EP0861086A1 - Typ ix kollagen und chimere - Google Patents

Typ ix kollagen und chimere

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Publication number
EP0861086A1
EP0861086A1 EP96939682A EP96939682A EP0861086A1 EP 0861086 A1 EP0861086 A1 EP 0861086A1 EP 96939682 A EP96939682 A EP 96939682A EP 96939682 A EP96939682 A EP 96939682A EP 0861086 A1 EP0861086 A1 EP 0861086A1
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EP
European Patent Office
Prior art keywords
collagen
die
type
expression
human
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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EP96939682A
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English (en)
French (fr)
Inventor
Eero Vuorio
George Martin
Thomas B. Neff
Leena Ala-Kokko
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Fibrogen Inc
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Fibrogen Inc
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Publication of EP0861086A1 publication Critical patent/EP0861086A1/de
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to novel human collagen proteins, polynucleotide sequences which encode these novel collagens proteins, and to the use of these novel proteins in die diagnosis and treatment of disease. More specifically, me present invention relates to polynucleotides encoding human ⁇ 3(LX) collagen and derivatives hereof, fusion proteins of type IX collagen with type II and/or type XI collagen subunits and derivatives, and use of these proteins and polynucleotides as diagnostic and therapeutic agents.
  • Type LX collagen is a heterotrimer composed of diree polypeptide subunits: ⁇ l(LX), ⁇ 2(IX) and ⁇ 3(IX), tiiat are products of distinct genes and tiiat contain alternating non-triple-helical or noncollagenous domains (NCI -4) and triple-helical or collagenous domains (COLI -3).
  • NCI -4 non-triple-helical or noncollagenous domains
  • COLI -3 triple-helical or collagenous domains
  • hyaline cartilage from a variety of sources also contains significant amounts of at least three other collagen molecules, types VI, X and XI. Thomas et al. , Ann. Rheumat. Diseases 55:488-496 (1994); Mayne and Brewton, Cartilage Degradation: Basic and Clinical Aspects (Woessner, J.F. and Howell, D.S. , eds) Marcel Dekker, Inc., New York, pp. 81-108 (1993).
  • Type XI collagen like type IX collagen, is a heterotrimer composed of three different polypeptide subunits, ⁇ l(XI), ⁇ 2(XI), and ⁇ 3(XI). Collagen types XII and XIV were also isolated from bovine articular cartilage. Watt et al. , J. Biol. Chem. 267:20093-20099 (1992). Native type IX collagen molecules interact with type II collagen molecules in a highly specific manner so that die domains NCI, COLI , NC2, COL2 and NC3 lie along the surface of the collagen fibril. The interactions between type IX and type II collagen are stabilized by multiple covalent crosslinks derived from specific lysine residues. See van der Rest and Mayne, J.
  • the genes encoding the three chains of type IX collagen are excellent candidates for chondrodysplasias and degenerative disorders that affect the joints and/or vitreous humor because type IX collagen is a significant structural molecule in both of these tissues. Therefore, cloning of the genes encoding the three type LX collagen subunits has been the object of intensive research.
  • Muragaki et al. Eur. J. Biochem. 792:703-8 (1990) presented the complete cDNA sequence of bom alternative transcripts from the human ⁇ l(IX) gene. The majority of the human ⁇ 2(LX) collagen cDNA was reported by Perala et al. , FEBS Lett.
  • the present invention relates to novel collagen derivative proteins and the polynucleotide sequences which encode them. Also described herein are methods for diagnosis of diseases resulting from abnormalities in collagen synthesis or structure.
  • One aspect of this invention is the discovery that fusion proteins of human type DC collagen may be produced in which a human type LX collagen subunit is covalently linked to human type II collagen and/or a human type XI collagen subunit.
  • the fusion proteins are recombinantly produced as chimeras by linking polynucleotide coding sequence for a human type LX collagen subunit in frame to polynucleotide coding sequence for human type II collagen and/or a human type XI collagen.
  • This chimeric coding sequence is inserted into an expression vector and used to transform appropriate host cells.
  • the host cells are then induced to express the chimeric coding sequence and thereby produce the chimeric collagen fusion proteins.
  • These fusion proteins are useful in the treatment of collagen related diseases and conditions.
  • the present invention also relates, in part, to nucleotide sequences and expression vectors encoding the chimeric collagens of the invention.
  • Such abnormalities can result in, for example, rheumatoid arthritis, osteoarthritis, reactive arthritis, autoimmune hearing disease, cartilage inflammation due to bacterial or viral infections (e.g. Lyme's disease), parasitic disease, bursitis, corneal diseases, and anky losing spondylitis (fusion of the spine).
  • the novel proteins of the invention are used in these methods of treating collagen related diseases.
  • the present invention relates to the polynucleotide and nucleic acid sequences encoding the recombinant fusion proteins of type IX collagen derivatives with type II collagen protein and/or type XI collagen protein and such fusion proteins. Also within the scope of the invention are methods of using these collagen fusion proteins to treat collagen related disorders and conditions.
  • collagen subunit refers to the amino acid sequence of one subunit of a collagen protein encoded by a single gene, as well as derivatives, including deletion derivatives, conservative substitutions, etc.
