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WO1998011241A1 - Systeme d'expression oligocistronique destine a la production de proteines heteromeres - Google Patents

Systeme d'expression oligocistronique destine a la production de proteines heteromeres Download PDF

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WO1998011241A1
WO1998011241A1 PCT/EP1997/004765 EP9704765W WO9811241A1 WO 1998011241 A1 WO1998011241 A1 WO 1998011241A1 EP 9704765 W EP9704765 W EP 9704765W WO 9811241 A1 WO9811241 A1 WO 9811241A1
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val
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Ilka Von Hoegen
Christa Burge
Wolfgang Brümmer
Reinhard Dunker
Erwin Rieke
Thomas Welge
Hansjörg HAUSER
Christian Mielke
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Merck Patent Gmbh
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Priority to AU42088/97A priority Critical patent/AU4208897A/en
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/525—Tumour necrosis factor [TNF]
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
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    • C07K14/55—IL-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32611—Poliovirus
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    • C12N2830/00—Vector systems having a special element relevant for transcription
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/15—Vector systems having a special element relevant for transcription chimeric enhancer/promoter combination
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/46—Vector systems having a special element relevant for transcription elements influencing chromatin structure, e.g. scaffold/matrix attachment region, methylation free island
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/60—Vector systems having a special element relevant for transcription from viruses
    • C—CHEMISTRY; METALLURGY
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES
    • C12N2840/206—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES having multiple IRES
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a mammalian expression system for the production of recombinant heteromeric proteins, preferably antibodies and antibody fusion proteins such as antibody-cytokine fusion proteins and fragments thereof, by means of tri- or oligocistronic expression vectors which are under the control of a strong promoter/enhancer unit and which contain a selection marker as one of the cistrons.
  • This selection marker guarantees together with at least two IRES elements a robust and stable production of the heteromeric proteins in excellent yields.
  • herteromeric proteins in mammalian cells such as antibody molecules
  • two vectors have been used which frequently leads to unpredictable overexpression of one of the protein chains in comparison with the second one. Where one chain is relatively overexpressed the cells begin to suffer resulting in instability of production and/or in purification problems (e.g. light chain dimers).
  • One traditional way to overcome this problem is to cotransfer the vectors in a well defined ratio into the host cells. This requires that the plasmid copies are accepted and integrated simultaneously and stable, and that the plasmid ratio remains constant during cell division. Only for a few systems satisfying results were obtained up to now.
  • Another traditional way is to use independent transcription units located on one plasmid.
  • the different genes are present on the vector in a correct ratio.
  • promoters of comparable strength are used equal amounts of the desired protein chains should be obtained.
  • different stability and translation efficiencies of the mRNAs which are coding for the different proteins, and different transcription efficiencies of the genes lead to an unequal synthesis of the desired protein chains.
  • di- and multicistronic vectors were developed recently.
  • the gene units used (coding for the desired proteins, cistrons) are under the control of one single promoter. Normally, only the first cistron located at the 5' terminus is translated efficiently in eucaryotes since the initiation of the translation occurs according to the "cap"- dependent mechanism.
  • the following cistrons are translated insufficiently or not at all. It has been found that the translation of the following cistrons in multicistronic systems can be initiated and pushed by using sequences having no "cap" structure. Such sequences are obtainable from non-translated sections of some viruses, such as poliovirus and encophalomyocarditis virus (Jang et al., 1988, J. Virol. 62:2636; Jang et al., 1989, J. Virol. 63: 1651 ; Pelletier und Sonnneberg, 1988, Nature 334:320).
  • viruses such as poliovirus and encophalomyocarditis virus (Jang et al., 1988, J. Virol. 62:2636; Jang et al., 1989, J. Virol. 63: 1651 ; Pelletier und Sonnneberg, 1988, Nature 334:320).
  • IRES internal ribosomal entry site
  • a bicistronic expression system is described using well defined genes, namely encoding PDGF chains A and B (platelet derived growth factor) separated by an IRES containing unit. No selection marker is used in this system. It has not been reported until now that heteomeric proteins such as antibody heavy and light chains have been expressed in stoichiometric and stable formation by tri- or oligocistronic systems. It has not been reported, furthermore, that the use of a selction marker as one of the cistrons leads to transformed cells which have an extraordinary ly high stability.
  • the biologically active ligand for an antibody-directed targeting should induce the destruction of the target cell either directly or through creating an environment lethal to the target cell.
  • the biologically active ligand can be a cytokine such as IL-1, IL-2, IL-4, IL- 6, IL-7, IL-10, IL-13, IFNs, TNF ⁇ or CSFs. These cytokines have been shown to elicit anti-tumor effects either directly or by activating host defense mechanisms (e.g. Mire-Sluis, TIBITECH, 1 1 :74). For instance, IL-2 is considered the central mediator of the immune response.
  • IL-2 has been shown to stimulate the proliferation of T- cells and NK-cells and to induce lymphokine-activated killer cells (LAK). IL-2 enhances the cytotoxicity of T-cells and monocytes.
  • TNF alpha has found a wide application in tumor therapy, mainly due to its direct cytotoxicity for certain tumor cells and the induction hemorrhagic regression of tumors.
  • TNF alpha potentiates the immune response: it is a costimulant of T-cell proliferation, it induces expression of MHC class I and II antigens and TNF alpha, IFN and IL-1 secretion by macrophages.
  • most of the known cytokines activate effector cells, but show no or only weak chemotactic activity.
  • Chemokines are chemotactic for many effector cells and enhance their presence at the tumor site and induce a variety of effector cell functions (e.g. Miller and Krangel, 1992, "Biology and Biochemistry of the Chemokines, --, Critical Reviews in Immunology 12:17).
