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EP0489830A1 - Expression des alpha-macroglobulines - Google Patents

Expression des alpha-macroglobulines

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Publication number
EP0489830A1
EP0489830A1 EP90913470A EP90913470A EP0489830A1 EP 0489830 A1 EP0489830 A1 EP 0489830A1 EP 90913470 A EP90913470 A EP 90913470A EP 90913470 A EP90913470 A EP 90913470A EP 0489830 A1 EP0489830 A1 EP 0489830A1
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EP
European Patent Office
Prior art keywords
val
leu
ser
glu
thr
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP90913470A
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German (de)
English (en)
Inventor
Boel Esper
Sottrup-Jensen Lars
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Novo Nordisk AS
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Novo Nordisk AS
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Priority claimed from DK423689A external-priority patent/DK423689D0/da
Priority claimed from DK423789A external-priority patent/DK423789D0/da
Priority claimed from DK423589A external-priority patent/DK423589D0/da
Application filed by Novo Nordisk AS filed Critical Novo Nordisk AS
Publication of EP0489830A1 publication Critical patent/EP0489830A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the expression of ⁇ -macroglobu- 1ins, derivatives and variants thereof, and especially the expression of the human ⁇ 2 -macroglobulin ( ⁇ 2 M) in an active form in mammalian cells, and the expression of genetically engineered variants thereof.
  • ⁇ -macroglobulins especially recombinant ⁇ 2 M(r ⁇ * 2 M) and variants is described with examples from the fields of medicine for therapeutic purposes, and the development of novel defined growth media for propagation of mammalian cells in culture.
  • BIOCHEMISTRY OF tt,-MACROGLOBULIN fa-M BIOCHEMISTRY OF tt,-MACROGLOBULIN fa-M.
  • the proteinase binding glycoprotein ⁇ 2 M which is synthesized in the liver, constitute together with the complement proteins C3, C4 and C5 a separate class of structurally and functionally related large plasma proteins.
  • the thiol este structure which in the active proteins can be slowly cleaved by a number o small nitrogen nucleophiles, constitutes a unique type of postsyntheti modification of proteins, and plays a prominent role in the biological properties of ⁇ 2 M.
  • the presence of the active thiol esters in ⁇ 2 M is reveale by a characteristic pattern of heat fragmentation (Harpel , P.C., et al . , (1979) J. Biol. Chem. 254: 8869-8878).
  • ⁇ 2 M forms complexes with a broad spectrum of proteinases differing in their substrate specifi ⁇ city and catalytic mechanism e.g.: trypsin, leucocyte elastase, chymotrypsin, pancreatic elastase, cathepsin G, plasmin, plasma kallikrein and thrombin. 5
  • trypsin trypsin
  • leucocyte elastase chymotrypsin
  • pancreatic elastase pancreatic elastase
  • cathepsin G plasmin
  • plasma kallikrein plasma kallikrein
  • thrombin The second-order rate constant for association between these proteinases and ⁇ 2 M varies by several orders of magnitude.
  • the main physiological targets may include proteinases of the coagulation and fibrinolysis systems and plasma kallikrein, and perhaps also proteinases like leucocyte elastase, cathepsin G and collagenases and other
  • ⁇ 2 M may be largely confined to the vasculature in healthy uninflamed tissues, the inhibitor and its proteinase complexes are found at near plasma levels in inflammatory exudates of rheumatoid joints and gingival crevicular fluids (Tollefsen, T. and Saltved, E. (1980) J. Periodont. Res. 15: 96-106; Borth, W., et al . , (1983) Ann. N. Y. Acad. Sci. 421: 377-381). While plasma ⁇ 2 M appear to be synthesized in the liver (Schreiber, G.
  • hepatocytes and Kupffer cells of the liver are most important for clearance of ⁇ 2 M-proteinase complexes in plasma (Davidsen, O., et al., (1985) Biochim. Biophys. Acta 846: 85-92), fibroblasts (Van Leuven, F., et al., (1979) J. Biol. Chem. 254: 5155-5160; Mosher, D.F. and Vaheri , A. (1980) Biochim. Biophys. Acta 627: 113-122) and macrophages (Debanne, M.T., et al., (1975) Biochim. Biophys. Acta ill: 295-304; Kaplan, J. and Nielsen, M.L. (1979) J. Biol. Chem. 254: 7323-7328) also possess receptors for ⁇ 2 M-proteinase complexes.
  • the present invention discloses a method for the production of recombinant ⁇ -macroglobulins, and especially human ⁇ 2 M, and variants thereof in an active form.
  • the cultured host cell is an eukaryotic cell such as a mammalian cell or cells derived from organisms such as insects, plants, yeast or other fungi, such as Aspergillus.
  • the invention further relates to DNA sequences comprising a gene encoding for the expression of human a 2 M and variants thereof, vectors comprising such DNA sequences, and suitable hosts transformed with such vectors.
  • Yet another aspect of the invention is the use of recombinant ⁇ . 2 M and variants thereof as a protein carrier in enzyme replacement therapy (ERT).
  • ERT enzyme replacement therapy
  • Further aspects of the invention relates to the use of recom ⁇ binant Qr-macroglobulins, especially human a 2 M, and variants thereof as constituents of growth media, either as an additive or co-expressed with a desired gene product.
