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WO1998029547A1 - Modulateurs de differenciation et de transformation radiale glie-astrocyte, et utilisations diagnostiques et therapeutiques - Google Patents

Modulateurs de differenciation et de transformation radiale glie-astrocyte, et utilisations diagnostiques et therapeutiques Download PDF

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Publication number
WO1998029547A1
WO1998029547A1 PCT/US1997/023818 US9723818W WO9829547A1 WO 1998029547 A1 WO1998029547 A1 WO 1998029547A1 US 9723818 W US9723818 W US 9723818W WO 9829547 A1 WO9829547 A1 WO 9829547A1
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WO
WIPO (PCT)
Prior art keywords
factor
radializing
antibody
astrocytes
radial
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PCT/US1997/023818
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English (en)
Inventor
Mary E. Hatten
Kim E. Hunter
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The Rockefeller University
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Publication of WO1998029547A1 publication Critical patent/WO1998029547A1/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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to modulators of radial glia-astrocyte differentiation and transformation, and to various diagnostic and therapeutic uses therefor.
  • Radial glial fibers provide the scaffolding that sets forth the pattern of the mammalian forebrain.
  • Postmitotic neurons leaving the ventricular zone use radial glia as a migratory substrate; cortical laminae form in a temporally regulated manner, the oldest neurons being positioned in deep layers, and the youngest, superficially (Angevine and Sidman, 1961; Caviness and Sidman, 1973; Rakic, 1972, 1974; Schmechel and Rakic, 1979a).
  • young neurons can migrate tangentially (Fishell et al., 1993; O'Rourke et al. 1992, 1995) and sibling cells can ultimately become dispersed over widespread areas of cortex (Luskin et al. , 1988; Austin and Cepko, 1990; Walsh and Cepko, 1992, 1993) it is recognized that the majority of neuronal migration is radial and glial-guided (O'Rourke et al. , 1995).
  • radial glia The functions of radial glia are exclusively developmental; correspondingly, in the early postnatal cortex they undergo a dramatic change of phenotype and function by transforming into mature astrocytes (Schmechel and Rakic, 1979; Voigt, 1989; Culican et al. , 1990). Though some of the molecules involved in neuronal migration along the glial fibers have recently been identified, such as astrotactin (Zheng et al. 1996), factors regulating radial glial cell development remain to be elucidated.
  • a radial glial differentiation signal was previously described which is present in embryonic cortex but absent postnatally; this signal is a protein which will induce astrocytes to express a radial glial phenotype (Hunter and Hatten, 1995), suggesting the radial glia-astrocyte developmental pathway is bidirectional.
  • the differentiation signal is therefore a 'maintenance' cue for the radial glia scaffold, and its perinatal downregulation precipitates the transformation of the elongated glial forms that support migration into the astrocytes of the mature cortex. The question remains as to how this signal functions to control the temporal program of glial development in vivo.
  • astroglial cells The response of astroglial cells to injury is based upon several basic assumptions about such cells.
  • the "reactive astroglia" seen in injured brain are considered to be generic cells, rather than a diverse family of glial cells descended from a variety of progenitors in different brain regions.
  • GFAP glial fibrillary acidic protein
  • modulators of radial glia-astrocyte differentiation and transformation comprising a secreted radial glial differentiation factor have been identified, isolated, purified and characterized.
  • This radializing factor herein called RF60, possesses an apparent molecular weight of approximately 60kD and functions as a radial glial maintenance signal, with the availability of the protein regulating the phenotype of cortical glia.
  • RF60 is present embryonically, but absent postnatally, so that its use in the mature mammal can reverse the glia to an embryonic-like state.
  • RF60 has been utilized to reverse the glial scarring occurring during CNS trauma, and thus reverse the glia to an embryonic-like state.
  • the present invention extends to a secreted radializing factor, its variants, functionally active fragments thereof, analogs thereof, and alleles thereof, the foregoing having the following characteristics: an apparent molecular weight of approximately 60kD; and the capacity to induce expression of a radial glial phenotype by cultured astrocytes.
  • the present invention extends to a radializing factor (RF60) which induces adult astrocytes to assume properties normally associated with their embryonic precursors.
  • RF60 radializing factor
  • astrocytes normally epithelioid in shape, become radial and elongated in the presence of RF60, express the embryonic glial marker RC2 and have dramatically reduced levels of GFAP.
  • the present invention relates to all members of the herein disclosed family of radializing factors (RF60).
  • the present invention also relates to a recombinant DNA molecule or cloned gene, or a degenerate variant thereof, which encodes a radializing factor (RF60); preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding the radializing factor (RF60).
  • RF60 radializing factor
  • the human and murine DNA sequences of the radializing factor (RF60) of the present invention or portions thereof may be prepared as probes to screen for complementary sequences and genomic clones in the same or alternate species.