  • suitable chemokines according to the invention are IL-8 and MIP 2 ⁇ and MIP 2 ⁇ which are members of the C-X-C chemokine superfamily (also known as small cytokine superfamily or intecrines).
  • Epidermal growth factor is a polypeptide hormone which is mitogenic for epidermal epithelial cells. When EGF interacts with sensitive cells, it binds to membrane receptors (EGFR).
  • EGFR membrane receptors
  • the EGFR is a trans-membrane glycoprotein of about 170 kD, and is a gene product of the c-erb-B proto- oncogene.
  • the murine monoclonal antibody mAb425 was raised against the human A431 carcinoma cell line (ATCC CRL 1555; US 5,470,571) and was found to bind to a polypeptide epitope on the external domaine of the EGFR. It was found to inhibit the binding of EGF and to mediate tumor cytotoxicity in vitro and to suppress tumor cell growth of epidermal and colorectal carcinoma-derived cell lines in vitro (Rodeck at al., 1987, Cancer Res., 47:3692).
  • oligocistronic expression units comprising at least two IRES elements where the different heteromeric chains, e.g the heavy and light protein chain of an antibody, are cotranslated from one mRNA molecule comprising a sequence encoding a selection marker.
  • the strength of the effect caused by the selection marker in this system is surprising and could not be expected compared with usual expression systems of the prior art.
  • the effect is especially strong when the gene encoding the selection marker is located at the end of all cistrons each separated by IRES units. This is not the case if the selection pressure is removed or if the selection marker is used in traditional expression vectors. Using the selection marker as last cistron forces the cell to produce the linked protein / proteins.
  • constructs according to the invention allow equimolar production of the heteromeric protein chains and guarantee selection and stable, long-term expression of the optimal production clones by concomittant expression of the selection marker, because only those clone will grow under selection pressure which express the entire cistronic expression unit.
  • IRES sequence located behind a bicistronic unit comprising the sequence coding for the light chain of an antibody, an IRES sequence and a sequence coding for a fusion protein consisting of the heavy chain of an antibody fused to another biologically active protein, such as a cytokine or chemokine, is very advantageous with respect to a stable expression in excellent yields.
  • the present invention relates to a mammalian expression system for the production of heteromeric proteins, preferably recombinant antibodies and more preferably antibody fusion proteins such as antibody-cytokine fusion proteins and fragments thereof.
  • the invention relates, preferably, to such a expression system which is able to produce antibody fusion proteins or fragments thereof, wherein the antibody binding sites are directed to the human EGF-receptor and the antibody is covalently linked to a biologically active ligand such as a growth and/or differentiation factor, above all TNF alpha, or IL-2.
  • the invention discloses a set of vectors which comprise oligocistronic, preferably tri- and tetracistronic expression units driven by a single strong promoter hybrid linked to genes encoding protein chains of the light chain, the heavy chain and the active ligand and, additionally a selection marker in the promoter- distal position.
  • oligocistronic expression vector suitable for the production of a heteromeric protein consisting of at least two protein chains in a mammalian host cell comprising (i) a promoter / enhancer sequence,
  • the order of the genes located in the vector construct is important with respect to the described advantageous effects.
  • the gene coding for the selection marker should be located as last cistron within the vector construct.
  • the gene encoding the light chain of the antibody should be located in upstream position before the gene coding for the heavy chain.
  • (6) optionally a sequence comprising the sequence encoding a third or further chain of the heteromeric protein or a fragment thereof (iv), a sequence comprising a third or further IRES element (vi) included,
  • the advantage of this system is also shown in Fig. 17 and 18. Under selection pressure the clones produce in a stable manner the different chains of the heteromeric protein but without selection pressure or "wrong" position of the selection marker the stable productivity is rapidly lost.
  • the greatest advantage of the system is that (heteromeric) proteins can be expressed which can be toxic to the host cells like proteases, glutamate receptor subtypes and serotonin receptor subtypes or antibody fusion proteins wherein the non-antibody partner is normally highly toxic for the host cells.
  • a corresponding expression system is object of the invention, wherein the sequence (ii) encodes the light chain and the sequence (iii) comprises a sequence encoding the heavy chain of a monclonal antibody or a fragment thereof.
  • the teaching of this invention is also applicable for heteromeric proteins other than antibodies, for heteromeric proteins having more than two chains, and even normal (one-chain) proteins having toxic activity against the host cell and, finally, heteromeric proteins (e.g. antibody fusion proteins) having strong toxic activity caused by a part of said heteromeric protein..
  • a corresponding expression system is object of the invention, wherein the sequence (iii) consists of two sequences (iiia, iiib), wherein (iiia) encodes the heavy chain of an antibody or a fragment thereof and (iiib) encodes a biologically active ligand, such as a cytokine or a chemokine or a fragment thereof, in order to form a fusion protein.
  • a biologically active ligand such as a cytokine or a chemokine or a fragment thereof
  • a special and preferred embodiment of the invention is a tricistronic expression vector as defined above and in the claims, wherein the sequence (iiia) and the sequence (iiib) are linked directly in order to encode a fusion protein.
  • the expression vector according to the invention may, optionally, contain eucaryotic sequence elements such as SAR/MAR elements to further increase production and stability of the system.
  • SAR/MAR elements eucaryotic sequence elements
  • the expression of certain genes has been reported to respond positively to butyrate.
  • the stimulatory effect of butyrate is largest if one or two scaffold matrix-attached regions (SAR/MAR elements) are present adjacent to the gene (Schlacke et al., 1994, Biochemistry 33:4197). Only after integration of the constructs in to the genome of the host cell these regions increase the expression of adjacent genes in an orientation- and position- independent fashion.
