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WO1993010150A1 - Expression de facteurs neurotrophiques au moyen de regions 'prepro' heterologues - Google Patents

Expression de facteurs neurotrophiques au moyen de regions 'prepro' heterologues Download PDF

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Publication number
WO1993010150A1
WO1993010150A1 PCT/US1992/009792 US9209792W WO9310150A1 WO 1993010150 A1 WO1993010150 A1 WO 1993010150A1 US 9209792 W US9209792 W US 9209792W WO 9310150 A1 WO9310150 A1 WO 9310150A1
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WIPO (PCT)
Prior art keywords
prepro
neurotrophin
chimeric
peptide
protein
Prior art date
Application number
PCT/US1992/009792
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English (en)
Inventor
Stephen P. Squinto
Nancy Ip
David Gies
George D. Yancopoulos
Shaw-Fen Sylvia Hu
Original Assignee
Regeneron Pharmaceuticals, Inc.
Amgen, Inc.
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Regeneron Pharmaceuticals, Inc., Amgen, Inc. filed Critical Regeneron Pharmaceuticals, Inc.
Priority to JP5509415A priority Critical patent/JPH07501220A/ja
Publication of WO1993010150A1 publication Critical patent/WO1993010150A1/fr

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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
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/48—Nerve growth factor [NGF]
    • 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
    • C07K14/475—Growth factors; Growth regulators
    • 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