  • Complementary DNA or cDNA A DNA molecule or sequence which have been lOenzy atically synthesized from sequences present in a mRNA template.
  • DNA Construct A DNA molecule, or a clone of such a molecule, either single- or double-stranded, which may be isolated in partial form from a naturally occurring gene or which has been modified to contain segments of DNA which
  • Plasmid or Vector A DNA construct containing genetic information which may provide for its replication when inserted into a host cell.
  • a plasmid 20 generally contains at least one gene sequence to be expressed in the host cell, as well as sequences encoding functions which facilitate such gene expression, including promoters and transcription initiation sites. It may be a linear or closed circular molecule.
  • DNA sequences are said to be joined when the 5' and 3' ends of one sequence are attached by phosphodiester bonds to the 3 ⁇ and 5' ends, respectively, of an adjacent sequence. Joining may be achieved by such methods as ligation of blunt or cohesive termini, by synthesis of joined sequences through cDNA cloning, or by removal of intervening sequences
  • Figure la illustrates the construction of plasmid pll36.
  • Figure lb illustrates the construction of plasmid pll67.
  • Figure 2 illustrates the structure of plasmid pll67.
  • Figure 3 illustrates a gel electrophoresis (10 - 20 % SDS-PAGE) of the thermal fragmentation products generated from ⁇ 2 M and r ⁇ 2 M.
  • Figure 4 illustrates a gel electrophoresis of the thermal fragmentation products generated from methylamine treated ⁇ 2 M and r ⁇ 2 M.
  • Figure 5 illustrates a gel electrophoresis (SDS-PAGE) of the reaction products generated from trypsin treatment of ⁇ 2 M and r ⁇ 2 M.
  • Figure 6 illustrates a gel electrophoresis of the reaction produ ⁇ cts generated from trypsin treatment of methylamine-treated a 2 M and r ⁇ 2 M.
  • Figure 7 illustrates a "rate gel” electrophoresis of unreacted native -and trypsin treated ⁇ 2 M and r ⁇ 2 M.
  • Figure 9 illustrates the chromatograms of ⁇ : 2 M and r ⁇ 2 M on a lOSuperose 6 column.
  • Figure 11 illustrates the gel electrophoresis (10 - 20 % reducing 15 SDS-PAGE) of the reaction products from elastase treated human ⁇ 2 M, human PZP and r ⁇ 2 M-PZP.
  • Figure 12 illustrates the gel electrophoresis (10 - 20 % reducing SDS-PAGE) of the reaction products from trypsin treated human ⁇ 2 M, human PZP and r ⁇ 2 M-PZP.
  • Figure 13 illustrates the gel electrophoresis (10 - 20 % reducing SDS-PAGE) of the reaction products from Staphylococcus aureus Glu-specific protease treated human ⁇ 2 M, human PZP and rcc 2 M-PZP.
  • ⁇ -macroglobulins especially human ⁇ 2 -macroglobulin, or fragments or derivatives, including variants thereof, wherein a functionally operative expression vector comprising a gene encoding for the expression of
  • a ⁇ -macroglobulin especially human ⁇ 2 -macroglobulin, or fragments or derivatives thereof, including variants, or alleles of such a gene, is intro ⁇ quizzed into a suitable host capable of expressing said gene, said host is cultured in a suitable nutrient medium containing sources of assimilable carbon and nitrogen and other essential nutrients, and the expressed a-
  • 35macroglobulin, especially human ⁇ 2 -macroglobulin, or fragments or derivatives thereof is recovered.
  • Human Platelet-derived Growth Factor AB heterodimer has been expres ⁇ sed in transformed CHO (Chinese Hamster Ovary) cells with correct processing of the A and B chain precursors and correct assembly of the AB heterodimer.
  • Human coagulation factor VIII has been expressed in transformed CHO cells with correct processing of the precursor leading to a two chain molecule that
  • thrombin and factor Xa can be activated by thrombin and factor Xa (Kaufman, R.J. et al . , (1988) J. Biol. Chem. 263: 6352-6362; Pittman, D.D. and Kaufman, R.J. (1988) Proc. Natl. Acad. Sci . USA 85: 2429-2433).
  • the C3 specific product could now incorporate methylamine.
  • concentration of the S-13 component(s) By increasing the concentration of the S-13 component(s), the incorporation of methylamine in C3 specific products was increased, and at the same time incorporation of iodoacetamide decreased. If the S-13 fraction was treated at 65 e C for 5 in, the activity was completely lost.
  • the results from this investigation strongly suggest an involve ⁇ ment of a transglutaminase-like or other type, of enzyme in the posttransla- tional formation of an active thiol ester in rabbit C3.
  • DNA sequences comprising a gene encoding for the expression of ⁇ - macroglobulins, especially human ⁇ 2 -macroglobulin, or fragments or deriva ⁇ tives and variants thereof as exemplified in SEQ ID NO:1 and SEQ ID N0:3.
  • Another aspect of the invention relates to functionally operative expression vectors comprising a gene encoding for the expression of at least one ⁇ -macroglobulin, especially human ⁇ 2 -macroglobulin or fragments or derivatives and variants thereof, or alleles of such a gene.
  • Such vectors preferably further comprise regulatory elements necessary for the stable maintenance of said vector in mammalian cells. Also, such vectors may further include sequences providing for the processing and secretion of the expressed product.