  • Gene activation causes the apparent loss of nucleosome structure ahead of the SAR element and a similar change has been demonstrated by the action of butyrate. Presence of both SARs and butyrate act synergistically in enhancing gene expression (Klehr et al. 1992, Biochemistry 31 :3223).
  • an expression vector defined above and in the claims is object of the invention, comprising, additionally, one or two, preferably two, SAR elements.
  • one SAR element is located in front of the promoter/enhancer region the second one behind the sequence encoding the selection marker.
  • other locations are also possible.
  • the invention relates to antibody fusion proteins, wherein the non- antibody protein is a biologically active protein.
  • expression vectors are object of the invention, wherein a sequence (iiib) is used which encodes a cytokine or chemokine such as TNF alpha, IL-2 and IL-8.
  • sequences (ii) and (iii) comprise sequences coding for the light and heavy chain of a monoclonal anti-EGFR antibody, preferably, humanized monoclonal antibody 425 (mAb425) or fragments thereof.
  • a monoclonal anti-EGFR antibody preferably, humanized monoclonal antibody 425 (mAb425) or fragments thereof.
  • the invention is not restricted to anti-EGFR antibody or mAb425, respectively, but includes also any other monoclonal antibodies directed to a variety of specificities, for example mAb361.
  • an expression vector comprising the following units in the given order: the CMV/MPSV promoter/enhancer sequence followed by the sequence encoding the mAb425 light chain, followed by the sequence from 5'-UTR poliovirus containing an IRES element, followed by a fusion gene encoding a fusion protein consisting of the heavy chain of humanized mAb425 and fused at its C-terminus the sequence encoding TNF alpha or IL-2, followed by another IRES element from 5'- UTR poliovirus, followed by a sequence coding for puromycin acetyl transferase as selection marker and, finally a nucleotide sequence derived from the polyadenylation signal of SV40.
  • an expression system comprising a mammalian host cell transformed with an expression vector specified above and in the claims, preferably, wherein the host cell is CHO or BHK.
  • a heteromeric protein preferably an antibody, especially an antibody fusion protein, especially a mAb425/TNF alpha or mAb425/IL-2 antibody fusion protein, or fragments thereof, by cultivating the host cells of an expression system as specified above and in the claims in a suitable nutrient and separating, if a tricistronic vector is used, the complete and active antibody fusion protein from the cells and / or the medium.
  • AmpR Ampicillin resistance gene
  • IRES Poliovirus derived internal ribosomal entry site
  • MPSV Promoter/Enhancer
  • CMV Cytomegalo virus promoter
  • Puromycin R Puromycin resistance gene
  • SV 40 pA SV 40 polyadenylation site.
  • Stability of BHK-21 mAb425CHl clones Stability of three different clones was determined over the time period indicated. The production of mAb425CHl fusion protein of 10 ⁇ cells/ml per 24 hrs was determined in an anti-Ig based ELISA. Cells were cultured in medium with (+P) or without (- P) Puromycin.
  • Fig. 3 Stability of a BHK21 mAb425CHl-TNF ⁇ clone. Cells were cultured in
  • Fig. 7 Determination of IL-2 activity of purified mAb425CH3-IL-2.
  • IL-2-dependent mouse CTLL2 cells were incubated with mAb425CH3-IL-2 or rec. human IL-2 (WHO Standard).
  • Concentrations of fusion protein are shown as mAb425 equivalents, determined by an ELISA based on an anti- idiotypic antibody specific for mAb425. 5x10 ⁇ were cultured for 2 days and pulsed with 0,5 ⁇ Ci ⁇ H-Thymidine 18 hrs before harvesting.
  • Fig. 8 pMCLDHAP tricistronic vector for the expression of mAb425CH3-TNF ⁇ .
  • AmpR Ampicillin resistance gene
  • IRES Poliovirus derived internal ribosomal entry site
  • MPSV Promoter/Enhancer
  • CMV Cytomegalo virus promoter
  • Puromycin R Puromycin resistance gene
  • SV 40 pA SV 40 polyadenylation site.
  • BHK-21 cell clones transfected with pMCLDHAP and expressing mAb425CH3-TNF ⁇ fusion protein were either cultivated under puromycin pressure (+) or grown in the absence of puromycin (-) for the indicated times.
  • Graph A shows antibody fusion protein secretion ( ⁇ g IgG/ml x 24 hr).
  • B is a Southern blot of chromosomal DNA prepared from cells which were taken at the indicated times. The DNA was restricted with PstI and hybridized with a labelled PstI fragment from pMCLDHAP (1231 bp) encompassing part of the heavy chain fusion protein encoding cDNA (he), mbhl represents a single copy DNA fragment ( 1900 bp) of a hamster c-myc gene which was cohybridized using a specific probe (see example 7). Since both probes are labelled with the same specific activity and their length is similar, the intensity of the he band corresponds to the copy number of the integrated expression plasmid.
  • TNF ⁇ activity of purified mAb425CH3-TNF ⁇ on MCF7 cells The TNF ⁇ -sensitive and EGF-R negative human breast adenocarcinoma cell line MCF7 was used to determine the TNF ⁇ activity of the mAb425CH3-TNFa fusion protein. Concentrations of fusion protein are shown as mAb425 equivalents, determined by an ELISA based on an anti- idiotypic antibody specific for mAb425. humanized mAb425 and rTNF ⁇ are mixed at a ratio of 6:1 reflecting the molecular ration of both parts in the fusion protein. 5x10 ⁇ were cultured for 4 days and pulsed with 0,5 ⁇ Ci 3H-
  • TNF ⁇ mediated cytotoxicity of purified mAb425CH3-TNF ⁇ is dependent on TNF ⁇ sensitivity.
  • the TNF ⁇ -resistant and EGF-R-positive human carcinoma cell line A431 was used to determine the specificity of the mAb425CH3-TNF ⁇ fusion protein. Concentrations of fusion protein are shown as mAb425 equivalents, determined by an ELISA based on an anti- idiotypic antibody specific for mAb425.