Definitions

  • the present invention relates to the construction and expression in eukaryotic host cells of novel chimeric prepro proteins or prepro peptides expressing bioactive neurotrophic factors.
  • the invention is based, in substantial part, on the discovery that chimeric prepro proteins or prepro peptides comprising the prepro region of a first neurotrophic factor fused to the mature protein, or portion thereof, of a second, different neurotrophic factor undergo efficient post-translational processing resulting in an increased level of expression of the bioactive second neurotrophic factor protein.
  • a neurotrophic factor is a cytokine, a protein which acts as a messenger and communicates with other cells in the ongoing
  • Neurotrophic factors promote the survival and/or differentiation of components of the nervous system. Widespread neuronal cell death accompanies normal development of the central and peripheral nervous systems, and apparently plays a crucial role in regulating the number of neurons which project to a given target field (Berg, D. K., 1982, Neuronal
  • neuronal cell death results from the competition among neurons for limiting amounts of survival factors ("neurotrophic factors”) produced in their projection fields.
  • nerve growth factor NGF
  • NT-3 neurotrophin-3
  • Hohn et al. 1990, Nature 344: 339
  • Maisonpierre et al. 1990, Science 247: 1446
  • brain-derived neurotrophic factor NT-3
  • BDNF neurotrophic factor
  • NT-4 neurotrophin-4
  • CNTF ciliary neurotrophic factor
  • Prepro neurotrophins are generally synthesized in vivo as "prepro" precursor proteins.
  • the "prepro” region refers to the NH 2 -terminus of the precursor which is proteolytically removed during biosynthesis of the mature, biologically active form of the
  • the "pre” region refers to the signal sequence normally removed by proteolytic processing during translocation across the cell membrane to yield a "pro”-protein; the "pro” region is then removed by proteolytic processing to yield the mature form (see e.g., Darnell et al., 1990, Molecular Cell Biology 2d ed., Scientific American Books, pp. 650-657).
  • Nerve growth factor is by far the most fully characterized of these neurotrophic molecules and has been shown, both in vitro and in vivo, to be essential for the survival of sympathetic and neural crest-derived sensory neurons during early development of both chick and rat (Levi-Montalcini and Angeletti, 1963, Develop. Biol. 7:653-659; Levi-Montalcini et al., 1968, Physiol. Rev. 48: 524-569).
  • NGF Nerve growth factor
  • NGF protein in mouse submaxillary gland allowed the primary amino acid sequence to be determined by relatively conventional protein chemistry (Angeletti and Bradshaw, 1971, Proc. Natl. Acad. Sci. (68.: 2417-2420).
  • the NGF gene has now been cloned from many species, including mouse (Scott et al., 1983, Nature 302: 538-540, human (Uilrich et al., 1983, Nature 303 : 821-825), cow and chick (Meier et al., 1986, EMBO J. 5 : 1489-1493), and rat
  • the mouse NGF gene encompasses approximately
  • the "long” precursor contains an additional "pro-region” at its NH 2 -terminus (see e.g., Suter et al, 1991,
  • mouse NGF The biologically active form of mouse NGF is a 7S complex, comprising a dimer of a fully processed mature form of ⁇ -NGF along with two members of the kallikrein family of serine proteases, the ⁇ -subunit and ⁇ -subunit of NGF (Varon et al., Biochemistry
  • the signal sequence of the prepro NGF precursor is removed via proteolytic processing to yield a pro-NGF species of approximately 31 kD.
  • the pro-region of the pro-NGF intermediate contains a pair of arginine residues known to be endoproteolytic processing sites. Proteolytic processing at either of these residues results in an additional major (21 kD) and minor (18.5 kD) intermediate species.
  • the mature form of NGF can be proteolytically derived from either of the above-mentioned intermediate species. At some point in the biosynthesis of the mature form of NGF, a COOH-terminal dipeptide (arg-gly) is proteolytically released.
  • the ⁇ -subunit has been shown in vivo to proteolytically cleave the pro-NGF precursor to the mature form of NGF (Edwards, et al., 1988, J. Biol.
  • the pro region may facilitate proper folding of the ⁇ -lytic protease precursor. Therefore, the pro region of the NGF precursor may also be required for proper folding prior to endoproteolytic processing to the mature form and association into the biologically active 7S NGF complex. Support for this hypothesis is documented in Suter et al. (1991, EMBO J. 10:2395-2400), who assigned functions for two partially conserved domains within the pro-region of NGF. Domain I was shown to be essential for NGF expression in COS cells. Additionally, Domain II, located in the NGF pro-region proximal to the mature coding region, was found to be involved in proteolytic processing.
  • initiation of biosynthesis of the active form of mouse NGF involves the transcription of the NGF gene and possible alternative splicing of the transcription product to generate mRNA's capable of translation of either a long or short NGF
  • the long or short prepro NGF The long or short prepro NGF
  • BDNF brain-derived neurotrophic factor
  • the neurotrophic activity was found to reside in a highly basic protein (isoelectric point, pi 10.1) which migrated during sodium dodecyl sulfate (SDS) gel electrophoresis as a single band of 12.3 kD. It was noted that the highly basic nature and
  • the first demonstration of neuronal specificity of BDNF distinct from that of NGF was the demonstration in vitro that purified BDNF supports the survival of 40-50% of sensory neurons dissociated from the neural placode-derived nodose ganglion of the chick embryo at E6, E9 or E12 (Lindsay et al., 1985, J. Cell. Sci. Supp. 3:115-129). NGF was without apparent effect on these neurons either by itself or in conjunction with BDNF. It was later shown in explant culture studies that BDNF appeared to support survival and neurite outgrowth from other neural placode-derived sensory ganglia, including the petrosal, geniculate and ventrolateral trigeminal ganglia (Davies et al., 1986, J.