  • the present invention extends to probes so prepared that may be provided for screening cDNA and genomic libraries for the radializing factor (RF60).
  • the probes may be prepared with a variety of known vectors, such as the phage ⁇ vector.
  • the present invention also includes radializing factor (RF60) proteins having the activities noted herein, and that display the characteristics set forth and described above.
  • RF60 radializing factor
  • the full DNA sequence of the recombinant DNA molecule or cloned gene so determined may be operatively linked to an expression control sequence which may be introduced into an appropriate host.
  • the invention accordingly extends to unicellular hosts transformed with the cloned gene or recombinant DNA molecule comprising a DNA sequence encoding the present radializing factor (RF60).
  • RF60 radializing factor
  • a recombinant expression system is provided to produce biologically active animal or human radializing factor (RF60).
  • radializing factor contemplates that specific factors exist for correspondingly specific ligands. Accordingly, the exact structure of each will understandably vary so as to achieve this ligand and activity specificity. It is this specificity and the direct involvement of the radializing factor (RF60) in the chain of events leading to glial scar formation, that offers the promise of a broad spectrum of diagnostic and therapeutic utilities.
  • the present invention naturally contemplates several means for preparation of the radializing factor (RF60), including as illustrated herein known recombinant techniques, and the invention is accordingly intended to cover such synthetic preparations within its scope.
  • the isolation of the cDNA and amino acid sequences disclosed herein facilitates the reproduction of the radializing factor (RF60) by such recombinant techniques, and accordingly, the invention extends to expression vectors prepared from the disclosed DNA sequences for expression of radializing factors such as RF60 in host systems by recombinant DNA techniques, and to the resulting transformed hosts.
  • the invention further extends to a class of cells, and particularly, to astrocytes, including adult astrocytes, that exhibit a radial glia phenotype, and more particularly, to astrocytes that exhibit such phenotype as a result of the presence of the radializing factor (RF60).
  • astrocytes possess a radial adn elongated shape, express the glial cell marker RC2 and have dramatically reduced levels of GFAP.
  • the invention includes an assay system for screening of potential drugs effective to modulate radializing factor (RF60) activity in target mammalian cells by modulating or potentiating the activity of the radializing factor (RF60).
  • the test drug could be administered to a cellular sample with radializing factor (RF60), or an extract containing the activated radializing factor (RF60), to determine its effect upon the activity of the radializing factor (RF60) to any chemical sample (including DNA), or to the test drug, by comparison with a control.
  • the assay system could more importantly be adapted to identify drugs or other entities that are capable of stimulating the production of radializing factor (RF60), either in the cytoplasm or in the nucleus, thereby increasing or potentiating radializing factor (RF60) activity.
  • RF60 radializing factor
  • Such assay would be useful in the development of drugs that would be specific against particular cellular activity, or that would potentiate such activity, in time or in level of activity.
  • RF60 radializing factor
  • the diagnostic utility of the present invention extends to the use of the present radializing factor (RF60) in assays to screen for the presence of radializing factor (RF60) agonists.
  • the present invention likewise extends to the development of antibodies against the radializing factor (RF60), including naturally raised and recombinantly prepared antibodies.
  • the antibodies could be used to screen expression libraries to obtain the gene or genes that encode the radializing factor (RF60).
  • Such antibodies could include both polyclonal and monoclonal antibodies prepared by known genetic techniques, as well as bi-specific (chimeric) antibodies, and antibodies including other functionalities suiting them for additional diagnostic use conjunctive with their capability of modulating radializing factor (RF60) activity.
  • the radializing factors (RF60), its variants and fragments, their analogs, and any antagonists or antibodies that may be raised thereto, are capable of use in connection with various diagnostic techniques, including immunoassay s, such as a radioirnmunoassay, using for example, an antibody to the radializing factor (RF60) that has been labeled by either radioactive addition, or radioiodination.
  • immunoassay s such as a radioirnmunoassay, using for example, an antibody to the radializing factor (RF60) that has been labeled by either radioactive addition, or radioiodination.
  • a control quantity of the antagonists or antibodies thereto, or the like may be prepared and labeled with an enzyme, a specific binding partner and/or a radioactive element, and may then be introduced into a cellular sample. After the labeled material or its binding partner(s) has had an opportunity to react with sites within the sample, the resulting mass may be examined by known techniques, which may vary with the nature of the label attached.
  • radioactive label such as the isotopes 3 H, 14 C, 32 P, 35 S, 36 C1, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, 131 I, and 186 Re
  • known currently available counting procedures may be utilized.
  • detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques known in the art.
  • the present invention includes an assay system which may be prepared in the form of a test kit for the quantitative analysis of the extent of the presence of the radializing factor (RF60), or to identify drugs or other agents that may mimic or block their activity.