  • Humanized mAb425 and rTNF ⁇ are mixed at a ratio of 6: 1 reflecting the molecular ratio of both parts in the fusion protein. 5x10 ⁇ were cultured for 4 days and pulsed with 0,5 ⁇ Ci ⁇ H-Thymidine 18 hrs before harvesting.
  • mAb425CH3-TNF ⁇ is highly cytotoxic for EGF-R-positive and TNF ⁇ - sensitive human tumor cell lines.
  • the human mamma carcinoma cell lines BT20 and the human melanoma cell line C8161 are both TNF ⁇ -sensitive and EGF-R-positive.
  • Concentrations of fusion protein are shown as mAb425 equivalents, determined by an ELISA based on an anti-idiotypic antibody specific for mAb425.
  • mAb425 and r TNF ⁇ are mixed at a ratio of 6:1 reflecting the molecular ratio of both parts in the fusion protein. 5x10 ⁇ were cultured for 4 days and pulsed with 0,5 ⁇ Ci 3H-Thymidine 18 hrs before harvesting.
  • Fig. 16 Hystory of relevant vectors of the invention.
  • ELISA detecting the antibody part Cells are cultured for the indicated days in medium with (+P) or without (-P) Puromycin.
  • Fig. 18 Stability of the cell clone rBHK21mAb425-CH3-IL2698-8 with (CHO-M +
  • heteromeric protein means a protein which natuarally consists of two or more chains. Only if the corresponding chains are associated and folded correctly the full biological activity of the heteromeric protein can be obtained.
  • mAb425CHl- means an antibody construction containing the light chain, the variable region of the heavy chain, and the
  • mAb425CH2- means an antibody construction containing the light chain, the variable region of the heavy chain, and the
  • mAb425CH3- means an antibody construction containing the light chain, the variable region of the heavy chain, and the
  • a sequence encoding does not mean exclusively the specific coding sequence, but may include also a sequence comprising said specific coding sequence, provided that no other statement is made.
  • Said additional sequences indicated above and coding for proteins [ii, iii (iiia, iib), iv, vi] can be prolonged or shortend each by 1 to 20 amino acids provided that the specific biological properties are not substantially amended.
  • Prolongation can be caused, for example, by linker or leader peptides.
  • the expression vector constructs according to the invention may contain introns which are not tranlated into amino acids.
  • Prolongations and deletions of coding regions may occur, preferably, at the C- and / or N-terminus of the corresponding specific peptide or protein.
  • Preferred deletions according to the invention may occur at the C-terminus of the heavy chain of the antibody and the N-terminus of the biological ligand.
  • the invention includes also mutations and varients of the sequences indicated in detail having the same or a very similar biological activity.
  • Such mutations and varients can be produced by accident (e.g. spontaneous mutations, natural radiation) or by intended chemical or physical activities.
  • antibody fragment means according to the invention an antibody fragment as defined above (mAb-CHl, mAb-CH2) as well as complete antibody (mAb-CH3) which is shortend by 1 to 20 amino acids at the C- terminus of its constant region.
  • biological active ligand means according to the invention any protein or peptide ligand which is effective against a target cell, above all, against a target cell which is recognized by the antibody part of the antibody fusion protein.
  • the effect of the biological ligand may be, for instance, a toxic and/ or lysing and / or inhibiting one against the target cell, preferably a tumor cell. Examples of suitable biological active ligands are given above.
  • biological activ ligand fragment means according to the present invention a biological ligand (cytokines, chemokines) which is usually shortened by 1 to 20 amino acids at its N-terminus which is connected directly, or optionally via a linker peptide, to the (optionally shortened) C- terminus of the constant region of the antibody heavy chain.
  • the selection marker according to the invention can be in principal any known selection marker suitable for high expression systems. Examples are enzymes such as puromycin-acetyl transferase or neomycin phosphotransferase. Puromycin-acetyl transferase is preferred according to this invention.
  • dominant acting genetic markers useful for monitoring gene transfer in mammalian cells that are based on procaryotic genes encoding key steps in the synthesis of the essential amino acids, such as tryptophane or histidine can be used. Under appropriate conditions, expression of these genes obviates the nutritional requirements for their respective amino acid products. Expression of the ⁇ subunit of tryptophan synthase (trpB, EC 4.2.1.20) of Escherichia coli allows mammalian cell survival and multiplication in medium containing indole in place of tryptophane.
  • the hisD gene of Salmonella typhimurium encodes histidinol dehydrogenase (EC 1.1.1.23), which catalyses the two-step NAD+-dependent oxidation of L- histidinol to L-histidine. In medium lacking histidine and containing histidinol only mammalian cells expressing the hisD gene survive.
  • Use of these markers is advantageous over the use of antibiotics because for either trp or his selection the substitute nutrients indole or histidinol are readily available, inexpensive, stable, permeable to cells and convertible to the end product in a step controlled by one gene (Bode et al. 1995, Int. Rev. Cytol., R. Berezney & K.W. Jeon eds. Academic Press, Vol 162A:389)
  • IRES sequences all sequences deriving from viral, synthetic origin or from cells can be used which allow an internal binding of ribosomes.
  • examples for such sequences are the 5'-UTRs elements from poliovirus type 1 , 2 or 3 (picorna virus), from “encephalomyocarditis virus” (EMCV) (Sugimoto et al., 1994, BioTechnol. 12:694), from “Theilers murine encephalomyelitis virus” (TMEV), from “foot and mouth disease virus” (FMDV), from “bovine enterovirus” (BEV), and from “coxsackie B virus” (CBV).