  • Neurosci. 6:1897- 1904 none of which have been found to be sensitive to NGF.
  • BDNF was found to stimulate survival and neuronal differentiation of cells cultured from quail neural crest (Kalcheim and Gendreau, 1988, Develop. Brain Res. 41:79-86).
  • BDNF BDNF-induced central nervous system
  • Neurosci. 6: 3031-3938) presented data indicating that BDNF supports the survival of retinal ganglion cells cultured from E17 rat embryos.
  • BDNF has been shown to have effects on cultured adult peripheral and central nervous system neurons.
  • neurotroph ⁇ n-3 Another member of the neurotrophin family, termed neurotroph ⁇ n-3, was discovered, and the NT-3 gene was cloned from mouse, rat, and human (see U.S. Patent Application Serial No. 07/490,004, filed
  • NGF and BDNF a putative signal sequence of 18 amino acids (showing 5 and 9 amino acid identities with BDNF and NGF, respectively) appears to be followed by a prosequence of 121 amino acids (as compared with a prosequence of 103 amino acids in mouse NGF and a prosequence of 112 amino acids in mouse BDNF).
  • rat NT-3 appears to share 57% amino acid homology with rat NGF, and 58% amino acid homology with rat BDNF; 57 of the 120 residues (48%) appear to be shared by all three proteins.
  • NT-3 is capable of promoting survival and neurite outgrowth of dissociated dorsal root ganglion neurons in culture. Furthermore, NT-3 was observed to promote neurite outgrowth from both nodose ganglion and sympathetic ganglion explants, whereas BDNF promoted outgrowth from nodose ganglion but not sympathetic ganglion, and NGF promoted
  • Neurotrophin-4 is a novel member of the NGF family that has recently been cloned and isolated (Hallbook et al., 1991, Neuron 6 :845-858). PCR fragments corresponding to the NT-4 gene from Xenopus and viper were obtained, and a genomic Xenopus clone was subsequently isolated. Nucleotide sequence analysis of this clone revealed an open reading frame for a protein of 236 amino acids, with several
  • N-glycosylation site near a proteolytic cleavage site N-glycosylation site near a proteolytic cleavage site.
  • the present invention relates to novel chimeric prepro proteins or prepro peptides comprising bioactive neurotrophic factors, and the use of such precursors and their nucleic acid sequences to produce proteins or peptides which have one or more biological activities of a neurotrophin.
  • the chimeric prepro molecules provided by the present invention contain a heterologous prepro region fused to a mature neurotrophin.
  • neurotrophin sequence or biologically active portion or derivative thereof are those of the NGF/BDNF family of
  • the prepro regions can be derived from those neurotrophin molecules of the NGF/BDNF family including but not limited to NGF, BDNF, NT-3 and NT-4.
  • the invention is based, in substantial part, on the discovery that chimeric prepro proteins or prepro peptides comprising the prepro region of nerve growth factor fused to the mature portion of brain-derived neurotrophic factor (prepro NGF/BDNF) are more efficiently processed by a eukaryotic host cell than homologous prepro brain- derived neurotrophic factor (prepro BDNF). It is further based on the discovery that stably transfected and amplified eukaryotic host cells expressing
  • chimeric prepro NGF/BDNF secrete only the mature form of BDNF into the media.
  • the "long" or “short” prepro regions of NGF can be utilized in the construction of chimeric neurotrophic genes.
  • the invention is also based on the. discovery that chimeric prepro proteins or prepro peptides comprising the prepro region of NT-3 fused to the mature coding region of brain-derived neurotrophic factor (prepro NT-3/BDNF) are more efficiently
  • prepro BDNF neurotrophic factor
  • the present invention provides for nucleic acids encoding chimeric neurotrophic prepro proteins or prepro peptides, and for methods of expressing these chimeric neurotrophic proteins and peptides by use of such nucleic acids.
  • Lane 1 wild-type control CHO-DG44 cells.
  • FIG. 1 Bioactivity of recombinant BDNF. Crude supernatants from transfected CHO cell lines were assayed with embryonic (E8) chick dorsal root ganglia and neurite outgrowth was scored. Closed diamonds: cell line DGC-N/B-2.5-#23 (containing long prepro NGF/BDNF chimeric construct). Dotted squares: cell line DGZ1000-B-3-2.5 (containing short prepro BDNF construct).
  • Lane 1 was loaded with 450 ng of purified mature human BDNF.
  • Lane 20 was loaded with prestained low molecular weight markers from BRL. Lane 8 and lane 16 represent non-producing clones from each transfection.
  • the present invention relates to novel chimeric prepro proteins or prepro peptides comprising bioactive neurotrophic factors, and the use of such precursors and their nucleic acid sequences to produce proteins or peptides which have one or more biological activities of a neurotrophin.
  • the chimeric prepro molecules provided by the present invention contain a heterologous prepro region fused to a mature neurotrophin.
  • neurotrophin sequence or biologically active portion or derivative thereof are those of the NGF/BDNF family of NGF/BDNF family of NGF/BDNF family of NGF/BDNF
  • the prepro regions can be derived from those neurotrophin molecules of the NGF/BDNF family including but not limited to NGF, BDNF, NT-3 and NT-4.
  • the invention is based, in substantial part, on the discovery that chimeric prepro proteins or prepro peptides comprising the prepro region of nerve growth factor and the mature portion of brain-derived neurotrophic factor (prepro NGF/BDNF) or the prepro region of NT-3 and the mature portion of brain-derived neurotrophic factor (prepro NT-3/BDNF) are more efficiently processed by a eukaryotic host cell than homologous prepro brain- derived neurotrophic factor (prepro BDNF). It is further based on the discovery that stably transfected and amplified eukaryotic host cells expressing chimeric prepro NGF/BDNF or NT-3/BDNF secrete only the mature form of BDNF into the media. The post-translational processing of homologous prepro BDNF is highly inefficient. In contrast, a member of the same neurotrophin gene family, NGF, is efficiently