  • the vectors of the invention may further comprise one or more other genes encoding for a desired gene product.
  • the invention further relates to transformed hosts comprising a functionally operative expression vector according to the invention compri ⁇ sing a gene encoding for the expression of human ⁇ 2 -macroglobulin or fragments or derivatives and variants thereof, or alleles of such a gene.
  • the host may be selected from the group comprising a bacterial strain, a fungal strain, a mammalian cell line, or a mammal, especially a fungus, such as belonging to the genus Asperqillus, or a yeast strain, pre ⁇ ferably belonging to the genus Saccharomvces.
  • Another preferred type of host is a mammalian cell line, preferably a Syrian Baby Hamster Kidney (BHK) cell line, and especially the one which is available from ATCC under No. CRL 1632.
  • the invention further relates to the recombinant human ⁇ 2 - macroglobulin or a variant thereof in an active form having the amino acid sequence of SEQ ID N0:2, or SEQ ID N0:4.
  • the present invention discloses applications of ⁇ -macroglobulins, and especially r ⁇ 2 M. These should be regarded not as limitations but as a few examples among many for the use of recombinant derived ⁇ -macroglobulins.
  • Degradation of specific heterologous products produced in either transformed or non-transformed mammalian cells is a potential problem in the production of recombinant products. This is due to the fact that many host cells secretes one or more different proteinases.
  • a production cell line is grown in the presence of e.g. 10
  • % fetal calf serum such proteolytic degradation of secreted recombinant or native protein products is a minor problem due to a buffering effect of the added serum proteins.
  • fetal calf serum in the large scale growth (fermentation) of mammalian production cell lines is not a desirable situation for a number of reasons.
  • First of all fetal calf serum is a very costly constituent of complex growth media;
  • third, the use of fetal calf serum constitutes a potential quality control problem in the production of pharmaceuticals intended for use in humans.
  • ⁇ -Macroglobulins and especially Human ⁇ 2 M, are proteinase inhibitors of broad specificity, and they are therefore according to the invention used as constituents of defined growth media for mammalian cells, either as a medium additive or as a product co-produced with the desired product.
  • the target sites for a number of different proteinases e.g.
  • bovine trypsin, Streptomvces qriseus trypsin, papain, porcine elastase, bovine chymosin, bovine chymotrypsin, Staphylococcus aureus strain V8 proteinase, human plasmin, bovine thrombin, thermolysin, subtilisin Novo and Streptomvces qriseus proteinase B have been mapped in the bait region of human ⁇ 2 M (Mortensen, S.B., et al., (1981) FEBS Lett.135: 295-300) and other ⁇ -macroglobulins (Sottrup-Jensen, L., Sand, 0., Kristensen, L.
  • ⁇ 2 M and the other ⁇ - acroglobulins as proteinase inhibitors have broad specificities.
  • the proteinase inhibitory spectrum of a ⁇ -macroglobulin, such as ⁇ 2 M is not sufficient for the prevention of product degradation, it is possible through site specific mutation, protein engineering, etc. to change the proteinase inhibitor specificity of the ⁇ - acroglobulin, such as ⁇ 2 M. Incorporation of desirable specific proteinase target sites in the bait region of recombinant ⁇ 2 M will change the inhibitor specificity of the mutated ⁇ 2 M.
  • the a ino acid sequence of the bait region of ⁇ -macroglobulins defines the specificity of the ⁇ -macroglobulin towards different proteina- ses.
  • a comparison of cleavage patterns for different proteinases and bait region sequences in five mammalian ⁇ -macroglobulins has recently been published (Sottrup-Jensen, L., Sand, 0., Kristensen, L. and Fey, G.H.
  • the ⁇ -macroglobulin bait region Sequence diversity and localization of cleavage sites for proteinases in five mammalian ⁇ -macroglobulins. J. Biol . Chem. 264, 15781-15789, 1989).
  • the bait region in each species of ⁇ -macroglobulin is the major determinant of proteinase inhibitor specificity.
  • the present invention demonstrates the possibility of modulating the inhibitor specificity of human ⁇ 2 M by 5 alterations of proteinase target sites in the bait region.
  • the invention thus demonstrates the possibility to design and produce proteinase inhibitors with altered and new inhibitor specificities at will .
  • ⁇ 2 M A different application of ⁇ is .its use as a carrier of macro- molecules such as proteins and nucleic acids.
  • ⁇ 2 M may bind other proteins (also non-proteinase proteins) present in that solution (Salvesen, G.S. et al . , (1981) Bioche . J. 195: 453-461).
  • Fabry's disease which is an X-chromosome linked disorder of glycosphingolipid metabolism, it has recently been demonstrated that ⁇ 2 M can function as a carrier in an in vitro model of enzyme replacement therapy (ERT) (Osada, T., et al., (1987) Biochem.
  • ERT enzyme replacement therapy
  • ERT Such a scheme in ERT provides a method of internalization to the lysosome of the enzyme in question and at the same time it might alleviate potential antigenicity problems arising from the use of heterologous enzymes in therapy.
  • One limitation in this type of ERT (Osada, T., et al., (1987) Biochem. Biophys. Res. Commu. 142: 100-106) would be the types of potential target cells that could be treated by this protocol. Obviously, they would have to express the ⁇ 2 M-receptor.