  • poliovirus type 1 , 2 or 3 picorna virus
  • EMCV encephalomyocarditis virus
  • FMDV foot and mouth disease virus
  • BEV bovine enterovirus
  • CBV coxsackie B virus
  • the tri- or oligocistronic expression vector according to the invention works with a single strong promoter/enhancer unit.
  • suitable promoters/enhancers are: CMV (Boshart et al., 1985, Cell 41:521); MPSV- LTR (Laker et al.,1987, Proc. Natl. Acad. Sci. USA 74,:8458); MPSV-CMV; RSV (Gorman et al., 1982, Proc. Natl. Acad. Sci. USA 79:6777); SV40 ( ⁇ rtelt et al., 1988, Gene 128: 247).
  • CMV (promoter of the cytomegalie virus) is the preferred unit according to the invention.
  • the fusion protein described in the examples contains a monoclonal antibody with specificity for the human EGF-receptor(EGFR).
  • the monoclonal mAb425 was raised against the human A431 carcinoma cell line and found to bind to a polypeptide epitope on the external domdin of the EGFR.
  • the heavy chain mAb425 antibody was fused C-terminally to cytokines/chemokines such as IL-2, IL-4, IL-7, TNF ⁇ and IL-8 as biologically active ligands.
  • the constructs encoding these immuno- conjugates were generated with recombinant DNA technologies.
  • the immuno-conjugates contain the variable region of the antibody heavy chain and the CHI domain of the constant region (antibody- CHI conjugates), or the CHI and CH2 domain of the constant region (antibody-CH2 conjugates) or the CHI, CH2 and CH3 domain of the constant region (antibody-CH3 conjugates) fused to the biologically active ligand.
  • the appropriate light chain immunoconjugates can be generated which target antigen-bearing cells and deliver an active ligand to to a specific site in the body.
  • the C-terminal amino acid sequence of the junctional region of CHI and CH3 fusion proteins is not involved in any secondary structure elements according to the hypothetical computer model. In these regions several putative sites for proteolytic cleavage are present.
  • N-terminal cytokine sequences are frequently involved in receptor binding and biological activity, e.g. in human TNF ⁇ amino acid sequences between positions 11 and 35 appear to be critical for receptor binding and triggering of biological responses (Goh & Porter, Prot. Eng. 4:385, 1991).
  • loss of activity is caused by inaccessibility of relevant amino acids due to interference of the antibody part linker sequences can be introduced which consist of repetitive units containing amino acids which do not interfere with chemical stability and biological activity, e.g. see Curtis et al. Proc. Natl. Acad. Sci. USA, 88:5809, 1991.
  • a system of expression vectors is provided, which allows easy generation of expression vectors for synthesis of three proteins from a tricistronic expression unit.
  • IRES internal ribosomal entry sites
  • the tricistronic mRNA is transcribed from any strong promoter such as a single hybrid MPSV/CMV promoter/enhancer.
  • the selection marker may be puromycin acetyl transferase, neomycin phosphotransferase or procaryotic genes such as the ⁇ -subunit of tryptophane synthase (trpB) derived from E. coli or the histidinol dehydrogenase (hisD) of Salmonella typhimurium or any resistance marker known in the art.
  • the selection marker is preferably located in the promoter-distal position to ensure stable expression of the entire cistron.
  • expression is further enhanced by inclusion of one or two, preferably two, scaffold/matrix- attached regions (SAR/MAR elements) into the expression vector.
  • SAR/MAR elements scaffold/matrix- attached regions
  • Expression can be synergistically by SAR/MAR elements and butyrat added to the medium.
  • the protein sequence between both parts of the fusion protein can be shortened up to a limit where the biologically active ligand looses its activity.
  • both parts of the fusion protein can be combined by introducing linker sequences which consist of repetitive units containing preferentially the amino acids alanin, glycin and serin.
  • said proteins such as immunoconjugates by transfering the expression vector which contains the tricistronic construct into appropriate host cells such as BHK-21 cells, CHO cells, SP2/0 cells or myeloma cells.
  • Fusion protein constructs consisting of mAb425 and cytokines or chemokines has been disclosed in EP 0659 439 and EP 0706 799, respectively. Fusion proteins have been constructed on the basis of chimeric and humanized mAb425 with cDNAs encoding cytokines such as IL-2, IL-4, IL-7 and TNF ⁇ or chemokines such as IL-8 and MIP-2 ⁇ and Mip2- ⁇ fused to the CHI, or CH2 or CH3 domain of the constant region of the mAb425 heavy chain, respectively.
  • the techniques used can be taken, for example from the two European patent publications indicated above which are incorporated in this application by reference.
  • the vector system according to the invention leads to an new and innovative production system for high expression of heterodimeric proteins in eucaryotic cells such as antibody-cytokine/chemokine fusion proteins.
  • Light chain and heavy-chain cytokine/chemokine fusion are transcribed together with a selectable marker from one tricistronic mRNA.
  • the advantage of this system is twofold: First, unpredictable overexpression of one of both chains which frequently leads to instability of production and purification problems will be avoided because both chains will be produced at equimolar amounts. Secondly, coupling of product and selection marker in the promoter-distal position guarantees stable and longterm expression of the product. Taken together, the system described herein represents a robust process for production of complex proteins in eucaryotic cells employing different fermentation techniques.
  • Plast cells for the expression of a monovalent immunoconjugate including only the CHI domain or divalent immunoconjugates including the CHI and CH2 and CH3 domains into host cells can be achieved by electroporation, DEAE dextrane, calcium phosphate, Lipofectin, protoplast fusion or any known method in the art. Any host cell type may be used provided that the recombinant DNA sequences encoding the immunoconjugate and the appropriate light chain are properly transcribed into mRNA in that cell type.