  • the system or test kit may comprise a labeled component prepared by one of the radioactive and/or enzymatic techniques discussed herein, coupling a label to the radializing factor (RF60), their agonists and/or antagonists, and one or more additional immunochemical reagents, at least one of which is a free or immobilized ligand, capable either of binding with the labeled component, its binding partner, one of the components to be determined or their binding partner(s).
  • the present invention relates to certain therapeutic methods which would be based upon the activity of the radializing factor, its (or their) variants, or active fragments thereof, or upon agents or other drugs determined to possess the same activity.
  • a first therapeutic method is associated with the prevention or inhibition of the formation of radial glial scar formation related to brain or central nervous system trauma or injury, and comprises administering a radializing factor, its variants, active fragments, or other agent or drug determined to possess the same activity, either individually or in mixture with other agents in an amount effective to prevent the development of such conditions in the host.
  • the therapeutic method generally referred to herein could include the method for the treatment of various pathologies or other cellular dysfunctions and derangements which occur in brain or central nervous system or trauma by the administration of pharmaceutical compositions that may comprise the radializing factor (RF60),its variants or fragments, or other equally effective drugs developed, for instance, by a drug screening assay prepared and used in accordance with a further aspect of the present invention.
  • pharmaceutical compositions that may comprise the radializing factor (RF60),its variants or fragments, or other equally effective drugs developed, for instance, by a drug screening assay prepared and used in accordance with a further aspect of the present invention.
  • drugs or other binding partners to the radializing factor (RF60) or proteins may be administered to increase or potentiate the glial scar inhibitory activity.
  • the agents of the present invention find broad utility in the treatment of brain or other central nervous system trauma, including use in the treatment of brain or spinal cord injury or surgery, treatment of neurological disorders or diseases where there has been a diminution of the normal activity of the brain or central nervous system, such as dementia, Alzheimer's Disease, multiple sclerosis, and situations where regeneration of brain or central nervous system tissue is therapeutic, such as surgery, trauma, stroke due to ischemic or hemorrhagic occurrences, bacterial and viral infections, neoplasms, and the like.
  • the proteins of radializing factor (RF60), their antibodies, agonists, antagonists, or active fragments thereof, could be prepared in pharmaceutical formulations for administration in instances wherein therapy is appropriate, such as to treat brain or spinal cord injury.
  • RF60 radializing factor
  • the specificity of the radializing factor (RF60) proteins hereof would make it possible to better manage the aftereffects of brain or central nervous system trauma or injury.
  • RF60 radializing factor
  • RF60 radializing factor
  • RF60 radializing factor
  • RF60 radializing factor
  • compositions for use in therapeutic methods which comprise or are based upon the radializing factor (RF60), its variants and fragments, their binding partner(s), or upon agents or drugs that potentiate or enhance the production, or that mimic or antagonize the activities of the radializing factor (RF60).
  • RF60 radializing factor
  • Other objects and advantages will become apparent to those skilled in the art from a review of the ensuing description which proceeds with reference to the following illustrative drawings.
  • FIGURE 1 is photograph of acrylamide gel utilized to show that a single gel band region defines the radial glial differentiation protein as RF60.
  • the protein was eluted from each gel slice, renatured, and screened for bioactivity on astrocytes which were scored for conversion to a radial glia phenotype; this is expressed as units of bioactivity per gel slice (1 unit of activity induces 25% of astrocytes to express a radial phenotype.
  • the bioactivity was found to be present only in one gel band in the molecular weight region of approximately 60kD (b).
  • FIGURES 2A-2C show comparisons of the results of administration of radializing factor (RF60) on normal and reeler astrocytes.
  • RF60 radializing factor
  • a cell-autonomous defect prevents reeler astrocytes from responding to RF60.
  • reeler astrocytes showed almost no increase in differentiation in response to greater or enriched amounts of bioactivity, rather, simply a flat, baseline response (FIGURE 2B).
  • FIG. 2C in a comparison screening of soluble factors secreted by reeler and normal E14 cortical cells, on reeler and normal astrocytes, both cortical sources were found to secrete comparable amounts of activity although reeler astrocytes were unresponsive to both (FIGURE 2C).
  • FIGURES 3A-3F are photographs illustrating the pattern of RC2 and GFAP protein distribution in developing normal and reeler cortex.
  • radial glia are well differentiated and form a dense, organized fiber scaffold (FIGURE 3A); in E14 reeler cortex, however, radial glia are poorly differentiated and present a looser, more disrupted scaffold (FIGURE 3B).
  • FIGURE 4 is a comparison of GFAP protein levels in developing normal and reeler cortex using a Western blot of cortex harvested from normal and reeler animals aged E14-P21, and probed with anti-GFAP.
  • normal cortex In normal cortex, only negligible amounts of GFAP are present through embryonic stages, until the time of birth when the protein is detectable.