  • a “fusion protein” is a protein in which peptide sequences from different proteins are covalently linked together.
  • the term “chimera” or “chimeric” refers to fusion protein produced by operably linking polynucleotide coding sequence for two or more collagen protein subunits in frame and recombinantly expressing the linked coding sequence as a single peptide chain.
  • Active human type IX collagen refers to the native trimeric protein complex, and may be recombinantly produced.
  • stringent conditions refers to those hybridizing conditions that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1 % SDS at 50°C; (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1 % bovine serum albumin 0.1 % Ficoll/0.1 % poly viny lpyrroIidone/50 mM sodium phosphate buffer at pH 6.5 witii 750 mM NaCl, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M Sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 g/ml), 0.1 % SDS, and 10% dextran sulfate at 42 °C, with washes at 42°C in 0.2 x S
  • formamide for example,
  • any nucleotide sequence which encodes d e amino acid sequence of claimed fusion protein can be used to generate recombinant molecules which direct die expression of the fusion protein.
  • purified as used herein in reference to collagens denotes that the indicated molecules are present in die substantial absence of otiier biological macromolecules, e.g. , polynucleotides, proteins, and the like.
  • purified as used herein preferably means at least 95% by weight, more preferably at least 99.8% by weight, of the indicated biological macromolecules present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000 daltons can be present).
  • isolated refers to a protein molecule separated not only from other proteins that are present in the natural source of the protein, but also from other proteins, and preferably refers to a protein found in die presence of (if anything) only a solvent, buffer, ion, or other component normally present in a solution of the same.
  • isolated and purified do not encompass proteins present in their natural source.
  • polynucleotide sequences which encode type IX, type IT, and type XI collagen proteins, or functional equivalents thereof may be used to generate recombinant DNA molecules that direct the expression of fusion proteins of type IX collagen subunit witii type II collagen and/or type XI collagen subunits, or a functional equivalent thereof, in appropriate host cells.
  • Such collagen polynucleotide sequences, as well as other polynucleotides which selectively hybridize to at least a part of such collagen polynucleotides or their complements may also be used in nucleic acid hybridization assays, Southern and Northern blot analyses, etc.
  • DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence, may be used in the practice of the invention for the cloning and expression of these collagen proteins.
  • DNA sequences include those which are capable of hybridizing to the appropriate human collagen sequence under stringent conditions.
  • Altered DNA sequences which may be used in accordance with the invention include deletions, additions or substitutions of different nucleotide residues resulting in a sequence that encodes the same or a functionally equivalent gene product.
  • the gene product itself may contain deletions, additions or substitutions of amino acid residues witiiin a collagen sequence, which result in a silent change thus producing a functionally equivalent collagen.
  • Such amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipatic nature of the residues involved.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; amino acids with uncharged polar head groups having similar hydrophilicity values include the following: leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine; serine, threonine; phenylalanine, tyrosine.
  • the DNA sequences of the invention may be engineered in order to alter die collagen coding sequence for a variety of ends including but not limited to alterations which modify processing and expression of the gene product.
  • alternative secretory signals may be substituted for the native human secretory signal and/or mutations may be introduced using techniques which are well known in the art, e.g. , site-directed mutagenesis, to insert new restriction sites, to alter glycosylation patterns, phosphorylation, etc.
  • die polynucleotides encoding the collagens of the invention may be modified in die silent position of any triplet amino acid codon so as to better conform to the codon preference of die particular host organism.
  • a collagen sequence may be ligated to a heterologous sequence to encode a fusion protein.
  • a fusion protein may be engineered to contain a cleavage site located between an ⁇ 3(IX) collagen sequence and the heterologous protein sequence, so that the ⁇ 3(IX) collagen may be cleaved away from the heterologous moiety.
  • chimeric fusion proteins are constructed by ligating sequence encoding a type IX collagen subunit, or derivative thereof, to sequence encoding type II collagen and/or type XI collagen subunit.
  • sequence encoding a type IX collagen subunit or derivative thereof, to sequence encoding type II collagen and/or type XI collagen subunit.
  • One of skill in the art will recognize that several techniques are available which allow one to ligate all or any part of the coding sequence of a type IX collagen subunit to all or any part of the coding sequences for type II and type XI collagens. For example, one may ligate coding sequences together at appropriately chosen restriction endonuclease sites. However, in order to ensure tiiat the coding sequences of the chosen collagens are ligated in the correct translational frame, it may be necessary to engineer restriction sites by site specific mutagenesis.
  • the protein itself could be produced using chemical methods to synthesize the desired collagen amino acid sequence at least in part.
  • peptides can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography. (e.g., see Creighton, Proteins Structures And Molecular Principles, W.H. Freeman and Co., N.Y. , pp. 50-60 (1983).
  • composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g. , the Edman degradation procedure; see Creighton, Proteins, Structures and Molecular Principles, W.H. Freeman and Co., N.Y. , pp. 34-49 (1983).
  • amino acid analysis or sequencing e.g. , the Edman degradation procedure; see Creighton, Proteins, Structures and Molecular Principles, W.H. Freeman and Co., N.Y. , pp. 34-49 (1983).