  • the possibility might exist to redesign the cell specificity of ⁇ 2 M internaliza- tion by exchanging the receptor binding domain of ⁇ 2 M with other receptor ligands.
  • ⁇ M-mutants could be designed to enter any cell type known to express a specific internalizable receptor.
  • the DNA carrier consisted of a galactose-terminal (asialo)glyco- protein and asialoorosomucoid covalently linked to poly-L-lysine.
  • the polycation poly-L-lysine can bind DNA in a strong non-covalent and nondamag- ing interaction. It was demonstrated that complex bound DNA was internalized by cell-surface asialoglycoprotein receptors that are unique to hepatocytes. The complex was injected intravenously, and upon analysis only the liver expressed the CAT activity.
  • r ⁇ 2 M as a carrier of DNA in gene therapy.
  • Reaction of r ⁇ 2 M with a proteinase such as trypsin or with methylamine in the presence of covalently closed circular plasmid DNA is likely to result in partial or total entrapment of DNA within the complexing ⁇ 2 M molecule.
  • the complex After intravenous injection of such complexes with exposed receptor binding domains, the complex will be rapidly cleared from the blood and internalized in specific target cells, such as hepatocytes and Kupffer cells.
  • target cells such as hepatocytes and Kupffer cells.
  • Through protein engineering on the receptor binding domain of r ⁇ 2 M it will be possible to design a DNA carrier specific for other cell types.
  • the advantage in this system as compared to the above described system using the asialoglycoprotein receptor is, that it will not be necessary to identify different DNA carrier systems for each new cell type.
  • E. coli K12 (MC1061) is available from e.g. Stratagene Inc., 11099 North Torrey Pines Rd., La Jolla, California 92037.
  • HepG2 Human hepatoblasto a cell line
  • HB 8065 American Type Culture Collection
  • BHK (Syrian Hamster Kidney cell line, thymidine kinase mutan line tk 'l sl3, (Waechter and Baserga (1982) Proc. Nat! . Acad. Sci . USA 79: 1106-1110); is freely available from American Type Culture Collection, under No. CRL 1632.
  • M13mpl8 is available from Pharmacia LKB Biotechnology (catalog # 27-1552-01) (Norrander, J., Ke pe, T. and Messing, J. Gene 26: 101-106, 151983).
  • M13mpl9 is available from e.g. International Biotechnologies, Inc., P.O. Box 9558, 275 Winchester Avenue, New Haven, Connecticut 06535, USA.
  • pDHFR-I is available from Dr. K.L.Berkner, ZymoGenetics Inc.,
  • pDHFR-I The backbone plasmid in pDHFR-I is pBR322 (Sutcliffe, J.G. (1979) Cold Spring Harbor Symp. Quant.
  • Anti- ⁇ 2 M A033 and peroxidase conjugated anti- ⁇ 2 M PE326 were from DAK0PATTS A/S, Copenhagen, Denmark.
  • HepG2 human hepatoblastoma cell line HepG2 (American Type Culture Collection No. HB 8065, freely available) was used as a source for mRNA preparation. HepG2 cells were grown to a total cell number of 15 * 10 7 in Dulbecco's Modified Eagle medium containing 10% fetal calf serum and antibiotics.
  • RNA was isolated by the guanidinium thiocyanate method (Chirgwin et al., (1979) Biochemistry 18: 5293-5299) and purified by CsCl gradient centrifugation. A total of 3000 ⁇ g RNA was obtained. mRNA was isolated by use of an oligo(dT)-cellulose column (Aviv & Leder (1972) Proc. Natl. Acad. Sci . USA 69: 1408-1412). 60 ⁇ g of mRNA was obtained after on cycle of affinity chromatography.
  • this preparation of mRNA was resuspended in 10 mM Tris-HCl pH 7.5, 0.1 mM EDTA- Na 2 at a final concentration of 1 ⁇ g/ ⁇ l and stored at -80°C for subsequen use in the construction of a cDNA library.
  • E. coli K12 (MC1061) (Casadaban & Cohen (1980) J. Mol. Biol.
  • the cells were plated onto L-broth plates containing ampicillin (50 ⁇ g/ml) and grown for 8 hrs at 37°C. A total of 2.9 5*10 5 individual colonies could be obtained from this library.
  • the insert contains sequences derived from the 5'- and 3' untranslated regions of the ⁇ 2 M mRNA molecule.
  • the amino acid sequence of the human ⁇ 2 M as deduced from the cDNA in p ⁇ 2 M is in total agreement with the published sequence (Sottrup-Jensen et al., (1984) J. Biol. Chem. 259: 8318-8327).
  • Codon number 1000 (numbered from the initiating methionine codon in the signal peptide) was found to be ATC encoding an isoleucine and not GTC (encoding a valine) as found in an ⁇ 2 M cDNA synthesized from human liver mRNA (Kan et al .
  • the position of the oligonucleotide mixture used as a hybridiza ⁇ tion probe in the colony screenings was from position 1574 to position 1594, and the position of the reactive thiol ester is from position 2939 to 2953 in SEQ ID N0:1.