  • the BDNF processing problem has carried through in the generation of stable host cell lines for the production of mature bioactive BDNF.
  • the present invention provides a novel solution to this processing problem by expression of chimeric
  • constructs which in a specific embodiment contains the long prepro region of NGF fused in frame to mature BDNF and in another specific embodiment contains the prepro region of NT-3 fused in frame to mature BDNF.
  • the present invention provides for nucleic acids encoding chimeric neurotrophic prepro proteins or prepro peptides, and for methods of expressing these chimeric neurotrophic proteins and peptides by use of such nucleic acids.
  • nucleic acids encoding chimeric neurotrophic prepro proteins or prepro peptides provide significant advantages relative to the use of nucleic acids encoding homologous neurotrophic prepro proteins or prepro peptides. Production of chimeric
  • neurotrophic prepro proteins or prepro peptides provides for increased expression levels of the bioactive neurotrophic factor. This increased level of expression should additionally provide for better bioactive neurotrophic factor purification schemes in that contaminating unprocessed forms of the expressed neurotrophic factors are not apparent in the crude supernatants.
  • bioactive proteins which can be obtained according to the present invention are the mature neurotrophic factors which are members of the
  • neurotrophin gene family or biologically active portions or derivatives thereof.
  • biologically active refers to the ability to express one or more biological activities of the full-length mature neurotrophin.
  • neurotrophins include but are not limited to mature BDNF, NT-3, NGF and NT-4 and such other members as are identified by those methods utilized to determine members of the neurotrophin gene family (e.g., using molecular probes, generated by PCR, corresponding to regions of homology within the family; see PCT
  • ATCC plasmid strain phBDNF-C-1 (Accession No. 4068) regarding a hBDNF cDNA clone and, e.g.. Leibrock et al., infra, regarding a pig BDNF cDNA
  • ATCC plasmid strain pC8-hN3 (Accession No. 40765) regarding a human NT-3 cDNA clone and Maisonpierre et al. (Science 247:1446 (1990)) and Hohn et al. (Nature 344:339 (1990)) regarding NT-3 coding sequences from various other species.
  • a neurotrophin gene from any organism may be identified using the regions of homology shared by any two members of the
  • BDNF/NGF/NT-3/NT-4 family of molecules using the methods set forth above.
  • a novel neurotrophin may be identified and cloned by BDNF/NGF/NT-3/NT-4 synthesizing
  • oligonucleotides corresponding to segments of protein sequences highly conserved between any two neurotrophins. These oligonucleotides can then be used as primers in polymerase chain reaction (PCR) with cDNA template prepared from cells suspecting of expressing the desired neurotrophin. The products of PCR can then be used as probes to permit cloning of complete cDNA and/or genomic genes, the sequences of which can be determined by standard methods. Novel neurotrophins can be identified by selecting those containing, in addition to the sequences homologous to other known neurotrophins, sequences non-homologous to other known neurotrophins (e.g., at least six
  • nucleotides differ).
  • oligonucleotides corresponding to sequences of a neurotrophin in one species can be used in PCR to generate probes to permit cloning of the neurotrophin gene from other species.
  • NGF and BDNF are basic proteins of approximately 120 amino acids that share about 50% amino acid sequence identity, including absolute conservation of six cysteine residues that, in active NGF, have been shown to form three disulfide bridges (Bradshaw, A., 1978, Ann. Rev. Biochem. 47:191-216: Leibrock et al., 1989, Nature 341:149-52). Comparison of the sequences of NGF from evolutionarily divergent species has revealed that the amino acids flanking these cysteine residues comprise the most highly conserved regions of the molecule (Meier et al., 1986, EMBO J. 5:1489-93; Selby et al., 1987, J. Neurosci. Res. 18:293-8). Strikingly, these are also the regions which are most similar between BDNF and NGF (Leibrock et al., 1989, Nature 341:149-52.
  • a mature human neurotrophin is produced by expression of a chimeric prepro molecule according to the present invention.
  • the chimeric prepro molecule is encoded by a nucleic acid containing the long prepro region of NGF fused in frame to the coding sequence for mature BDNF.
  • the chimeric prepro molecule is encoded by a nucleic acid containing the prepro region of NT-3 fused in frame to the coding region for mature BDNF.
  • the long prepro region of NGF is fused in frame to the coding region for NT-3.
  • either the "long” or “short” prepro region may be utilized in the construction of chimeric neurotrophic genes.
  • One of ordinary skill in the art can utilize either a "short” NGF prepro region or a "long” NGF prepro region when constructing chimeric fusions of the present invention comprising an NGF prepro region.
  • the mature neurotrophin molecules which can be expressed as chimeric prepro precursors according to the present invention also include substantially equivalent sequences, and fragments or derivatives which are biologically active.
  • the neurotrophin nucleic acid sequences can be altered by substitutions, additions or deletions that provide for functional molecules. Due to the degeneracy of nucleotide coding sequences, other DNA sequences that encode substantially the same neurotrophin amino acid sequence may be used in the practice of the present invention. These include but are not limited to nucleotide sequences comprising all or portions of the neurotrophin genes that are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change.
  • neurotrophin proteins, or fragments or derivatives thereof, of the invention include, but are not limited to, those containing, as part of their primary amino acid sequence, altered sequences in which functionally equivalent ammo acid residues are substituted for residues within the sequence resulting in a silent change.