  • GFAP is present in reeler cortex at the earliest developmental stage probed (El 4); levels continue to be greater in reeler than in normal cortex, even in mature cortex (P21).
  • FIGURES 5A and 5B are photographs depicting the results of stab wound experiments in cortex, presenting a comparison of GFAP protein levels in injured, untreated cortex (Figure 5A), and injured but treated with RF60 ( Figure 5B), 5 days after injury.
  • the stab wound is to the left side of each figure. Note the dense, strongly GFAP+ reactive astrocytes in the untreated cortex (see arrow, Figure 5A). In contrast, in the RF60 infused cortex, GFAP levels are much lower and the astrocytes assume a radial morphology (see arrow, Figure 5B).
  • radializing factor may be used herein interchangeably, and as used throughout the present application and claims refer to proteinaceous material including single or multiple proteins, and extends to those proteins having the profile of activities set forth herein and in the Claims. Accordingly, proteins displaying substantially equivalent or altered activity are likewise contemplated. These modifications may be deliberate, for example, such as modifications obtained through site-directed mutagenesis, or may be accidental, such as those obtained through mutations in hosts that are producers of the complex or its named subunits. Also, the terms “radializing factor,” “RF60,” and “radial glial differentiation factor(s)” are intended to include within their scope proteins specifically recited herein as well as all substantially homologous analogs and allelic variations.
  • amino acid residues described herein are preferred to be in the "L" isomeric form.
  • residues in the "D” isomeric form can be substituted for any L- amino acid residue, as long as the desired functional property of immunoglobulin- binding is retained by the polypeptide.
  • NH 2 refers to the free amino group present at the amino terminus of a polypeptide.
  • COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide.
  • amino-acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino- terminus to carboxy-terminus. Furthermore, it should be noted that a dash at the begmning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino-acid residues.
  • the above Table is presented to correlate the three-letter and one-letter notations which may appear alternately herein.
  • a “replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control.
  • a “vector” is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • a “DNA molecule” refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double- stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes.
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
  • An "origin of replication” refers to those DNA sequences that participate in DNA synthesis.
  • a DNA "coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy 1) terminus.
  • a coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences.
  • a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
  • Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the mmimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT” boxes.
  • Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.
  • An “expression control sequence” is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence.
  • a coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.
  • a "signal sequence” can be included before the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that cornmunicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.
  • oligonucleotide as used herein in referring to the probe of the present invention, is defined as a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide.
  • primer refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH.
  • the primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent.
  • the exact length of the primer will depend upon many factors, including temperature, source of primer and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.
  • the primers herein are selected to be “substantially" complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product.
  • restriction endonucleases and “restriction enzymes” refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.
  • a cell has been "transformed” by exogenous or heterologous DNA when such DNA has been introduced inside the cell.
  • the transforming DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
  • the transforming DNA may be maintained on an episomal element such as a plasmid.
  • a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA.
  • a "clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a "cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • Two DNA sequences are "substantially homologous" when at least about 75% (preferably at least about 80%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis et al., DNA Cloning, Volumes. I & II, supra; Nucleic Acid Hybridization, supra.
  • DNA sequences encoding radializing factor (RF60) which code for a radializing factor (RF60).
  • RF60 radializing factor
  • other DNA sequences which are degenerate to such sequences.
  • degenerate to is meant that a different three-letter codon is used to specify a particular amino acid. It is well known in the art that the following codons can be used interchangeably to code for each specific amino acid:
  • Leucine Leu or L
  • UUA or UUG or CUU or CUC or CUA or CUG Isoleucine (He or I)
  • AUU or AUC or AUA Leucine (Leu or L) UUA or UUG or CUU or CUC or CUA or CUG Isoleucine (He or I) AUU or AUC or AUA
  • Aspartic Acid Aspartic Acid (Asp or D) GAU or GAC Glutamic Acid (Glu or E) GAA or GAG Cysteine (Cys or C) UGU or UGC Arginine (Arg or R) CGU or CGC or CGA or CGG or AGA or AGG Glycine (Gly or G) GGU or GGC or GGA or GGG Tryptophan (Trp or W) UGG Termination codon UAA (ochre) or UAG (amber) or UGA (opal)
  • codons specified above are for RNA sequences.
  • the corresponding codons for DNA have a T substituted for U.
  • Mutations can be made such that a particular codon is changed to a codon which codes for a different amino acid. Such a mutation is generally made by making the fewest nucleotide changes possible.
  • a substitution mutation of this sort can be made to change an amino acid in the resulting protein in a non-conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (i.e. , by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping).
  • Such a conservative change generally leads to less change in the structure and function of the resulting protein.
  • a non-conservative change is more likely to alter the structure, activity or function of the resulting protein.
  • the present invention should be considered to include seguences containing conservative changes which do not significantly alter the activity or binding characteristics of the resulting protein.