  • the nucleotide sequence encoding d e collagen, or a functional equivalent is inserted into an appropriate expression vector, i.e. , a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • a variety of host-expression vector systems may be utilized to express a collagen coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing a collagen coding sequence; yeast transformed witii recombinant yeast expression vectors containing a collagen coding sequence; insect cell systems infected witii recombinant virus expression vectors (e.g. , baculovirus) containing sequence encoding the collagens of the invention; plant cell systems infected with recombinant virus expression vectors (e.g.
  • the collagens of the invention may be expressed in transgenic non-human animals wherein the desired collagen product may be recovered from d e milk of d e transgenic animal.
  • the expression elements of these systems vary in their strength and specificities. Depending on the host/vector system utilized, any of a number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used in die expression vector.
  • inducible promoters such as pL of bacteriophage 1, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used; when cloning in insect cell systems, promoters such as die baculovirus polyhedron promoter may be used; when cloning in plant cell systems, promoters derived from the genome of plant cells (e.g. , heat shock promoters; the promoter for the small subunit of RUBISCO; the promoter for the chlorophyll a/b binding protein) or from plant viruses (e.g.
  • the 35S RNA promoter of CaMV; the coat protein promoter of TMV may be used; when cloning in mammalian cell systems, promoters derived from d e genome of mammalian cells (e.g. , metallothionein promoter) or from mammalian viruses (e.g. , the adenovirus late promoter; the vaccinia virus 7.5 K promoter) may be used; when generating cell lines tiiat contain multiple copies of a collagen DNA, SV40-, BPV- and EBV-based vectors may be used with an appropriate selectable marker. In bacterial systems a number of expression vectors may be advantageously selected depending upon die use intended for the collagen expressed.
  • promoters derived from d e genome of mammalian cells e.g. , metallothionein promoter
  • mammalian viruses e.g. , the adenovirus late promoter; the vaccinia virus 7.5 K promoter
  • vectors which direct die expression of high levels of fusion protein products that are readily purified may be desirable.
  • Such vectors include but are not limited to the E. coli expression vector pUR278 (Ruther et al. , EMBO J. 2: 1791 (1983)), in which the collagen coding sequence may be ligated into the vector in frame with the lac Z coding region so that a hybrid AS-lac Z protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 75:3101- 3109 (1985); Van Heeke & Schuster, . Biol. Chem.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in die presence of free glutatiuone.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from die GST moiety.
  • a preferred expression system is a yeast expression system.
  • yeast a number of vectors containing constitutive or inducible promoters may be used.
  • Current Protocols in Molecular Biology Vol. 2, Ed. Ausubel et al. , Greene Publish. Assoc. & Wiley Interscience, Ch. 13 (1988); Grant et al. , Expression and Secretion Vectors for Yeast, in Methods in Enzymology, Ed. Wu & Grossman, Acad. Press, N.Y. 755:516-544 (1987); Glover, DNA Cloning, Vol. 77, IRL Press, Wash. , D.C, Ch.
  • a particularly preferred system useful for cloning and expression of die collagen proteins of the invention uses host cells from the yeast Pichia.
  • Species of non-Saccharomyces yeast such as Pichia pastoris appear to have special advantages in producing high yields of recombinant protein in scaled up procedures.
  • a Pichia expression kit is available from Invitrogen Corporation (San Diego, CA).
  • methanol responsive regulatory regions also referred to as promoters. Any of such methanol responsive promoters are suitable for use in the practice of the present invention.
  • Examples of specific regulatory regions include the promoter for the primary alcohol oxidase gene from Pichia pastoris AOX1, die promoter for the secondary alcohol oxidase gene from P. pastoris AXO2, the promoter for the dihydroxyacetone syntiiase gene from P. pastoris (DAS), the promoter for the P40 gene from P. pastoris, the promoter for the catalase gene from P. pastoris, and die like.
  • Typical expression in Pichia pastoris is obtained by the promoter from die tightly regulated AOX1 gene. See Ellis et al., Mol. Cell. Biol. 5: 1111 (1985) and U.S. Patent No. 4,855,231. This promoter can be induced to produce high levels of recombinant protein after addition of metiianol to the culture. By subsequent manipulations of the same cells, expression of genes for the collagens of the invention described herein is achieved under conditions where die recombinant protein is adequately hydroxylated by prolyl 4-hydroxylase and, therefore, can fold into a stable helix tiiat is required for the normal biological function of the proteins in forming fibrils.
  • Another particularly preferred yeast expression system makes use of the methylotrophic yeast Hansenula polymorpha.
  • Growth on methanol results in the induction of key enzymes of the methanol metabolism, namely MOX (methanol oxidase), DAS (dihydroxyacetone synthase) and FMHD (formate dehydrogenase). These enzymes can constitute up to 30-40% of the total cell protein.
  • MOX methanol oxidase
  • DAS dihydroxyacetone synthase
  • FMHD formate dehydrogenase
  • the genes encoding MOX, DAS, and FMDH production are controlled by very strong promoters which are induced by growtii on methanol and repressed by growth on glucose. Any or all three of these promoters may be used to obtain high level expression of heterologous genes in H. polymorpha.
  • the gene encoding a collagen of the invention is cloned into an expression vector under die control of an inducible H. polymorpha promoter. If secretion of die product is desired, a polynucleotides encoding a signal sequence for secretion in yeast, such as die S. cerevisiae prepro-mating factor ⁇ l, is fused in frame witii the coding sequence for die collagen of d e invention.