  • one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent
  • Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.
  • the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • the polar neutral amino acids include glycine, serine,
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • negatively charged amino acids include aspartic acid and glutamic acid.
  • neurotrophin proteins or fragments or derivatives thereof which are obtained through modification during or after translation, e.g., by glycosylation, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, acetylation, phosphorylation, reduction, cleavage, etc.
  • a given neurotrophin sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification.
  • Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis
  • the present invention also relates to expression of the nucleic acids encoding chimeric prepro neurotrophin molecules, and recovery of the mature neurotrophin product.
  • Nucleic acids encoding chimeric neurotrophic prepro proteins or prepro peptides may be constructed using standard recombinant DNA technology, for
  • nucleic acid sequences which encode the desired prepro and mature regions.
  • nucleic acid sequences may be constructed using chemical synthesis, such as solid-phase phosphoramidate
  • polymerase chain reaction (PCR; Saiki et al., 1985, Science 230:1350-1354) may be used to accomplish splicing of nucleic acid sequences by overlap extension (Horton et al., 1989, Gene 77:61-68) and thereby produce nucleic acids encoding the
  • chimeric neurotrophic prepro proteins or prepro peptides of the invention see e.g., Section 6,
  • the nucleic acids of the invention are produced by use of two separate PCR reactions, each with a different template.
  • PCR is first carried out with one template, for example, X, using a probe completely homologous to X, and a probe with a region homologous to X and a region homologous to Y.
  • the PCR reaction product is then isolated and used as probe in a second PCR reaction, with Y as a template, and a second probe completely homologous to Y. It may further be desirable to incorporate useful restriction endonuclease cleavage sites in the primers.
  • chimeric neurotrophic factors may be produced by one-step PCR utilizing three oligonucleotide primers.
  • a nucleic acid encoding at least a portion of a desired prepro region (X) may be ligated to a nucleic acid sequence encoding a mature neurotrophic protein or peptide (Y) by creating three oligonucleotide primers, one of which corresponds to a portion of the X sequence (the "X primer”), another which corresponds to a portion of the Y sequence (the "Y primer”), and a third which contains a portion of both X and Y sequences ("the XY primer”).
  • the template utilized in the PCR may be a mixture of nucleic acids encoding the desired prepro region and the mature neurotrophic protein or peptide.
  • the position of the splice site is
  • bridging nucleotide e.g. the XY primer
  • Amplification conditions routinely used in the art may be used, for example, 1 minute at about 94oC, 2 minutes at about 43°C and 3 minutes at about 72oC for 35 cycles, using standard PCR reaction solutions and methods.
  • the resulting PCR fragment may then be gel purified using gel electrophoresis, digested with the appropriate restriction endonuclease and ligated into a suitable cloning vector.
  • DNA reaction products may be cloned using any method known in the art. Any number of vectorhost systems known in the art may be used. Possible vectors include, but are not limited to, cosmids, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Such vectors include, but are not limited to,
  • bacteriophages such as lambda derivatives, or plasmids such as pBR322, pUC, or Bluescript ® (Stratagene) plasmid derivatives.
  • Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc.
  • peptide can be ligated into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription of the cloned chimeric
  • the necessary transcriptional and translation signals can also be supplied by one of the neurotrophin genes and/or its flanking regions
  • eukaryotic host-vector systems may be utilized to express the cloned chimeric DNA sequence and resulting mRNA transcript. These include but are not limited to mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.), transfected with other vectors, containing chromosomally integrated nucleic acids of the invention, etc., but the host system used must have the appropriate cell machinery to process the prepro chimera to the mature
  • the expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used.
  • any of the methods previously described for the insertion of DNA fragments into a vector may be used to construct expression vectors containing a sequence encoding a chimeric neurotrophic prepro protein or prepro peptide, consisting of appropriate transcriptional/translational control signals upstream of the chimeric DNA sequences. These methods may include in vitro recombinant DNA and synthetic
  • nucleic acid sequences encoding chimeric neurotrophic prepro protein or prepro peptide may be regulated by a second nucleic acid sequence so that chimeric neurotrophic prepro protein or prepro peptide is expressed in a host transformed with the recombinant DNA molecule.
  • expression may be controlled by any combination of nucleic acid sequences encoding chimeric neurotrophic prepro protein or prepro peptide.
  • Promoters which may be used to control chimeric neurotrophic factor
  • CMV cytomegalovirus
  • elastase I gene control region which is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol.
  • mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45: 485-495), albumin gene control region which is active in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol.
  • alpha 1-antitrypsin gene control region which is active in the liver (Kelsey et al, 1987, Genes and Devel. 1:161-171), beta-globin gene control region which is active in myeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94; myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712); myosin light chain-2 gene control region which is active in skeletal muscle (Sani, 1985, Nature 314:283-286), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al., 1986, Science