  • Another grouping may be those amino acids with phenyl groups:
  • Another grouping may be according to molecular weight (i.e., size of R groups):
  • substitutions are: - Lys for Arg and vice versa such that a positive charge may be maintained;
  • Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property.
  • a Cys may be introduced a potential site for disulfide bridges with another Cys.
  • a His may be introduced as a particularly "catalytic" site (i.e., His can act as an acid or base and is the most common amino acid in biochemical catalysis).
  • Pro may be introduced because of its particularly planar structure, which induces -turns in the protein's structure.
  • Two amino acid sequences are "substantially homologous" when at least about 70% of the amino acid residues (preferably at least about 80%, and most preferably at least about 90 or 95%) are identical, or represent conservative substitutions.
  • a "heterologous" region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature.
  • the gene when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism.
  • Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g. , a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.
  • an “antibody” is any immunoglobulin, including antibodies and fragments thereof, that binds a specific epitope.
  • the term encompasses polyclonal, monoclonal, and chimeric antibodies, the last mentioned described in further detail in U.S. Patent Nos. 4,816,397 and 4,816,567.
  • an "antibody combining site” is that structural portion of an antibody molecule comprised of heavy and light chain variable and hypervariable regions that specifically binds antigen.
  • antibody molecule in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule.
  • Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contains the paratope, including those portions known in the art as Fab, Fab' , F(ab') 2 and F(v), which portions are preferred for use in the therapeutic methods described herein.
  • Fab and F(ab') 2 portions of antibody molecules are prepared by the proteolytic reaction of papain and pepsin, respectively, on substantially intact antibody molecules by methods that are well-known. See for example, U.S. Patent No. 4,342,566 to Theofilopolous et al. Fab' antibody molecule portions are also well- known and are produced from F(ab') 2 portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoethanol, and followed by alkylation of the resulting protein mercaptan with a reagent such as iodoacetamide.
  • An antibody containing intact antibody molecules is preferred herein.
  • the phrase "monoclonal antibody” in its various grammatical forms refers to an antibody having only one species of antibody combining site capable of immunoreacting with a particular antigen.
  • a monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts.
  • a monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different antigen; e.g., a bispecific (chimeric) monoclonal antibody.
  • phrases “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • terapéuticaally effective amount is used herein to mean an amount sufficient to prevent, and preferably reduce by at least about 30 percent, more preferably by at least 50 percent, most preferably by at least 90 percent, the formation of glial scar formation in the situation where brain or central nervous system trauma or injury has occurred.
  • a DNA sequence is "operatively linked" to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that DNA sequence.
  • the term "operatively linked” includes having an appropriate start signal (e.g., ATG) in front of the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence. If a gene that one desires to insert into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene.
  • standard hybridization conditions refers to salt and temperature conditions substantially equivalent to 5 x SSC and 65 °C for both hybridization and wash. However, one skilled in the art will appreciate that such “standard hybridization conditions” are dependent on particular conditions including the concentration of sodium and magnesium in the buffer, nucleotide sequence length and concentration, percent mismatch, percent formamide, and the like. Also important in the determination of “standard hybridization conditions” is whether the two sequences hybridizing are RNA-RNA, DNA-DNA or RNA-DNA. Such standard hybridization conditions are easily determined by one skilled in the art according to well known formulae, wherein hybridization is typically 10-20°C below the predicted or determined T m with washes of higher stringency, if desired.
  • the present invention concerns the identification, isolation and purification of a radializing factor (RF60).
  • RF60 radializing factor
  • the present invention relates to all members of the herein disclosed radializing factor (RF60).
  • the present invention also relates to a recombinant DNA molecule or cloned gene, or a degenerate variant thereof, which encodes a radializing factor (RF60), or a fragment thereof, that possesses a molecular weight of about 60 kD and that exhibits the structural and mophological characteristics of a radial glial cell; preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding the 60 kD radializing factor.
  • RF60 radializing factor
  • the present invention contemplates pharmaceutical intervention in the cascade of reactions in which the radializing factor (RF60) is implicated, to utilize the activity initiated by the radializing factor (RF60), and consequently affect the formation of glial scar formation and its deleterious effects.
  • RF60 radializing factor
  • the radializing factor (RF60), its active fragments, or analogs thereof may be prepared in pharmaceutical compositions, with a suitable carrier and at a strength effective for administration by various means to a patient experiencing an adverse medical condition associated with specific brain or central nervous system trauma or injury.
  • a suitable carrier such as subcutaneous, intravenous and intraperitoneal injections, catheterizations and the like.
  • Average quantities of the radializing factor (RF60), its variants and fragments may vary and in particular should be based upon the recommendations and prescription of a qualified physician or veterinarian.