  • p ⁇ 2 M was digested (fig. la) with Xbal and EcoRI, and a 1.2 kb fragment containing the 5' part of the ⁇ 2 M cDNA together with the multiple cloning site of pSP62-K2 was isolated on an agarose gel and cloned in an Xbal/EcoRI digested M13mpl9 vector to generate M13mpl9A.
  • ⁇ 2 M cDNA The mutated 5' end of ⁇ 2 M cDNA was isolated from M13mpl9A repli- cative form through digestion with Hindlll and EcoRI and agarose gel electro- ⁇ phoresis. The isolated DNA fragment was then joined to Hindlll/EcoRI digested p ⁇ 2 M through ligation to generate pll36. In this plasmid the ⁇ 2 M cDNA is reassembled in its total length, but now with a unique EcoRV site at the 5' end.
  • pi136 was digested with EcoRV/Dral, and the ⁇ 2 M fragment was isolated on an agarose gel and cloned in a mammalian expression vector under control of 0 the adenovirus 2 major late promoter (Ad 2 MLP).
  • the adenovirus-promoter based vector was constructed by K.L.Berk- ner (ZymoGenetics Inc., Seattle, WA.), and a detailed description of the functional elements in the mammalian expression vector is given in: Powell, J.S. et al., (1986) Proc. Natl. Acad. Sci. USA 83: 6465-6469 and in: Boel 5 et al., (1987) FEBS Lett. 219: 181-188).
  • the expression vector used for expression of human ⁇ 2 M was generated from the mammalian expression vector pPP (Boel, E. et al . , (1987) FEBS Lett. 219: 181-188), in which human pancreatic polypeptide cDNA was cloned under control of Ad 2 MLP.
  • pPP mammalian expression vector
  • 0 pPP was digested (fig. lb) with BamHI and the resulting stag ⁇ gered ends were repaired with DNA polymerase (Klenow fragment and the four deoxynucleotide triphosphates).
  • the 4.5 kb EcoRV/Dral ⁇ 2 M cDNA fragment was joined to this vector through ligation, and correct recombinants were characterized through restriction enzyme analysis on isolated iniprep. 5 plasmids.
  • the materials used in the ELISA were: Catching antibody A033 anti- ⁇ 2 M,
  • 1,2-Phenylenediamine, dihydrochloride (0PD) all from DAK0PATTS A/S, Copenhagen, Denmark.
  • Urea peroxide 125 g was from Organon Teknika.
  • 96 well ELISA plates were from NUNC, Copenhagen.
  • Citric acid-phosphate buffer pH 4.9:
  • the buffer was used for a maximum of 14 days, stored at 4°C.
  • Urea peroxide solution 125 mg urea peroxide was dissolved in 8.93 ml water. The solution was kept in the dark at 4°C.
  • the 96 well plate was coated with 175 ⁇ l of the DAKO A033 antibody diluted 1:1000 in the coating buffer. The plate was incubated over night at 4°C. Before use the plate was washed 4 times in washing buffer.
  • the r ⁇ 2 M was purified according to published procedures (Sottrup- Jensen et al., (1983) Ann. N. Y. Acad. Sci. 421: 41-60). Briefly the conditioned medium was loaded onto a 10 ml Zn-Chelate column (Zn 2+ - iminodiacetic acid Sepharose 4B (Porath, J. et al . , (1975) Nature 258: 598- 599) equilibrated with 25 mM Tris-HCl pH 8.0, and washed with 100 ml phosphate buffered saline (PBS) pH 7.2 until A 280 ⁇ 0.036.
  • PBS phosphate buffered saline
  • the flow rate was 100 ml/hr and 3 ml fractions were collected.
  • r ⁇ 2 M was eluted with 100 mM EDTA pH 7.0 at a flow rate of 40 ml/hr. During elution 1 ml fractions were collected.
  • r ⁇ 2 M Recovery of r ⁇ 2 M was 44%.
  • the r ⁇ 2 M containing fractions were con ⁇ centrated to 1 ml on an Amicon devise equipped with a PM 10 membrane and then loaded onto a Superose 12 gelfiltration column (25 M Tris-HCl, 150 M NaCl pH 8.0).
  • the r ⁇ 2 M containing fractions were pooled and stored at -20°C until analysis.
  • ⁇ 2 M An important structural feature of ⁇ 2 M is the presence of the 5 thiol ester. When heated to 95 ⁇ C for 15 min, the thiol ester will induce a peptide bond cleavage in the backbone of ⁇ 2 M at the position of the thiol esterified Glx-residue. This results in the fragmentation of the 180 kD ⁇ 2 M monomer into two polypeptides of 120 kD and 60 kD.
  • Fig. 3 shows an analysis of both the purified r ⁇ 2 M (from two transformed BHK cell lines) and the
  • Lanes 6 and 7 indicated the presence in the recombinant material of a simila faster migrating fragment. It is possible that this fragment represented slightly underglycosylated variant of the 60 kD fragment.
  • Fig. 4 shows a SDS-PAGE run similar to that shown in Fig. 3 (wit
  • trypsin will cleave at its target site(s) in the bait region of ⁇ 2 M, and the resulting reduced cleavage products (85 kD) will migrate as a double band. Under nonreducing conditions the trypsin- ⁇ 2 M complexes will migrate as high molecular weight products.
  • Fig. 6 shows the results of a set of experiments that were run in parallel to the experiments described above and shown in Fig. 5.