  • CDM8 Seed, 1987, Nature 329:840-842; Seed and Aruffo, 1987, Proc. Natl. Acad. Sci. USA 84:3365-3369; Aruffo & Seed, Proc. Natl. Acad. Sci. USA 84: 8573-8577
  • pCMX see copending application Serial No. 678,408, filed
  • Expression vectors containing chimeric neurotrophic prepro protein or prepro peptide gene inserts can be identified by three general approaches: (a) DNA-DNA hybridization, (b) presence or absence of "marker" gene functions, and (c) expression of
  • the presence of a foreign gene inserted in an expression vector can be detected by DNA-DNA hybridization using probes comprising sequences that are homologous to at least a portion of an inserted chimeric neurotrophic prepro protein or prepro peptide gene.
  • the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g., thymidine kinase activity, transformation phenotype, etc.) caused by the insertion of foreign genes in the vector.
  • recombinants containing the chimeric insert can be identified by the absence of the marker gene function.
  • expression vectors can be identified by assaying the foreign gene product expressed by the recombinant.
  • Such assays can be based, for example, on the physical or functional properties of the neurotrophic factor gene product in bioassay systems as described infra, in Section 5.4.
  • recombinant DNA molecule Once a particular recombinant DNA molecule is identified and isolated, several methods known in the art may be used to propagate it. Once a suitable host system and growth conditions are established, recombinant expression vectors can be propagated and prepared in quantity.
  • Expression from certain promoters can be elevated in the presence of certain inducers; thus, expression of the genetically engineered chimeric neurotrophic prepro protein or prepro peptide may be controlled.
  • different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation, cleavage) of proteins. Appropriate cell lines or host systems should be chosen to ensure the necessary processing (e.g., removal by cleavage of the prepro region) and any desired modification. Mammalian host cells, such as monkey, human, or bovine, are thus preferred.
  • DNA encoding chimeric neurotrophins may be expressed in a CHO cell system according to methods set forth infra. Once a recombinant which expresses the chimeric neurotrophin is identified, the mature gene product should be analyzed. This can be achieved by assays based on the physical or functional properties of the product. See infra Section 5.4.
  • the mature neurotrophic factor protein or peptide may be isolated and purified by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential
  • solubility or by any other standard technique for the purification of proteins.
  • neurotrophin proteins and peptides produced according to the invention are able to exhibit one or more biological activities including but not limited to neurotrophic activity, binding by antibodies to neurotrophins, binding to cognate receptors, etc.
  • neurotrophic activity should be construed to refer to a biological effect on nervous system cells, including, but not limited to, neurons, astrocytes, glial cells, oligodendrocytes, microglia and Schwann cells.
  • the biological effect is an alteration in the structure and/or physiology of a nervous system cell which does not occur absent direct or indirect exposure to the chimeric neurotrophic factor. Examples of a
  • biological effects are the prolongation of survival, neurite sprouting, the maintenance or development of differentiated functions (such as expression of an enzyme e.g. choline acetyltransferase or tyrosine hydroxylase) or, conversely, cell death or senescence, or dedifferentiation.
  • differentiated functions such as expression of an enzyme e.g. choline acetyltransferase or tyrosine hydroxylase
  • neurotrophic activity may be determined using any known assay for such activity as well as systems which may be developed in the future.
  • Assay systems may include in vitro testing systems, such as tissue culture bioassay systems using tissue explants, cells prepared from tissue, or immortalized cell lines, for example, derived from the brain, spinal cord, or peripheral nervous system, as well as in vivo testing systems in which neurotrophic factor may be administered to an animal; neurotrophic effects may be detected in such an animal by performing, chemical, histologic, or behavioral tests using said animal.
  • a neurotrophic factor may be incorporated as a transgene in a non-human transgenic animal, and its biological effects may be measured in said animal.
  • neurotrophic activity may be measured using any of the following well known bioassay systems:
  • spinal cord neurons may be removed aseptically from a test animal, severed caudal to the bulb, and freed of sensory ganglia and meninges.
  • the cord may then be subdivided into ventral and mediodorsal segments for separate cultures, and the tissues minced into small pieces and dissociated by trituration through a Pasteur pipet in 50 percent DMEM
  • Ham's nutrient mixture F12 (Gibco) and 50 percent Ham's nutrient mixture F12 (Gibco) supplemented with 33 mM glucose, 2 mM glutamine, 15 mM NaHCO 3 , 10 mM HEPES, 25 ⁇ g/ml insulin, 100 ⁇ g/ml transferrin, 60 ⁇ m putrescine, 20 nM progesterone, 30 nM Na selenite, 0.5 ⁇ g/ml penicillin G, 0.5 ⁇ g/nl streptomycin, and 2.5 ⁇ g/ml bovine serum albumin. Trituration may then be repeated twice and supernatants may be pooled and filtered through a 40 ⁇ m Tetko filter.
  • Dissociated ventral cells may then be plated in on poly-D-lysine coated (10 ⁇ g/ml) culture dish at a density of 0.5 million cells per 35 mm dish.
  • Dissociated mediodorsal cells may be plated at a density of 1.5 million cells per 35 mm dish coated with poly-D-lysine (10 ⁇ g/ml),
  • the 5' primer (5'- CTC-GTC-GAC-AGC-CGG- CAC-TCT-GAC-CCT-GCG-CGC-CGA-3') [SEQ ID NO: 17] encoded the first 7 amino acids of BDNF and included two unique restriction sites, Nael and BssH2 which were generated by modifying codon usage.