  • the expression vector preferably contains an auxotrophic marker gene, such as URA3 or LEU2, which may be used to complement d e deficiency of an auxotrophic host.
  • the expression vector is tiien used to transform H. polymorpha host cells using techniques known to those of skill in the art.
  • An interesting and useful feature of H. polymorpha transformation is d e spontaneous integration of up to 100 copies of die expression vector into die genome.
  • the integrated DNA forms multimers exhibiting a head-to- tail arrangement.
  • the integrated foreign DNA has been shown to be mitotically stable in several recombinant strains, even under non-selective conditions. This phenomena of high copy integration further adds to me high productivity potential of the system.
  • die expression of sequences encoding the collagens of the invention may be driven by any of a number of promoters.
  • viral promoters such as d e 35S RNA and 19S RNA promoters of CaMV (Brisson et al , Nature 570:511-514 (1984), or die coat protein promoter of TMV (Takamatsu et al. , EMBO J. 3:17-311 (1987)) may be used; alternatively, plant promoters such as die small subunit of RUBISCO (Conizzi et al. , EMBO J. 5: 1671-1680 (1984); Broglie et al.
  • An alternative expression system which could be used to express the collagens of the invention is an insect system.
  • Autographa calif ornica nuclear polyhidrosis virus (AcNPV) is used as a vector to express foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • Coding sequence for the collagens of the invention may be cloned into non- essential regions (for example die polyhedron gene) of the virus and placed under control of an AcNPV promoter (for example, die polyhedron promoter).
  • Successful insertion of a collagen coding sequence will result in inactivation of d e polyhedron gene and production of non-occluded recombinant virus (i.e. , virus lacking die proteinaceous coat coded for by the polyhedron gene).
  • viruses are tiien used to infect Spodoptera frugiperda cells in which the inserted gene is expressed. (e.g. , see Smith et al. , J. Virol. 46:584 (1983); Smith, U.S. Patent No. 4,215,051).
  • a number of viral based expression systems may be utilized.
  • coding sequence for die collagens of die invention may be ligated to an adenovirus transcription/translation control complex, e.g. , die late promoter and tripartite leader sequence.
  • This chimeric gene may tiien be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non- essential region of the viral genome (e.g. , region El or E3) will result in a recombinant virus that is viable and capable of expressing collagen in infected hosts, (e.g. , See Logan & Shenk, Proc.
  • the vaccinia 7.5 K promoter may be used.
  • Mackett et al. Proc. Natl. Acad. Sci. (USA) 79:7415- 7419 (1982); Mackett et al. , J. Virol. 49:857-864 (1984); Panicali et al. , Proc. Natl. Acad. Sci. 79:4927-4931 (1982).
  • Specific initiation signals may also be required for efficient translation of inserted collagen coding sequences. These signals include die ATG initiation codon and adjacent sequences. In cases where d e entire collagen gene, including its own initiation codon and adjacent sequences, is inserted into d e appropriate expression vector, no additional translational control signals may be needed. However, in cases where only a portion of a collagen coding sequence is inserted, exogenous translational control signals, including the ATG initiation codon, must be provided. Furthermore, die initiation codon must be in phase witii the reading frame of the collagen coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and syntiietic. The efficiency of expression may be enhanced by d e inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al. , Methods in Enzymol. 755:516-544 (1987)).
  • One preferred expression system for the recombinant production of the collagens of the invention is in transgenic non-human animals, wherein the desired collagen may be recovered from the milk of the transgenic animal.
  • a system is constructed by operably linking the DNA sequence encoding the collagens of the invention to a promoter and other required or optional regulatory sequences capable of effecting expression in mammary glands.
  • required or optional post-translational enzymes may be produced simultaneously in the target cells, employing suitable expression systems, as disclosed in, inter alia, U.S. Application, Serial No. 08/037,728, operable in the targeted milk protein producing mammary gland cells.
  • the promoter of choice would preferably be from one of the abundant milk- specific proteins, such as alpha Sl-casein, or b- lactoglobulin.
  • alpha Sl-casein 5' and 3' regulatory sequences of alpha Sl-casein have been successfully used for the expression of the human lactoferrin cDNA, and similarly, die b-lactoglobin promoter has effected the expression of human antitrypsin gene fragments in sheep milk producing cells.
  • the whey acid promoter has been used for die expression of human tissue plasminogen activator, resulting in the secretion of human tissue plasminogen activator in the milk of the transgenics.
  • the gene encoding the desired collagen chain can simply be ligated to suitable control sequences which function in d e mammary cells of die chosen animal species.
  • Expression systems for the genes encoding the required post-translational enzymes are constructed analogously.
  • the collagens of the invention are expressed as secreted proteins.
  • the engineered cells used for expression of the proteins are non- human host cells, it is often advantageous to replace die human secretory signal peptide of the collagen protein with an alternative secretory signal peptide which is more efficiently recognized by die host cell's secretory targeting machinery.