  • Host cells may be mouse myeloma cells which do not produce immunoglobulin such as Sp2/0-AG14 (ATCC CRL 1581), NSO (Gaffe & Milstein, 1991, Meth. Enzymol. 73(B):3), P3X63Ag8.653 (ATCC CRL 1580) or hamster cells such as CHO-
  • the vectors pSBC-1 and pSBC-2 have been developed as monocistronic expression vectors. Both vectors contain the SV40 origin of replication, the SV40 early promoter, the SV40 19s splice donor and 19s acceptor, the SV40 polyadenylation signal, procaryotic sequences such as the origin of replication from ColEl and the Ampicillin resistance gene.
  • pSBC-1 contains the internal ribosomal entry site sequence (IRES) of polio virus for the generation of dicistronic messenger RNAs when appropriately combined with pSBC-2.
  • pSBC vectors were altered by replacing the promoter fragment (Clal/Xhol) by a hybrid promoter/enhancer composed of an MPSV enhancer of 300 bp (Clal/Xbal)
  • pMC-1 Fig. 1A
  • pMC-2 Fig. IB
  • pMC-2 and pMCC- 2 are also identical except for the multi-cloning site and allow expression of one protein chain, but do not contain a selection marker.
  • the vector pMC-2P (Fig. IE) was created in several steps. First, the blunt- ended fragment of the puromycin resistance gene from pSV2pac (Vara et al. 1986, Nucl. Acid Res. 14:4617) was cloned into the NotI site of pMCC-1. In the resulting plasmid the Xbal/EcoRI was replaced by the analogous fragment from pMCC-2, thereby inserting a new NotI site.
  • pMCC-2P The resulting plasmid is called pMCC-2P (Fig. IF).
  • pMC-2P was created by exchanging the polylinker into an Hindlll/EcoRI polylinker.
  • pMC-2PS (Fig. 1G) was created by insertion of a scaffold-attached region sequence (SAR) of 800 bp from the human Interferon- ⁇ gene as described (Mielke et al. 1990,
  • All three vectors contain an IRES sequence followed by the selection marker, in this case Puromycin resistance.
  • generation of a tricistrion expression vector is performed as follows: A Clal/NotI restriction fragment containing the promoter-proximal cistron followed by an IRES sequence is derived from the vectors pMC-1 or pMCC-1, respectively. A Notl/Clal restriction fragment containing the second cistron followed by an IRES sequence and the selection marker is derived from the vectors pMCC-2P, pMCC-2, pMC-2P, and pMC-2PS. By combination of these two fragments a complete expression vector is generated.
  • DMEM fetal calf serum
  • FCS fetal calf serum
  • BHK-21 (ATCC CCL 10) were transfected with the tricistronic expression vector encoding mAb425CHl-IL2 fusion protein by the calcium phosphate method with a kit commercially available (InVitrogen) according to the manufacturer's instructions. Selection for transfected BHK-21A cells was done in the presence of 5 ⁇ g/ml Puromycin (Sigma). Clones are analyzed for expression of immunoconjugates by EGF-receptor or cytokine-specific ELISAs. Selected clones are then further purified by limiting dilution cloning. In the presence of Puromycin a lot of clones could be isolated which stably express the mAb425CHl-IL2 fusion protein. Three examples are shown in Fig. 2).
  • the heavy chain-TNF ⁇ fusion gene construct was generated on the basis of the heavy chain-IL-2 fusion gene.
  • the KpnI/EcoRI fragment containing part of the heavy chain variable region, the CHI domain and IL-2 was subcloned into pUC19.
  • the NcoI/EcoRI fragment containing the IL-2- encoding sequences was exchanged with the NcoI/EcoRI fragment containing the TNF ⁇ -encoding sequences.
  • the KpnI/EcoRI fragment of this construct was combined in pUC18 with the Hindlll/Kpnl fragment containing the 5 'part of the heavy chain variable region to generate the full length heavy chain-TNF ⁇ fusion gene.
  • the Hindlll/EcoRI fragment was ligated into the multi-cloning site of the pMC2PS ⁇ H vector.
  • the Notl/Clal fragment of this construct was ligated with the Clal/NotI fragment from pMCL ⁇ HAP containing the mAb425 light chain.
  • the gene contains the light chain in the promoter-proximal position followed by the heavy-chain-TNF ⁇ fusion and the Puromycin resistance.
  • the genes are interspersed by two IRES sequences to allow transcription of all three cistrons into one messenger RNA.
  • Transfected BHK cells (rBHK21A-CH3-IL2/K69-8) were fermented in a semicontinous manner and the fusion protein was isolated from the collected, cell free supernatant.
  • the first purification step was performed by affinity chromatography on carrier bound ProteinA (Pharmacia) using the extended bed technology.
  • the starting conditions were 0,1 M phosphate buffer, pH 8,5. Impurities were removed with 0,2 M glycin buffer, pH 5,0 and subsequently, the fusion protein was eluted from the sedimented gel bed with 0,2 M glycin buffer, pH
  • Detection of protein-containing fractions was done by SDS-PAGE and an ELISA specific for human Ig with affinity-purified goat anti-human Fc as catcher antibody and affinity-purified goat anti human anti F(ab)2 coupled to alkaline phospatase for detection (both Dianova).
  • the protein content of the preparation was about 1 mg/ml.
  • the endotoxin content was ⁇ 1 EU/mg fusion protein.
  • the purity of the protein preparation could be demonstrated by SDS Page (Fig. 5). In Western Blots identity of heavy and light chain could be verified (data not shown).
  • the PCR amplified coding region of the recombinant light chain (Hindlll- EcoRI) gene was inserted into pMC-1 at the polylinker site.
  • the puromycin resistance gene coding sequence was inserted between the IRES sequence and the polyadenylation site of pMC-2 to give pMC-2P.