  • the native human ⁇ 2 M and the r ⁇ 2 M used in this experiment had been treated with methylamine (Sottrup-Jensen, L., et al., (1980) FEBS Lett. .121: 275-280).
  • both the native ⁇ 2 M and the r ⁇ 2 M show a marked decrease in reactivity towards trypsin (80% or more of the ⁇ 2 M and r ⁇ 2 M monomers were migrating as a 180 kD polypeptide) . This indicates that trypsin does not rapidly cleave at the bait region in methylamine treated human ⁇ 2 M or in BHK cell derived r ⁇ 2 M.
  • BHK cell derived r ⁇ z M has shown characteristics similar to those of native human ⁇ 2 M.
  • ⁇ 2 M will undergo a conformational change both through complex formation with proteinases and through methyl ⁇ amine induced cleavage of the thiol ester.
  • the change in structure results in an altered mobility on rate gels (Sottrup-Jensen, L. (1987) in: The Plasma Proteins (Putnam, F.W., ed.) 2nd Ed., 5: 191-291, Academic Press, Orlando, FL; Van Leuven, F., Cassiman, J.-J. and Van Den Berghe, H. (1981) J. Biol. Chem. 256: 9016-9022); unreacted ⁇ 2 M will migrate as a "slow” form, while reacted ⁇ 2 M will migrate as a "fast” form.
  • Fig. 7 and Fig. 8 show these conformational changes, as they appear after reaction with trypsin and methylamine, respectively (analyzed on 5-10% rate gels).
  • Lanes 1 on both gels contain purified human pregnancy zone protein (PZP) (Sand, 0. et al . , (1985) J. Biol. Chem. 260: 15723-15735), which is known to appear in both a dimeric (D) and a tetrameric (T) configuration.
  • PZP human pregnancy zone protein
  • Lanes 2 on both gels contain unreacted human ⁇ 2 M preparation LSJ39. Lanes 3 on both gels show the fast migrating form, resulting fro reaction with trypsin and methylamine, respectively. Lanes 4 on both gels show the unreacted r ⁇ z M preparation K16-6, and lanes 5 show the corresponding fast forms. Lanes 6 on both gels show the unreacted r ⁇ 2 M preparation K17- 6, and lanes 7 show the corresponding fast forms.
  • a Superose 6 column can partially resolve ⁇ 2 M molecules in the dimeric configuration from molecules in the tetrameric configuration
  • K16-6 and K17-6 derived r ⁇ 2 M was compared with human plasma ⁇ 2 M in such a protection assay.
  • 100 ⁇ l ⁇ 2 M (in 25 mM Tris-HCl, 125 mM NaCl , pH 8.0) was mixed with 30 ⁇ l trypsin (0.5 mg/ml in 20 mM sodium acetate pH 5.0). After incubating for 2 min. 30 ⁇ l 1 mg/ml STI (in PBS) was added. 10 ⁇ l ali- quots were removed after 2 and 4 min. and each mixed with 750 ⁇ l 0.12 mM S- 2222 (dissolved 0.1 M sodiumphosphate pH 8.0, 5% dimethylsulfoxide) . The change in absorbance at 405 nm was recorded for 2 min.
  • Table II The results of the assay are given in the following Table II:
  • the ⁇ 2 M characterized in the present investiga ⁇ tion could only be either bovine (contaminant from serum), from hamster (endogenous product from the BHK cell) or derived from expression of the transfected plasmid pll67.
  • the ELISA assay used never recognized any ⁇ 2 M in BHK cell conditioned medium, whether with or without added fetal calf serum.
  • amino terminal amino acid sequence determination was carried on out K16-6 and K17-6 r ⁇ 2 M as described (Sottrup-Jensen, L. et al., (1984) J. Biol. Chem.
  • EXAMPLE 4 Construction and expression of a bait region mutant of human ⁇ ? M.
  • the bait region of human ⁇ 2 M can be substituted by the bait region of human pregnancy zone protein (PZP) (Sottrup Jensen, L., Folkersen, J., Kristensen, T. and Tack, B.F. Partial primary structure of human pregnancy zone protein: extensive sequence homology with human alpha 2-macroglobul n. Proc. Natl. Acad. Sci. U.S.A. 81. 7353-7357, 1984; Sand, 0., Folkersen, J., Westergaard, J.G. and Sottrup Jensen, L. Characterization of human pregnancy zone protein. Comparison with human alpha 2-macroglobulin. J.Biol.Chem. 260, 15723-15735, 1985).
  • the resulting ⁇ 2 M bait region mutant exhibited a proteinase inhibitor profile similar to that of human pregnancy zone protein.
  • target sites for the restriction enzymes Pstl and SacII were introduced at the 5' and at the 3' end of the cDNA region encoding the bait region.
  • the human ⁇ 2 M expression plasmid pll67 was digested with BamHI and Clal, and a 2660 bp fragment, which carried the central part of the human ⁇ 2 M cDNA, was subcloned in the BamHI and Clal digested vector pSX191.
  • This vector which had previously been constructed, is a derivative of pUC19. It was constructed as described: pUC19 was digested with EcoRI and Hindlll, and a synthetic linker with the following sequence
  • the linker which was an annealing product from the two synthetic oligonucleotides N0R781 and N0R782, has cohesive ends that will ligate to the EcoRI and the Hindlll sites of pUC19 in such a way that these ligation sites are not regenerated in the pSX191 vector.
