates and in this form also binds to its ligand with high affinity.

Example 3: Erythropoietin fusion proteins

Mature EPO (EPO) is a glycoprotein of 166 amino acids that is essential for the development of erythrocytes. It stimulates the maturation and terminal differentiation of erythroid progenitor cells. The cDNA for human EPO has been cloned (EPA-0267 678) and encodes for the 166 amino acids of mature EPO and a signaling peptide essential for secretion of 22 amino acids.

According to the invention, the synthesis of EPO as an immunoglobulin infusion protein is particularly advantageous due to its easy purification and improved pharmacokinetic properties.

Design of a hybrid plasmid pEPOFc coding for erythropoietin fusion protein.

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Claims (7)

1. A process for preparing soluble fusion proteins 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, which comprises introducing the DNA coding for these constructs into a mammalian cell expression system and, after expression, purifying the fusion protein which has been formed by affinity chromatography via the immunoglobulin portion.
2. A process 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 process 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 process as claimed in claim 3, wherein the portion of the immunoglobulin molecule consists of the constant region of the heavy chain of human IgG1 or a protein A-binding fragment thereof.