  • the 3' PCR primer was a 3' pCDM8 oligo corresponding to a region downstream from the polylinker sequence at the 3' end of the BDNF sequence in pC8hB (5'-CAA-AGA-TCC-TCT-AGA- GTC-G-(C)-3') [SEQ ID NO: 18].
  • the polylinker contains a Notl restriction site. These two primers were used in PCR with pC8hB (hBDNF in pCDM8) DNA as template. 5 micrograms of pC8hB was used with 500 ng of each primer for 5 PCR cycles. The PCR product was digested with both Nael and Notl simultaneously and a 365 bp digestion product was isolated by gel electrophoresis. The preparation of the vector was carried out by digesting pC8lmN (long mouse NGF in pCDM8) with both Eco47 and Notl and isolating the 4.6 kb vector
  • CHO-DG44 cells were used to generate stable lines for the production of bioactive BDNF. CHO-DG44 cells (obtained from Dr. L. Chasin at Columbia
  • WO 91/03568 published March 21, 1991. These lines were generated by transfection with pC8hB DNA which encodes the human BDNF gene including the prepro region cloned into the expression vector pCDM8.
  • CHO-DG44 cells (1 ⁇ 10 6 cells/100 mm plate) were transfected by the calcium phosphate coprecipitation method with 20 ⁇ g of the NGF/BDNF chimera (pC81mN/B) along with 0.2 ⁇ g of plasmid p410 which encodes a weakened dihydrofolate reductase gene (dhfr).
  • Bioactivity was assessed by scoring neurite outgrowth of embryonic (E8) chick dorsal root ganglia (DRG) (Maisonpierre et al., 1990, Science 247: 1446-1451).
  • CHO-DG44 cell lines stably transfected with either pC8hB or pC81mN/B and amplified with 2.5 ⁇ M methotrexate were compared by metabolic labeling
  • the cells were then labeled with both 35 S-cysteine and 35 S-methionine for 4 hours under serum-free conditions.
  • 30 ⁇ l aliquots of labeled cell supernatants were resolved by SDS polyacrylamide gel electrophoresis (15% gel) and labeled proteins were transferred to nylon membranes and visualized by autoradiography.
  • Unprocessed proBDNF 31 kD
  • the pro-portion of the processed proBDNF precursor (16 kD)
  • the mature form 14 kD
  • the short preproBDNF protein was detected in the stably transfected cell line DGZ1000-B-3-2.5 (obtained after similar MTX selection and amplification as used for cell line DGC-N/B-2.5-#23) ( Figure 1, lane 3). Only the proteolytically
  • COS cells were transfected with prepro NGF containing either the long (“lmNGF”) or short
  • smNGF NGF prepro region with the mature NGF coding region.
  • Culture supernatants were harvested 48 hours after transfection and assayed on DRG explants, along with purified NGF and a mock transfected COS cell supernatant. Results using three different
  • concentrations of each construct reveal significant bioactivity of NGF expressed with either the long or short form of the prepro region.
  • a Hindlll-Xhol DNA fragment containing the entire coding region of prepro and mature human BDNF was obtained from digestion of plasmid pC8hB with corresponding restriction enzymes.
  • the plasmid pC8hB was derived by cloning the human BDNF coding
  • pDSR ⁇ 2(BDNF) The plasmid pDSR ⁇ 2 had been previously digested to make available the cloning sites 5'-Hindlll and 3'-Sail for ligation of the human BDNF containing fragment.
  • the resulting plasmid was designated pDSR ⁇ 2(BDNF).
  • the plasmid pC8hN3 was derived by cloning the human NT-3 coding
  • DNA fragment No. 3 was prepared, which was an oligonucleotide linker synthesized to regenerate the aforementioned missing 35 amino acid residues ( Figure 6 and SEQ ID NO: 15-17).
  • the linker also contained the half sites of the 5'-SacII and 3'-Narl restriction sites to promote ligation to DNA fragments Nos. 1 and 2 disclosed supra. This ligation resulted in the expression vector pDSR ⁇ 2(NT-3/BDNF), in which the prepro region of NT-3 (fragment No. 2) is joined with mature BDNF (fragment No. 1) by the oligonucleotide linker (fragment No. 3; Figure 6 and SEQ ID NO: 15).
  • CHO-D(-) cells (ATCC accession number CCL 61) were used to generate stable lines for the
  • CHO-D(-) cells are defective in the gene encoding dihydrofolate reductase and are maintained in the medium of Dulbecco's
  • D-MEM modified Eagle media
  • CHO-D(-) cells (0.8 ⁇ 10 6 /60 mm plate) were transfected by the calcium phosphate coprecipitation method, using 2.5 ⁇ g of the NT-3/BDNF chimeric
  • reductase minigene which, when expressed, enables the transfected CHO-D(-) to overcome the deficiency of the dhfr gene and become capable of growing in the absence of the nucleotides hypoxanthine and thymidine.
  • Parental CHO-D(-) cells or cells not successfully transfected by the vector pDSR ⁇ 2 will not survive in the selection media, which has the
  • the cells were trypsinized and seeded 48 hours after transfection at 1 ⁇ 10 5 cells/100 mm plate in selection media. Individual colonies were picked two weeks later using cloning cylinders. Each clone was then expanded to 100 mm plates. When the cultures reached confluency, the original serum- containing media were aspirated and replaced with 3 ml of serum free media.
  • the conditioned media (CM) were collected and 50 ⁇ l each was loaded on a 15% SDS- polyacrylamide gel and subjected to gel
  • COS-7 cells (ATCC accession number CRL 1651) were used as a transient expression system to test the production of bioactive BDNF. COS-7 cells are
  • COS-7 cells (5 ⁇ 10 6 cells/ml) were transfected by electroporation at 1600 volts for 0.4 msec with, individually, 20 ⁇ g each of pDSR ⁇ 2, pDSR ⁇ 2(BDNF) and pDSR ⁇ 2(NT-3/BDNF). Transfected COS-7 cells were plated at 2 ⁇ 10 6 cells/60 mm plate. Conditioned medium accumulated between 24 and 72 hours post transfection was collected. Bioactivity was assessed by scoring neurite outgrowth of embryonic (E8) chick dorsal root ganglia (as with the NGF/BDNF chimera).
  • NT-3/BDNF chimeric pDSR ⁇ 2
  • BDNF pDSR ⁇ 2
  • chimeric NT-3/BDNF gene construct results for higher levels of processed BDNF on a per cell basis in mammalian cells, and it should also allow for better purification schemes by elimination or minimization of contaminating unprocessed forms.