  • the appropriate secretory signal sequence is particularly important in obtaining optimal fungal expression of mammalian genes. For example, in methylotrophic yeasts, a DNA sequence encoding the in-reading frame S. cerevisiae a- mating factor pre-pro sequence may be inserted at the aniino-terniinal end of die coding sequence.
  • the aMF pre-pro sequence is a leader sequence contained in die aMF precursor molecule, and includes the lys-arg encoding sequence which is necessary for proteolytic processing and secretion (see, e.g. , Brake et al , Proc. Nat'l Acad. Sci. USA, 57:4642 (1984)).
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes d e gene product in the specific fashion desired. Such modifications (e.g. , glycosylation) and processing (e.g. , cleavage) of protein products may be important for d e function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post- translational processing and modification of proteins.
  • Appropriate cells lines or host systems can be chosen to ensure die correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of die gene product may be used.
  • mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, WI38, etc.
  • host cells may be engineered to express various enzymes to ensure the proper processing of the collagen molecules.
  • the gene for prolyl-4-hydroxylase may be coexpressed witii the collagen gene in the host cell. For long-term, high-yield production of recombinant proteins, stable expression is preferred.
  • cell lines which stably express die collagens of the invention may be engineered.
  • host cells can be transformed witii collagen encoding DNA controlled by appropriate expression control elements (e.g. , promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • expression control elements e.g. , promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and tiien are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • This metiiod may advantageously be used to engineer cell lines which express a desired collagen.
  • a number of selection systems may be used, including but not limited to the he ⁇ es simplex virus thymidine kinase (Wigler et al , Cell 77:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci.
  • adenine phosphoribosyltransferase genes can be employed in tk-, hgprt- or aprt- cells, respectively.
  • antimetabolite resistance can be used as die basis of selection for dhfr, which confers resistance to memotrexate (Wigler et al. , Natl Acad. Sci. USA 77:3567 (1980); O'Hare et al , Proc. Natl. Acad. Sci. USA 78:1521 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc.
  • the host cells which contain the coding sequence and which express die biologically active gene product may be identified by at least four general approaches; (a) DNA-DNA or DNA-RNA hybridization; (b) the presence or absence of "marker" gene functions; (c) assessing the level of transcription as measured by die expression of collagen mRNA transcripts in the host cell; and (d) detection of the gene product as measured by immunoassay or by its biological activity.
  • the presence of the collagen coding sequence inserted in the expression vector can be detected by DNA-DNA or DNA-RNA hybridization using probes comprising nucleotide sequences that are homologous to the collagen coding sequence, respectively, or portions or derivatives thereof.
  • the recombinant expression vector/host system can be identified and selected based upon die presence or absence of certain "marker" gene functions (e.g. , tiiymidine kinase activity, resistance to antibiotics, resistance to methotrexate, transformation phenotype, occlusion body formation in baculovirus, etc.).
  • certain "marker" gene functions e.g. , tiiymidine kinase activity, resistance to antibiotics, resistance to methotrexate, transformation phenotype, occlusion body formation in baculovirus, etc.
  • certain "marker” gene functions e.g. , tiiymidine kinase activity, resistance to antibiotics, resistance to methotrexate, transformation phenotype, occlusion body formation in baculovirus, etc.
  • a marker gene can be placed in tandem witii the collagen sequence under the control of the same or different promoter used to control die expression of the collagen coding sequence
  • transeriptional activity of the collagen coding region can be assessed by hybridization assays.
  • RNA can be isolated and analyzed by Northern blot using a probe homologous to the collagen coding sequence or particular portions thereof.
  • total nucleic acids of die host cell may be extracted and assayed for hybridization to such probes.
  • die expression of a collagen protein product can be assessed immunologically, for example by Western blots, immunoassays such as radioimmuno-precipitation, enzyme-linked immunoassays and d e like.
  • the expressed collagen of the invention which is preferably secreted into the culture medium, is purified to homogeneity, e.g., by chromatography.
  • die recombinant collagen protein is purified by size exclusion chromatography.
  • odier purification techniques known in the art can also be used, including ion exchange chromatography, and reverse-phase chromatography.
  • antibodies to epitopes of the recombinantly produced collagens.
  • Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library.
  • various host animals may be immunized by injection witii a collagen protein including but not limited to rabbits, mice, rats, etc.
  • adjuvants may be used to increase d e immunological response, depending on die host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacilli Calmette-Guerin) and Corynebacterium parvum.
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum bacilli Calmette-Guerin
  • Monoclonal antibodies to a collagen may be prepared by using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Koehler and Milstein, (Nature, 256:495-497 (1975)), die human B-cell hybridoma technique (Kosbor et al , Immunology Today, 4:12 (1983)); Cote et al , Proc. Natl. Acad. Sc , 50:2026-2030 (1983) and the EBV- hybridoma technique (Cole et al , Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985).
  • such fragments include but are not limited to: the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and die Fab fragments which can be generated by reducing die disulfide bridges of the F(ab')2 fragments.
  • Fab expression libraries may be constructed (Huse et al , Science 246: 1275-1281 (1989)) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for the collagen of interest. 2. Therapeutic Uses Of The Collagen Proteins Of The Invention
  • Another aspect of the invention is to provide methods of treating immune system-mediated diseases using the collagen proteins of the invention.