  • the heavy chain- cytokine fusion protein genes were inserted into the polylinker sequence of pMC-2P.
  • the Xmnl/Notl fragments of both Immunoglobulin chain vectors were combined to give e.g. pMCLDHAP, a 8298 bp tricistronic expression vector for IgG-TNF-alpha and puromycin acetyltransferase (Fig. 8).
  • BHK-21 cells were transfected with the tricistronic expression vector encoding mAb425CH3-TNF ⁇ fusion protein using the calcium phosphate precipitation method as detailed by Mielke et al. (1990, Biochemistry 29:7475). 5 ⁇ g of uncut plasmid were used without the addition of carrier DNA. Stable transfectants were selected and cultivated in medium containing Puromycin (Sigma) at a final concentration of 5 ⁇ g/ml. Clones are analysed for expression of immunoconjugates by IgG-specific ELISA.
  • Fig. 9 Chromosomal DNA analysis Isolation of genomic DNA: Cells from a 141 cm 2 culture dish were harvested in 20 ml TEN buffer [40mM Tris/HCl (pH 7.5), ImM EDTA, 150 mM NaCL], split into two portions and pelleted for 5 min at 1000 rpm in a table top centrifuge.
  • Southern Blots/Methylation pattern 20 ⁇ g of genomic DNA was digested over night with the indicated restriction enzyme in a total volume of 500 ⁇ l, precipitated by addition of 300 ⁇ l 2-propanol and pelleted at 13000 rpm, 4 ° C in a microcentrifuge. DNA pellets were carefully resuspended in 40 ⁇ l of lx loading buffer [2.5 % Ficoll (Type 400), 17 mM EDTA, o.ol % Xylene Cyanol FF), 20 ⁇ l were applied on a 0.8 % TAE agarose gel and electrophoresed.
  • lx loading buffer 2.5 % Ficoll (Type 400), 17 mM EDTA, o.ol % Xylene Cyanol FF
  • Transformed BHK cells (rBHK21A-CH3-TNF ⁇ /SC7.4) were fermented in a semicontinous manner and the fusion protein was isolated from the collected, cell free supernatant.
  • the first purification step performed by affinity chromatography on carrier bound ProteinA (Pharmacia) using the extended bed technology.
  • the starting conditions were 0,1 M phosphate buffer, pH 8,5. Impurities were removed with 0,2 M glycin buffer, pH 5,0 before the fusion protein was eluted from the sedimented gel bed with 0,2 M glycin buffer, pH 3,3.
  • the pH of the eluat was immediately brought up to pH 8 - 8,5 by adding 10 % (vol./vol.) 1 M TRIS solution.
  • the second purification step was done by chromatography on hydroxyapatite (Merck).
  • the starting conditions were 5 mM phosphate, pH 7,0.
  • the elution was performed with a phosphate gradient (5 - 500 mM).
  • the final purification step was done by size exclusion chromatography on
  • Detection of protein-containing fractions was done by SDS-PAGE and an ELISA specific for human Ig with affinity-purified goat anti-human Fc as catcher antibody and affinity-purified goat anti human anti F(ab)2 coupled to alkaline phospatase for detection (both Dianova).
  • the protein content of the preparation was about 1 mg/ml.
  • the endotoxin content was ⁇ I EU/mg fusion protein.
  • EGF-R-positive cells with the same quality as the mAb425 control antibody.
  • TNFa activity of the mAb425CH3-TNF ⁇ fusion protein was investigated on different human tumor cell lines.
  • MCF7 is a human mamma carcinoma cell line which is not EGF-R positive. The inhibition of proliferation is therefore exclusively based on TNF ⁇ activity.
  • the growth inhibition induced by the mAb425CH3-TNF ⁇ fusion protein is virtually identical to that of recombinant TNF ⁇ .
  • mAb425 alone does not have any effect on proliferation of MCF7.
  • mAb425 was raised against the human carcinoma cell line A431 which is highly positive for EGF-R expression (Rodeck et al.).
  • mAb425 is internalized upon binding to A431 cells.
  • A431 is not TNF ⁇ sensitive and neither mAb425CII3-TNF ⁇ fusion protein nor the combination of mAb425 and recombinant TNF ⁇ does have any effect on the growth of A431 cells (Fig. 13) indicating that the growth inhibition specifically requires expression of TNF ⁇ receptors. Lack of TNF ⁇ receptors cannot be overcome through internalization of mAb425CH3-TNF ⁇ fusion protein mediated by EGF-R receptor.
  • BT20 a human mamma carcinoma cell line and C8161, a human melanoma cell line, are both EGF-R positive and TNF ⁇ sensitive.