  • antibodies including both polyclonal and monoclonal antibodies, and drugs that modulate the production or activity of the radializing factor (RF60) and/or its variants and fragments, or their subunits may possess certain diagnostic applications and may for example, be utilized for the purpose of detecting and/or measuring the extent of the formation of the glial scar formation.
  • the radializing factor (RF60) or its variants and fragments, or subunits may be used to produce both polyclonal and monoclonal antibodies to themselves in a variety of cellular media, by known techniques such as the hybridoma technique utilizing, for example, fused mouse spleen lymphocytes and myeloma cells.
  • RF60 radializing factor
  • small molecules that mimic or antagonize the activity (ies) of the radializing factor (RF60) of the invention may be discovered or synthesized, and may be used in diagnostic and/or therapeutic protocols.
  • the general methodology for making monoclonal antibodies by hybridomas is well known.
  • Immortal, antibody-producing cell lines can also be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., M. Schreier et al.
  • Panels of monoclonal antibodies produced against radializing factor (RF60) peptides can be screened for various properties; i.e. , isotype, epitope, affinity, etc.
  • monoclonal antibodies that neutralize the activity of the radializing factor (RF60), its variants and analogs can be readily identified in radializing factor (RF60) activity assays.
  • High affinity antibodies are also useful when immunoaffinity purification of native or recombinant radializing factor (RF60) is possible.
  • the anti-radializing factor (RF60) antibody used in the diagnostic methods of this invention is an affinity purified polyclonal antibody. More preferably, the antibody is a monoclonal antibody (mAb).
  • mAb monoclonal antibody
  • the anti-radializing factor (RF60) antibody molecules used herein be in the form of Fab, Fab', F(ab' 2 or F(v) portions of whole antibody molecules.
  • the diagnostic method of the present invention comprises examining a cellular sample or medium by means of an assay including an effective amount of an antagonist to a radializing factor (RF60)/protein, such as an anti- radializing factor (RF60) antibody, preferably an affinity-purified polyclonal antibody, and more preferably a mAb.
  • a radializing factor (RF60)/protein such as an anti- radializing factor (RF60) antibody, preferably an affinity-purified polyclonal antibody, and more preferably a mAb.
  • RF60 radializing factor
  • the anti- radializing factor (RF60) antibody molecules used herein be in the form of Fab, Fab', F(ab') 2 or F(v) portions or whole antibody molecules.
  • patients capable of benefiting from this method include those suffering from various forms of brain or central nervous system trauma or injury.
  • a myeloma or other self-perpetuating cell line is fused with lymphocytes obtained from the spleen of a mammal hyperimmunized with a radializing factor (RF60)- binding portion thereof, or radializing factor (RF60), or an origin-specific DNA- binding portion thereof.
  • RF60 radializing factor-binding factor
  • Splenocytes are typically fused with myeloma cells using polyethylene glycol (PEG) 6000.
  • Fused hybrids are selected by their sensitivity to HAT.
  • Hybridomas producing a monoclonal antibody useful in practicing this invention are identified by their ability to immunoreact with the present radializing factor (RF60) and their ability to inhibit specified radializing factor (RF60) activity in target cells.
  • a monoclonal antibody useful in practicing the present invention can be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes antibody molecules of the appropriate antigen specificity.
  • the culture is maintained under conditions and for a time period sufficient for the hybridoma to secrete the antibody molecules into the medium.
  • the antibody-containing medium is then collected.
  • the antibody molecules can then be further isolated by well-known techniques.
  • Media useful for the preparation of these compositions are both well-known in the art and commercially available and include synthetic culture media, inbred mice and the like.
  • An exemplary synthetic medium is Dulbecco's minimal essential medium (DMEM; Dulbecco et al., Virol. 8:396 (1959)) supplemented with 4.5 gm/1 glucose, 20 mm glutamine, and 20% fetal calf serum.
  • An exemplary inbred mouse strain is the Balb/c.
  • RF60 monoclonal anti-radializing factor
  • Methods for producing monoclonal anti-radializing factor (RF60) antibodies are also well-known in the art. See Niman et al., Proc. Natl. Acad. Sci. USA, 80:4949-4953 (1983).
  • the present radializing factor (RF60) or a peptide analog is used either alone or conjugated to an immunogenic carrier, as the immunogen in the before described procedure for producing anti-radializing factor (RF60) monoclonal antibodies.
  • the hybridomas are screened for the ability to produce an antibody that immunoreacts with the radializing factor (RF60) peptide.
  • a subject therapeutic composition includes, in admixture, a pharmaceutically acceptable excipient (carrier) and one or more of a radializing factor (RF60), polypeptide analog thereof or fragment thereof, as described herein as an active ingredient.
  • RF60 radializing factor
  • the composition comprises radializing factor (RF60) together with any necessary pharmaceutical adjuvants.
  • compositions which contain polypeptides, analogs or active fragments as active ingredients are well understood in the art.
  • such compositions are prepared as injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.