  • treatment or “treating” as used herein witii reference to a disease refer both to prophylaxis and to the amelioration of symptoms already present in an individual. It will be appreciated by die person of ordinary skill in die art that a treatment need not be completely effective in preventing the onset of a disease or in reducing die symptoms associated widi die disease. Any reduction of the severity of symptoms, delay in the onset of symptoms, or delay in die progression of severity of symptoms is desirable to a patient.
  • Persons at risk of developing a given immune system-mediated disease may be treated prophylactically based on any of a variety of factors suggesting the possible onset of an immune system- mediated disease, e.g. , family history, genetic markers, early symptoms, and die like.
  • Immune system-mediated diseases that may be treated by the subject methods include, but are not limited to, e.g. , rheumatoid arthritis, osteoarthritis, reactive arthritis, autoimmune hearing disease, cartilage inflammation due to bacterial or viral infections (e.g. Lyme's disease), parasitic disease, bursitis, comeal diseases, and anky losing spondylitis (fusion of the spine).
  • the subject methods of the invention comprise the step of administering an effective amount of a composition of the invention, e.g., collagens, collagen derivatives.
  • compositions for use in treating specific immune system-mediated diseases are fusion proteins of type IX collagen, preferably chimeras of a type IX collagen subunit with type 13 collagen and/or type XI collagen, and derivatives and subunits thereof, as well as as described in the preceding sections.
  • the compositions administered to the subject comprise variably glycosylated collagens.
  • the compositions administered in die subject methods are administered so that the active components, i.e. , collagens and/or collagen derivatives, contact the lymphoid tissue of the gut, e.g. , Peyer's patches or other similar sites, so that immune tolerance is induced.
  • Such administration may be effected, by among many possible methods, dirough the use of formulations comprising the subjected compositions that are designed for oral administration, i.e. , the active components are not destroyed or inactivated in the mouth, stomach, or other portions of the digestive system prior to contacting the appropriate gut lymphoid tissue.
  • the treatment med ods of the invention may also comprise the steps of administering additional pharmaceutical compounds for d e treatment of immune system-mediated diseases, such as anti-inflammatory agents and d e like.
  • the dosage at which d e subject compositions are administered may vary within a wide range and will depend on various factors such as for example die severity of die inflammation, the age of the patient, etc., and may have to be individually adjusted.
  • the amount of collagen(s) and/or collagen(s) derivatives which may be ad'ministered per day may be in the range of from about 0.001 mg to about 200 mg.
  • d e amount of collagen and/or collagen derivatives administered is low, tiiereby favoring die induction of immune tolerance by suppression rather than clonal anergy.
  • the pharmaceutical compositions containing the collagen(s) and/or collagen(s) derivatives may suitably be formulated so diat they provide doses witiiin these ranges either as single dosage units or as multiple dosage units.
  • the optimal dosage of tolerance inducing compositions for use in d e methods of the invention will vary in accordance witii a number of factors.
  • the terms "dosage” and “dose” as used herein, unless indicated otiierwise, may refer not only to a single administration of a composition but may be used to refer to die total amount of a given pharmaceutical composition administered over a selected period of time and involving multiple individual administrations.
  • Factors affecting d e optimal dosage include d e choice of collagen molecule or molecules (and/or collagen derivatives) administered to the patient, the specific mucosa binding molecules selected, d e age of the patient, the severity of the disease, odier diseases tiiat may be present in the patient, inert components in die formulation, adjuvants, and the like. There may be considerable variation in the range of dosages that are effective in treating a given immune disorder. Different dosages of die same pharmaceutical composition may produce the desired tolerance effect by different mechanisms. Although the operation of the invention is not dependent upon a particular theory of operation, the person of ordinary skill in the art will better understand the invention and provide additional embodiments by appreciating that there are believed to be two primary mechanisms by which oral tolerance is mediated.
  • Oral tolerance may be mediated by active cellular suppression in which regulatory T cells that suppress the activation and proliferation of lymphocytes specific for tolerized antigen.
  • Anotiier mechanism of oral tolerance induction is clonal anergy in which T lymphocytes having a suitable receptor are rendered unresponsive.
  • active suppression tolerance is favored by "low" doses of a tolerizing antigen and clonal anergy is favored by comparatively "high” doses of die same tolerizing antigen.
  • compositions may be formulated as pharmaceutical compositions so as to be adapted for certain types of administration to mucosal surfaces, e.g. , oral, topical, and inhalation.
  • compositions of the invention may be administered topically, orally, intranasally, by injection or by inhalation in die form of a pharmaceutical compositions comprising a collagen(s) and/or collagen(s) derivatives in the form of the original compound or optionally in die form of a pharmaceutically acceptable salt thereof, in association with a pharmaceutically acceptable carrier which may be a solid, semi-solid or liquid diluent or an ingestible capsule, and such preparations comprise a further aspect of the invention.
  • a pharmaceutically acceptable carrier which may be a solid, semi-solid or liquid diluent or an ingestible capsule, and such preparations comprise a further aspect of the invention.
  • the collagen(s) and/or collagen(s) derivatives and mucosa binding collagen conjugates may also be used with carrier material.
  • d e collagen(s) and/or collagen(s) derivatives will comprise between 0.05 and 99%, or between 0.1 and 99% by weight of d e preparation, for example between 0.5 and 20% for preparations intended for injection and between 0.1 and 50% for preparations intended for oral administration.