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Abstract

L'invention se rapporte à des préproprotéines ou à des prépropeptides chimériques contenant des facteurs neurotrophiques, et à l'utilisation de ses précurseurs et de leurs séquences d'acides nucléiques pour produire des protéines ou des peptides qui possèdent une ou plusieurs des activités biologiques d'une neurotrophine. Les molécules 'prépro' chimériques décrites dans cette invention contiennent une région 'prépro' hétérologue fusionnée à une séquence de neurotrophine mature ou à une partie ou un dérivé biologiquement actif de cette séquence. Les séquences de neurotrophine matures qui peuvent être utilisées selon la présente invention sont celles qui appartiennent à la famille NGF/BDNF des molécules homologues, y compris mais pas exclusivement NGF, BDNF, NT-3 et NT-4. De même, des régions 'prépro' peuvent être dérivées de ces molécules de neurotrophine de la famille NGF/BDNF, y compris mais pas exclusivement NGF, BDNF, NT-3 et NT-4.
PCT/US1992/009792 1991-11-14 1992-11-13 Expression de facteurs neurotrophiques au moyen de regions 'prepro' heterologues WO1993010150A1 (fr)

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Cited By (4)

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US5441937A (en) * 1988-10-31 1995-08-15 Houston Biotechnology Incorporated Neurotrophic factor
WO1996008562A1 (fr) * 1994-09-12 1996-03-21 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V., Berlin Molecules biologiquement actives derivees de neurotrophines
EP1997900A2 (fr) 1996-04-05 2008-12-03 Novartis Vaccines and Diagnostics, Inc. Vecteurs à base d'alpha virus recombinants avec inhibition réduite de synthèse macromoléculaire cellulaire
US7972778B2 (en) 1997-04-17 2011-07-05 Applied Biosystems, Llc Method for detecting the presence of a single target nucleic acid in a sample

Families Citing this family (1)

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Publication number Priority date Publication date Assignee Title
EP0894867A4 (fr) * 1996-11-19 2000-11-15 Yamasa Corp Procede pour produire des nucleosides 5'-triphosphates et utilisation desdits nucleosides

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US4923808A (en) * 1985-03-12 1990-05-08 Genentech, Inc. Method for identifying mutants secreting high levels of heterologous proteins

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US4923808A (en) * 1985-03-12 1990-05-08 Genentech, Inc. Method for identifying mutants secreting high levels of heterologous proteins

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Title
DNA, Volume 7, No. 9, issued November 1988, B.R. CULLEN, "Expression of a Cloned Human Interleukin-2 cDNA is Enhanced by the Substitution of a Heterologous mRNA Leader Region", pages 645-650. *
J. OF CELL BIOLOGY, Volume 108, No. 5, issued May 1989, T.J. STOLLER et al., "The Propeptide of Preprosomatostatin Mediates Intracellular Transport and Secretion of Alpha-Globin from Mammalian Cells", pages 1647-1656. *
J. OF FERM AND BIOENG, Volume 68, No. 4, issued 1989, H. OYAMA et al., "Secretion of Escherichia Coli Aminopeptidase P in Bacillus Subtilis Using the Prepro-Structure Coding Region of Subtilisin Amylosacchariticus", pages 289-292. *
PROC. NATL. ACAD. SCI. USA, Volume 80, No. 23, issued December 1983, S.D. EMR, "An MFalpha1-SUC2 (Alpha-Factor-Invertase) Gene Fusion for Study of Protein Localization and Gene Expression in Yeast", pages 7080-7084. *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5441937A (en) * 1988-10-31 1995-08-15 Houston Biotechnology Incorporated Neurotrophic factor
WO1996008562A1 (fr) * 1994-09-12 1996-03-21 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V., Berlin Molecules biologiquement actives derivees de neurotrophines
EP1997900A2 (fr) 1996-04-05 2008-12-03 Novartis Vaccines and Diagnostics, Inc. Vecteurs à base d'alpha virus recombinants avec inhibition réduite de synthèse macromoléculaire cellulaire
US7972778B2 (en) 1997-04-17 2011-07-05 Applied Biosystems, Llc Method for detecting the presence of a single target nucleic acid in a sample
US8067159B2 (en) 1997-04-17 2011-11-29 Applied Biosystems, Llc Methods of detecting amplified product
US8257925B2 (en) 1997-04-17 2012-09-04 Applied Biosystems, Llc Method for detecting the presence of a single target nucleic acid in a sample
US8278071B2 (en) 1997-04-17 2012-10-02 Applied Biosystems, Llc Method for detecting the presence of a single target nucleic acid in a sample
US8551698B2 (en) 1997-04-17 2013-10-08 Applied Biosystems, Llc Method of loading sample into a microfluidic device
US8563275B2 (en) 1997-04-17 2013-10-22 Applied Biosystems, Llc Method and device for detecting the presence of a single target nucleic acid in a sample
US8822183B2 (en) 1997-04-17 2014-09-02 Applied Biosystems, Llc Device for amplifying target nucleic acid
US8859204B2 (en) 1997-04-17 2014-10-14 Applied Biosystems, Llc Method for detecting the presence of a target nucleic acid sequence in a sample
US9506105B2 (en) 1997-04-17 2016-11-29 Applied Biosystems, Llc Device and method for amplifying target nucleic acid

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CN1078493A (zh) 1993-11-17
ZA928776B (en) 1993-05-13

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