  • pSX191 carried sites for K ⁇ nl, Pstl, EcoRI, Hindlll. Clal, Sphl and BamHI.
  • 5'(AGCCACCCCCGCGGAGTTTACCAC)3 / was used to introduce a SacII site at position 2271 (SEQ ID NO:1) in the cDNA sequence. These sites were chosen because they did not introduce alterations in the encoded amino acid sequence, and they were within a convenient distance of the bait region in human ⁇ 2 M cDNA. Both primers were used in the same mutagenesis experiment (Kunkel , T.A., Roberts, J.D. and Zakour, R.A. Rapid and Efficient Site-Specific Mutagenesis without Phenotypic Selection. Methods in Enzvmol . 154.
  • dsDNA was isolated from mutated M13mpl8 ⁇ 2 M plaques, and the DNA was digested with the restriction enzymes Pstl and SacII. Correctly mutated recombinants, which had an insert of 160 bp, were further analyzed by DNA sequencing (Tabor, S. and Richardson, C.C DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 84, 4767-4771, 1987).
  • a 2.6 kb BamHI/Hindlll fragment from a correct ⁇ 2 M cDNA mutant (M13mpl8 ⁇ 2 M#212.1) was subcloned in a BamHI/Hindlll digested pUC13 vector, and a correct subclone pl308 was isolated and characterized with BamHI/Hindlll and Pstl/SacII double digestions and DNA electrophoresis.
  • the Pstl/SacII fragment in pl308 can be excised and replaced with a different DNA fragment, which encodes bait region variants.
  • the resulting new variants (bait region mutants or analogs) of ⁇ 2 M cDNA can be isolated as BamHI/Clal fragments and subcloned back into BamHI/Clal digested expression vector pll67.
  • DNA encoding the amino acids of the bait region for human PZP was obtained fro ligation, annealing and cloning of 8 synthetic oligonucleotide...
  • the DNA sequence of the synthetic fragment and the encoded amin acids as inserted into the ⁇ 2 M clone are given in SEQ ID N0:3, and comprises positions 2107 to 2305 and the corresponding amino acids.
  • This synthetic 0.2 kb DNA fragment was cloned in a Pstl/Ba H digested M13mpl8 vector for DNA sequencing.
  • DNA from a clone containing th correct sequence was digested with Pstl and SacII, and the purified 0.2 k fragment was cloned in a Pstl/SacII digested and gel purified pl308 vector.
  • nucleotides 2102 to 2275 in SEQ ID N0:1 was removed and replaced with nucleotides 2102 to 2305 in SEQ ID N0:3.
  • the purified recombinant ⁇ 2 M mutant, r ⁇ 2 M-PZP was characterized with respect to its inhibitor specificity profile against various proteina ⁇ ses by the use of previously described methods (Sand et al .1985) .
  • human plasma derived ⁇ 2 M and PZP were treated with the same set of proteinases in parallel reactions.
  • the proteinases used were chymotryp- sin, elastase, trypsin and Staphylococcus aureus Glu-specific proteinase.
  • Figure 10 illustrates the gel electrophoresis (10 - 20 % reducing SDS-PAGE) of the reaction products from chymotrypsin treated human ⁇ 2 M, human PZP and r ⁇ 2 M-PZP.
  • Molecular weight markers (from top to bottom: 180, 120, 92, 60, 43, 26, 14 and 6 kD) were applied to lanes 1 and 8. All samples were reduced.
  • Lanes 2, 3 and 4 show the cleavage products obtained from reaction of chymotrypsin with human plasma derived PZP, r ⁇ 2 M-PZP and human plasma derived ⁇ 2 M, respectively.
  • the ratio of proteinase to inhibitor was 1:1.
  • Lanes 5, 6 and 7 show cleavage products from similar reactions at a ratio of 2:1 UTE SHEET between proteinase and the three tested inhibitors. In all 6 lanes cleavage products (85 kD) could be identified. This indicated that r ⁇ 2 M-PZP reacted with chymotrypsin with similar characteristics as did human plasma derived ⁇ 2 M and PZP.
  • Figure 13 illustrates the gel electrophoresis (10 - 20 % reducing
  • the ratio of proteinas to inhibitor was 1:1.
  • Lanes 5, 6 and 7 show cleavage products from simila reactions at a ratio of 2:1 between proteinase and the three teste inhibitors.
  • cleavage products 85 kD
  • cleavage products could be identifie from the reaction between Staphylococcus aureus Glu-specific protease an ⁇ 2 M.
  • lanes 2, 3, 5 and 6 much less cleavage product could be identified from the reaction of this proteinase with .PZP and r ⁇ 2 M-PZP. This result demonstrated that r ⁇ 2 M-PZP reacted poorly with the Staphylococcus aureus proteinase as did human plasma derived PZP, while ⁇ 2 M was cleaved in the reaction with this proteinase.
  • r ⁇ 2 M-PZP showed the same pattern of reaction with four proteinases as did human plasma derived PZP. This pattern of reaction was different from the corresponding pattern obtained from reaction with ⁇ 2 M.