  • the active ingredient may be mixed widi a solid, pulverulent carrier, for example lactose, saccharose, sorbitol, mannitol, a starch such as potato starch, co starch, amylopectin, laminaria powder or citrus pulp powder, a cellulose derivative or gelatine and also may include lubricants such as magnesium or calcium stearate or a Carbowax " or other polyethylene glycol waxes and are compressed to form tablets or cores for dragees.
  • a solid, pulverulent carrier for example lactose, saccharose, sorbitol, mannitol, a starch such as potato starch, co starch, amylopectin, laminaria powder or citrus pulp powder, a cellulose derivative or gelatine and also may include lubricants such as magnesium or calcium stearate or a Carbowax " or other polyethylene glycol waxes and are compressed to form tablets or cores for dragees.
  • die cores may be coated, for example, witii concentrated sugar solutions which may contain gum arabic, talc and/or titanium dioxide, or alternatively with a film forming agent dissolved in easily volatile organic solvents or mixtures of organic solvents.
  • Dyestuffs can be added to these coatings, for example, to distinguish between different contents of active substance.
  • die active substance may be admixed witii a Carbowax " or a suitable oil as e.g. sesame oil, olive oil, or arachis oil.
  • Hard gelatine capsules may contain granulates of die active substance witii solid, pulverulent carriers such as lactose, saccharose, sorbitol, mannitol, starches (for example) potato starch, com starch or amylopectin), cellulose derivatives or gelatine, and may also include magnesium stearate or stearic acid as lubricants.
  • pulverulent carriers such as lactose, saccharose, sorbitol, mannitol, starches (for example) potato starch, com starch or amylopectin), cellulose derivatives or gelatine, and may also include magnesium stearate or stearic acid as lubricants.
  • compositions of the invention may also be formulated so as to provide a sustained release.
  • sustained release tablets By using several layers of die active drug, separated by slowly dissolving coatings sustained release tablets may be obtained.
  • Anotiier way of preparing sustained release tablets is to divide die dose of d e active drug into granules witii coatings of different thicknesses and compress the granules into tablets together with the carrier substance.
  • the collagen(s) and/or collagen(s) derivatives and mucosa binding collagen conjugates may also be inco ⁇ orated in slowly dissolving tablets made, for instance, of fat and wax substances or evenly distributed in a tablet of an insoluble substance such as a physiologically inert plastic substance.
  • die tablets, dragees etc. may be enteric- coated, that is provided witii a layer of gastric juice-resistant enteric film or coating having such properties that it is not dissolved at the acidic pH in the gastric juice.
  • enteric coatings may be mentioned cellulose acetate phthalate, hydroxypropyl-methylcellulose phthalates such as those sold under the trade names HP 55 and HP 50, and Edragir»L and Eudragit*S.
  • Liquid preparations for oral application may be in the form of elixirs, syrups or suspensions, for example solutions containing from about 0.1 % to 20% by weight of active substance, sugar and a mixture or ethanol, water glycerol, propylene glycol and optionally aroma, saccharine and/or carboxymethylcellulose as a dispersing agent.
  • PCR primers for the amplification of the ⁇ 3(IX) collagen cDNA coding sequence from d e plasmid p545 and die cDNA library clone RB410 are prepared.
  • the primers are designed such tiiat they introduce an Eco RI site at d e
  • a Primer 1 and a Primer 2 are used to amplify the mature amino-terminal coding sequence for ⁇ 3(IX) collagen from plasmid p545 using standard PCR conditions as described in Ausubel et al , Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y. (1990).
  • a Primer 3 and Primer 4 are used to amplify tiie remaining cDNA coding sequence, including die stop codon, from die cDNA clone RB410 as described above.
  • the resulting PCR product is digested widi die chosen unique restriction endonuclease and witii EcoR I.
  • Commercially available expression vector pPIC9 Invitrogen, San Diego,
  • CA which directs secreted expression in Pichia pastoris is digested with restriction endonuclease EcoR I, followed by calf intestinal phosphatase (Pharmacia), and en heat denaturation at 70°C for 5 minutes.
  • the digested PCR products and d e pPIC9 vector are gel purified as described in Example 3 and a tirree-way ligation is performed. After transformation into competent Escherichica coli, correctly ligated plasmids are identified by restriction analysis and confirmed by sequencing using the commercially available Pichia sequencing primers (Invitrogen, San Diego, CA).
  • the ⁇ 3(IX) Pichia expression vector is linearized and used to transform spheroblasts of a his4 Pichia pastoris strain which also expresses prolyl-4- hydroxylase. Transformants are identified on histidine deficient media and are confirmed by assaying for die loss of die AOX1 gene by slow growth on methanol media. Expression of the ⁇ 3(IX) gene is induced by growing cells on methanol as the sole carbon source. ⁇ 3(IX) collagen subunit protein is secreted into die growtii medium and subsequently purified using standard centrifugation, filtration, and chromatographic techniques.
  • die Pichia pastoris strain which produces c.3(IX) collagen subunit is engineered to coexpress the ⁇ l(IX) and ⁇ 2(IX) collagen subunits in die same cell.