  • the density of EGF- R on the cell surface is higher on BT20 than on C8161 as determined by
  • GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG GTAGGCGTGT 780 ACGGTGGGAG GTCTATATAA GCAGAGCTCG TTTAGTGAAC CGTCAGATCG CCTGGAGACG 840
  • CAGCACTCAA CCCCAGAGTG TAGCTTAGGC TGATGAGTCT GGACATCCCT CACCGGTGA: 2273 GGTGGTCCAG GCTGCGTTGG CGGCCTACCT ATGGCTAACG CCATGGGACG CTAGTTGTGA 2333
  • AAG CCC TGG TAT GAG CCC ATC TAT CTG GGA GGG GTC TTC CAG CTC GAG 4447 Lys Pro Trp Tyr Glu Pro He Tyr Leu Gly Gly Val Phe Gin Leu Glu 610 615 620
  • AAACCAGTAC CACCACGAAC AAGCACTTCT GTTTCCCCGG TGATGTCGTA TAGACTGCTT 4777
  • G C G GGTGTCG GGGCGCAGCC ATGACCCAGT CACGTAGCGA TAGCGGAGTG TATACTGGCT 6366
  • AGA GGTGGCG AAACCCGACA GGACTATAAA GATACCAGGC GTTTCCCCCT GGAAGCTCCC 6546 TCGTGCGCTC TCCTGTTCCG ACCCTGCCGC TTACCGGATA CCTGTCCGCC TTTCTCCCTT 6606
  • ACTCGTGCAC CCAACTGATC TTCAGCATCT TTTACTTTCA CCAGCGTTTC TGGGTGAGCA 7986 AAAACAGGAA GGCAAAATGC CGCAAAAAAG GGAATAAGGG CGACACGGAA ATGTTGAATA 8046
  • MOLECULE TYPE protein

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Abstract

La présente invention concerne un système d'expression mammifère, destiné à la production de protéines hétéromères recombinées, de préférence des anticorps, et plus préférablement des protéines de fusion contenant un anticorps, telles que des protéines de fusion contenant un anticorps dirigé contre une cytokine, ainsi que de fragments de ces protéines, au moyen de vecteurs d'expression oligocistroniques, lesquels sont régis par un activateur/promoteur très actif, par un gène marqueur de sélection, et par au moins deux éléments IRES (site d'entrée ribosomique interne). On peut produire ces protéines de fusion hétéromères au moyen d'un procédé fiable et stable et avec d'excellents rendements.
PCT/EP1997/004765 1996-09-16 1997-09-02 Systeme d'expression oligocistronique destine a la production de proteines heteromeres WO1998011241A1 (fr)

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WO2003031630A1 (fr) * 2001-10-12 2003-04-17 Keryos Spa Vecteurs multicistroniques destines a des protocoles de transfert genique
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EP2322214A1 (fr) 2003-04-17 2011-05-18 Altropus Gmbh Anticorps recombinant immunogène
US7968334B2 (en) 2004-01-23 2011-06-28 Virxsys Corporation Expression of apoAI and variants thereof using spliceosome mediated RNA trans-splicing
US8053232B2 (en) 2004-01-23 2011-11-08 Virxsys Corporation Correction of alpha-1-antitrypsin genetic defects using spliceosome mediated RNA trans splicing
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EP1108780A1 (fr) * 1998-08-27 2001-06-20 Fuso Pharmaceutical Industries Ltd. Sequence d'acide nucleique utilisee pour potentialiser l'expression d'un gene utile et procede associe
EP1108780A4 (fr) * 1998-08-27 2003-01-02 Fuso Pharmaceutical Ind Sequence d'acide nucleique utilisee pour potentialiser l'expression d'un gene utile et procede associe
US6869779B1 (en) 1998-08-27 2005-03-22 Fuso Pharmaceutical Industries, Ltd. Nucleic acid sequence for potentiating the expression of useful gene and method therefor
WO2000029583A3 (fr) * 1998-11-19 2000-09-08 Incyte Pharma Inc Proteines de la superfamille des immunoglobulines
WO2000065074A1 (fr) * 1999-04-23 2000-11-02 Gesellschaft Fuer Biotechnologische Forschung Mbh (Gbf) Systeme de promoteur-transactivateur destine a une expression inductible de genes mammaliens de haut niveau avec option de regulation de la croissance cellulaire
US7319139B2 (en) 2001-01-29 2008-01-15 Biogen Idec, Inc. TAG-72 specific CH2 domain deleted antibodies
WO2003031630A1 (fr) * 2001-10-12 2003-04-17 Keryos Spa Vecteurs multicistroniques destines a des protocoles de transfert genique
WO2003048306A2 (fr) 2001-11-16 2003-06-12 Idec Pharmaceuticals Corporation Expression polycistronique d'anticorps
EP1456386A2 (fr) * 2001-11-16 2004-09-15 Idec Pharmaceuticals Corporation Expression polycistronique d'anticorps
EP1456386A4 (fr) * 2001-11-16 2006-05-03 Biogen Idec Inc Expression polycistronique d'anticorps
US8206977B2 (en) 2002-07-30 2012-06-26 Morphosys Ag Tricistronic vectors and uses therefor
EP2322214A1 (fr) 2003-04-17 2011-05-18 Altropus Gmbh Anticorps recombinant immunogène
US7968334B2 (en) 2004-01-23 2011-06-28 Virxsys Corporation Expression of apoAI and variants thereof using spliceosome mediated RNA trans-splicing
US8053232B2 (en) 2004-01-23 2011-11-08 Virxsys Corporation Correction of alpha-1-antitrypsin genetic defects using spliceosome mediated RNA trans splicing
US7879321B2 (en) 2004-10-08 2011-02-01 Virxsys Corporation Use of RNA trans-splicing for antibody gene transfer and antibody polypeptide production
US7871795B2 (en) 2004-10-08 2011-01-18 Virxsys Corporation Targeted trans-splicing of highly abundant transcripts for in vivo production of recombinant proteins
EP2163563A1 (fr) * 2006-03-31 2010-03-17 Massachusetts Institute of Technology Traitement des tumeurs exprimant des récepteurs EGF mutants
WO2007123661A3 (fr) * 2006-03-31 2008-11-20 Massachusetts Inst Technology Traitement de tumeurs exprimant les recepteurs mutants de l'egf
EP2700713A1 (fr) 2012-08-21 2014-02-26 Miltenyi Biotec GmbH Système d'enrichissement et de criblage pour l'expression de protéines dans des cellules eucaryotes à l'aide d'une cassette d'expression tricistronique
EP2700713B1 (fr) * 2012-08-21 2016-07-13 Miltenyi Biotec GmbH Système d'enrichissement et de criblage pour l'expression de protéines dans des cellules eucaryotes à l'aide d'une cassette d'expression tricistronique

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