  • the preparation can also be emulsified.
  • the active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents which enhance the effectiveness of the active ingredient.
  • a polypeptide, analog or active fragment can be formulated into the therapeutic composition as neutralized pharmaceutically acceptable salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • the therapeutic polypeptide-, analog- or active fragment-containing compositions are conventionally administered intravenously, as by injection of a unit dose, for example.
  • unit dose when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for humans, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e. , carrier, or vehicle.
  • compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount.
  • the quantity to be administered depends on the subject to be treated, and the severity of the condition under treatment. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosages may range from about 0.1 to 20, preferably about 0.5 to about 10, and more preferably one to several, milligrams of active ingredient per kilogram body weight of individual per day and depend on the route of administration.
  • Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain concentrations of ten nanomolar to ten micromolar in the blood are contemplated.
  • the therapeutic compositions may further include an effective amount of the radializing factor (RF60) or analog thereof, and one or more of the following active ingredients: an antibiotic, a steroid.
  • RF60 radializing factor
  • active ingredients an antibiotic, a steroid.
  • Intravenous Formulation II Ingredient mg/ml ampicillin 250.0 radializing factor (RF60) 10.0 sodium bisulfite USP 3.2 disodium edetate USP 0.1 water for injection q.s.a.d. 1.0 ml Intravenous Formulation III
  • gentamicin charged as sulfate
  • RF60 radializing factor 10.0 sodium bisulfite USP 3.2 disodium edetate USP 0.1 water for injection q.s.a.d. 1.0 ml
  • Intravenous Formulation IV Ingredient mg/ml radializing factor (RF60) 10.0 dextrose USP 45.0 sodium bisulfite USP 3.2 edetate disodium USP 0.1 water for injection q.s.a.d. 1.0 ml
  • pg means picogram
  • ng means nanogram
  • ug means microgram
  • mg means milligram
  • ul means microliter
  • ml means milliliter
  • 1 means liter.
  • DNA sequences disclosed herein may be expressed by operatively linking them to an expression control sequence in an appropriate expression vector and employing that expression vector to transform an appropriate unicellular host.
  • Such operative linking of a DNA sequence of this invention to an expression control sequence includes, if not already part of the DNA sequence, the provision of an initiation codon, ATG, in the correct reading frame upstream of the DNA sequence.
  • a wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention.
  • Useful expression vectors may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E.
  • coli plasmids col El, pCRl, pBR322, pMB9 and their derivatives, plasmids such as RP4; phage DNAs, e.g., the numerous derivatives of phage ⁇ , e.g., NM989, and other phage DNA, e.g.
  • yeast plasmids such as the 2 ⁇ plasmid or derivatives thereof
  • vectors useful in eukaryotic cells such as vectors useful in insect or mammalian cells
  • vectors derived from combinations of plasmids and phage DNAs such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.
  • any of a wide variety of expression control sequences sequences that control the expression of a DNA sequence operatively linked to it — may be used in these vectors to express the DNA sequences of this invention.
  • useful expression control sequences include, for example, the early or late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lac system, the trp system, the TAC system, the TRC system, the 1-77? system, the major operator and promoter regions of phage ⁇ , the control regions of fd coat protein, the promoter for
  • 3-phosphoglycerate kinase or other glycolytic enzymes the promoters of acid phosphatase (e.g., Pho5), the promoters of the yeast o.-mating factors, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
  • a wide variety of unicellular host cells are also useful in expressing the DNA sequences of this invention.
  • These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animal cells, such as CHO, Rl.l, B-W and L-M cells, African Green Monkey kidney cells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (e.g., Sf9), and human cells and plant cells in tissue culture.
  • eukaryotic and prokaryotic hosts such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animal cells, such as CHO, Rl.l, B-W and L-M cells, African Green Monkey kidney cells (e.g., COS 1, COS 7, BSC1, BSC40, and
  • astrocytes of a host may be transformed to incorporate the nucleic acid molecules of the present invention within their genome, to facilitate the expression by such transformed astrocytes of the radializing factor (RF60), and to thereby promote the adoption by such cells of the characteristics of radial glia.
  • RF60 radializing factor
  • an expression control sequence a variety of factors will normally be considered. These include, for example, the. relative strength of the system, its controllability, and its compatibility with the particular DNA sequence or gene to be expressed, particularly as regards potential secondary structures. Suitable unicellular hosts will be selected by consideration of, e.g., their compatibility with the chosen vector, their secretion characteristics, their ability to fold proteins correctly, and their fermentation requirements, as well as the toxicity to the host of the product encoded by the DNA sequences to be expressed, and the ease of purification of the expression products. Considering these and other factors a person skilled in the art will be able to construct a variety of vector/expression control sequence/host combinations that will express the DNA sequences of this invention on fermentation or in large scale animal culture.