  • Three recombinant viruses were generated by cotransfection of recombinant ⁇ l(IX), ⁇ 2(IX), and ⁇ 3(IX) constructs and a modified Autographa califomica nuclear polyhedrosis virus DNA into Spodoptera frugiperda Sf9 insect cells using the Baculogold transfection kit (Pharmingen).
  • the sequences used to construct tiie three a(IX) chains are disclosed in van der Rest and Mayne, Structure and Function of Collagen Types (Mayne, R. and Burgeson, R. eds.) Academic Press, Orlando, FL, pp. 185-221 (1987).
  • the resultant viral pools were collected, amplified, and plaque- purified as described by Gruenwald, S. and Heitz, J.,
  • Baculovirus Expression System Procedures & Methods Manual, Pharmingen, San Diego, CA (1993). Spodoptera frugiperda Sf9 insect cells were cultured in TNH-FH medium supplemented witii 10% fetal bovine serum (BioClear) as monolayers at 27°C. Approximately, 5 x IO 6 insect cells were infected with recombinant human ⁇ l(IX), c-2(IX), and ⁇ 3(IX) constructs and widi die a ⁇ virus for human prolyl 4-hydroxylase (manuscript in preparation). Viruses for the type IX collagen ⁇ - chains were used in 2 to 3 fold excess over the prolyl 4-hydroxylase virus.
  • Ascorbate of 80 ⁇ g/ml was added to the culture medium daily.
  • the culture media was removed 72 hours after infection, and the cell layer was washed once with a solution of 0.15 NaCl and 0.02 M phosphate at pH of 7.4.
  • the cells were harvested by scraping diem in 1.4 ml of ice cold solution of 0.5 acetic acid, 0.75 M NaCl, lOmM EDTA, and ImM PMSF at pH of 2.5. The cells were then homogenized, and centrifuged at 15000 x g for 20 minutes. Supernatant was precipitated widi NaCl at the final concentration of 1.2 M by mixing the sample for 12 hours at 4°C.
  • the precipitate was centrifuged at 15000 x g for 20 minutes at 4°C.
  • the resulting pellet was dissolved in 500 ⁇ l of cold 50mM acetic acid for tliree hours at 4°C.
  • a sample of 15 ⁇ l was analyzed by eitiier non-reducing or reducing SDS-PAGE followed by staining witii Coomassie Brilliant Blue.
  • the material was also digested with pepsin for 4 hours at 22 °C, and the thermal stability of the pepsin-resistant recombinant type IX collagen was measured by rapid digestion witii a mixture of trypsin and chymotrypsin as described in Buckner et al, Anal Biochem 110:360-368 (1981).
  • the resulting material was analyzed on reducing SDS-PAGE followed by Westem Blotting with an antibody to a triple-helical collagen.
  • the ⁇ 3(IX) Pichia expression vector described above is modified so as to direct expression of a chimeric type 11/ type IX/type XI molecule.
  • die vector is cleaved eitiier 5' or 3' to the ⁇ 3(IX) collagen coding sequence and coding sequence for type II collagen is inserted in-frame.
  • the vector may be again cleaved, eitiier 5', 3', or between the type II and type IX coding sequence, and sequence encoding type XI collagen is also inserted in die correct reading frame so as to express a chimeric type II/IX XI collagen molecule.
  • Transformants of competent E. coli are screened for plasmids witii the desired orientation by restriction digestion and confirmed by sequencing as described above.

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US5773248A (en) * 1995-11-13 1998-06-30 Uab Research Foundation Nucleic acid encoding a human α3(IX) collagen protein and method of producing the protein recombinantly
CA2269801A1 (en) * 1996-10-29 1998-05-07 Commonwealth Scientific And Industrial Research Organisation Stable expression of triple helical proteins
AUPO331096A0 (en) * 1996-10-29 1996-11-21 Commonwealth Scientific And Industrial Research Organisation Collagen products
KR20020059719A (ko) * 1999-11-12 2002-07-13 추후보정 재조합 젤라틴
AU1770201A (en) * 1999-11-17 2001-05-30 Philadelphia Health & Education Corporation Altered type ix collagen gene sequences associated with intervertebral disc disease
US7268116B2 (en) * 2003-10-02 2007-09-11 Genhunter Corp. Methods and compositions for producing secreted trimeric receptor analogs and biologically active fusion proteins
CN102212126B (zh) * 2010-04-08 2013-06-19 上海普洛康裕药物研究院有限公司 具有抑制内皮细胞生长活性的重组EDI-8t蛋白
CA2800595C (en) 2010-05-26 2018-04-24 Shriners Hospitals For Children Compositions comprising the nc2 domain of collagen ix and methods of using same
US11377490B2 (en) 2017-05-31 2022-07-05 Sichuan Clover Biopharmaceuticals, Inc Method for treating cancer using disulfide-linked trimeric 4-1BBL
CN109456989B (zh) * 2018-10-31 2022-03-29 陕西慧康生物科技有限责任公司 一种提高毕赤酵母分泌表达的载体的构建方法
WO2021249116A1 (en) 2020-06-10 2021-12-16 Sichuan Clover Biopharmaceuticals, Inc. Coronavirus vaccine compositions, methods, and uses thereof

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