  • r ⁇ 2 M-PZP has been demonstrated to have a proteinase inhibitor profile similar to native PZP and dissimilar to ⁇ 2 M.
  • the proteinase inhibitor profile of ⁇ 2 M can be modulated by substitution of DNA fragments encoding the bait region.
  • ⁇ 2 M can be engineered (mutated in the bait region) to obtain the desired specificity.
  • saturation mutagenesis or random synthesis of the bait region will lead to an indefinite number of target sequences that can be introduced and expressed in hybrid macroglobulins.
  • These hybrids can be screened for proteinase inhibition, and the target sequence(s) can be identified.
  • the resulting ⁇ 2 M analog can be produced and purified as described elsewhere in this invention. Upon injection into the circulation such ⁇ 2 M analogs will inhibit and clear from the blood any proteinase of the given specificity.
  • GGT GGC CTC AAG CAA TTT TCT TTT CCC CTC TCA TCA GAG CCC TTC CAG 628 Gly Gly Leu Lys Gin Phe Ser Phe Pro Leu Ser Ser Glu Pro Phe Gin 185 190 195 200
  • AAG GGC CAT TTT TCC ATC TCA ATC CCT GTG AAG TCA GAC ATT GCT CCT 1636 Lys Gly His Phe Ser He Ser He Pro Val Lys Ser Asp He Ala Pro 525 530 535
  • AAC TAC AAA CAC TAT GAT GGC TCC
  • AGC ACC TTT GGG GAG CGA TAT 3124 Asn Tyr Lys His Tyr Asp Gly Ser Tyr Ser Thr Phe Gly Glu Arg Tyr 1020 1025 1030
  • GAT CCC AAA GGA AAT CGC ATC GCA CAA TGG CAG AGT TTC CAG TTA GAG 580 Asp Pro Lys Gly Asn Arg He Ala Gin Trp Gin Ser Phe Gin Leu Glu 170 175 180 GGT GGC CTC AAG CAA TTT TCT TTT CCC CTC TCA TCA GAG CCC TTC CAG 628 Gly Gly Leu Lys Gin Phe Ser Phe Pro Leu Ser Ser Glu Pro Phe Gin 185 190 195 ⁇ 200
  • GGC TCC TAC AAG GTG GTG GTA CAG AAG AAA TCA GGT GGA AGG ACA GAG 676 Gly Ser Tyr Lys Val Val Val Gin Lys Lys Ser Gly Gly Arg Thr Glu 205 210 215
  • AAG GGC CAT TTT TCC ATC TCA ATC CCT GTG AAG TCA GAC ATT GCT CCT 1636 Lys Gly His Phe Ser He Ser He Pro Val Lys Ser Asp He Ala Pro 525 530 535
  • GGC TCC TAC AGC ACC TTT GGG GAG CGA TAT GGC AGG AAC CAG GGC AAC 3172 Gly Ser Tyr Ser Thr Phe Gly Glu Arg Tyr Gly Arg Asn Gin Gly Asn 1035 1040 1045
  • GCT TAT CTC ACG GCC CAG CCA GCC CCA ACC TCG GAG GAC CTG ACC TCT 3748 Ala Tyr Leu Thr Ala Gin Pro Ala Pro Thr Ser Glu Asp Leu Thr Ser 1225 1230 1235 1240

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Abstract

Des alpha-macroglobulines, notamment les alpha2-macroglobulines humaines et leurs variantes, leurs fragments ou leurs dérivés, sont produites au moyen de la technologie de la recombinaison. Les produits sont utiles comme additifs aux milieux de croissance, comme inhibiteurs de la protéinase, comme porteurs lors du traitement de substitution d'enzymes, et comme porteurs ADN lors de la thérapie des gènes.
EP90913470A 1989-08-29 1990-08-29 Expression des alpha-macroglobulines Withdrawn EP0489830A1 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
DK423689A DK423689D0 (da) 1989-08-29 1989-08-29 Anvendelse af et humant plasmaglycoprotein
DK4235/89 1989-08-29
DK4236/89 1989-08-29
DK4237/89 1989-08-29
DK423789A DK423789D0 (da) 1989-08-29 1989-08-29 Ekspression og anvendelse af et humant plasmaglycoprotein
DK423589A DK423589D0 (da) 1989-08-29 1989-08-29 Ekspression af et humant plasmaglycoprotein

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EP0489830A1 true EP0489830A1 (fr) 1992-06-17

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US6342350B1 (en) 1997-09-05 2002-01-29 The General Hospital Corporation Alpha-2-macroglobulin diagnostic test
US6472140B1 (en) 1997-09-05 2002-10-29 The General Hospital Corporation α-2- macroglobulin therapies and drug screening methods for Alzheimer's disease.
US7741029B2 (en) 2001-03-07 2010-06-22 Andre Schuh Diagnosis and treatment of blood disorders
EP1409676A2 (fr) * 2001-03-07 2004-04-21 Andre Schuh Molecules d'acides nucleiques cd109, polypeptides et methodes d'utilisation associees
GB0224116D0 (en) * 2002-10-16 2002-11-27 Ares Trading Sa Proteins
WO2008024767A2 (fr) * 2006-08-21 2008-02-28 Wisconsin Alumni Research Foundation Procédé et composition permettant de traiter un trouble fibrotique

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