  • radializing factor (RF60) analogs may be prepared from nucleotide sequences of the protein complex/subunit derived within the scope of the present invention. Analogs, such as fragments, may be produced, for example, by pepsin digestion of radializing factor (RF60) material. Other analogs, such as muteins, can be produced by standard site-directed mutagenesis of radializing factor (RF60) coding sequences. Analogs exhibiting "radializing factor (RF60) activity" such as small molecules, may be identified by known in vivo and/or in vitro assays.
  • a DNA sequence encoding radializing factor (RF60) can be prepared synthetically rather than cloned.
  • the DNA sequence can be designed with the appropriate codons for the radializing factor (RF60) amino acid sequence. In general, one will select preferred codons for the intended host if the sequence will be used for expression.
  • the complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge, Nature, 292:756 (1981); Nambair et al., Science, 223:1299 (1984); Jay et al., J. Biol. Chem. , 259:6311 (1984).
  • RF60 radializing factor
  • muteins can be made by site-directed mutagenesis of native radializing factor (RF60) genes or cDNAs, and muteins can be made directly using conventional polypeptide synthesis.
  • the present invention also relates to a variety of diagnostic applications, including methods for detecting the extent of brain or central nervous system trauma or injury, by reference to the ability to elicit the activities which are mediated by the present radializing factor (RF60).
  • the radializing factor (RF60) can be used to produce antibodies to itself by a variety of known techniques, and such antibodies could then be isolated and utilized as in tests for the presence of particular radializing factor (RF60) activity in suspect target cells.
  • antibody (ies) to the radializing factor (RF60) can be produced and isolated by standard methods including the well known hybridoma techniques.
  • the antibody (ies) to the radializing factor (RF60) will be referred to herein as Ab, and antibody (ies) raised in another species as Ab 2 .
  • radializing factor (RF60) The presence of radializing factor (RF60) in cells can be ascertained by the usual immunological procedures applicable to such determinations. A number of useful procedures are known. Three such procedures which are especially useful utilize either the radializing factor (RF60) labeled with a detectable label, antibody Ab, labeled with a detectable label, or antibody Ab 2 labeled with a detectable label. The procedures may be summarized by the following equations wherein the asterisk indicates that the particle is labeled, and "RF60" stands for the radializing factor:
  • the radializing factor (RF60) forms complexes with one or more antibody(ies) or binding partners and one member of the complex is labeled with a detectable label.
  • RF60 radializing factor
  • Abj a characteristic property of Abj is that it will react with Ab,.
  • Ab raised in one mammalian species has been used in another species as an antigen to raise the antibody Ab 2 .
  • Ab 2 may be raised in goats using rabbit antibodies as antigens. Ab 2 therefore would be anti-rabbit antibody raised in goats.
  • Ab will be referred to as a primary or anti-radializing factor (RF60) antibody, and Ab 2 will be referred to as a secondary or anti-Ab, antibody.
  • RF60 primary or anti-radializing factor
  • the labels most commonly employed for these studies are radioactive elements, enzymes, chemicals which fluoresce when exposed to ultraviolet light, and others.
  • fluorescent materials are known and can be utilized as labels. These include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow.
  • a particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate.
  • the radializing factor (RF60) or its binding partner(s) can also be labeled with a radioactive element or with an enzyme.
  • the radioactive label can be detected by any of the currently available counting procedures.
  • the preferred isotope may be selected from 3 H, 14 C, 32 P, 35 S, 36 C1, 51 Cr, 57 Co, 58 Co, 59 Fe, * > Y, 125 I, 131 I, and 186 Re.
  • Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques.
  • the enzyme is conjugated to the selected particle by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like.
  • bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like.
  • Many enzymes which can be used in these procedures are known and can be utilized.
  • the preferred are peroxidase, ⁇ -glucuronidase, ⁇ -D-glucosidase, ⁇ -D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase.
  • U.S. Patent Nos. 3,654,090; 3,850,752; and 4,016,043 are referred to by way of example for their disclosure of alternate labeling material and methods.
  • a particular assay system developed and utilized in accordance with the present invention is known as a receptor assay.
  • the material to be assayed is appropriately labeled and then certain cellular test colonies are inoculated with a quantity of both the labeled and unlabeled material after which binding studies are conducted to determine the extent to which the labeled material binds to the cell receptors. In this way, differences in affinity between materials can be ascertained.
  • a purified quantity of the radializing factor (RF60) may be radiolabeled and combined, for example, with antibodies or other inhibitors thereto, after which binding studies would be carried out. Solutions would then be prepared that contain various quantities of labeled and unlabeled uncombined radializing factor (RF60), and cell samples would then be inoculated and thereafter incubated. The resulting cell monolayers are then washed, solubilized and then counted in a gamma counter for a length of time sufficient to yield a standard error of ⁇ 5 % . These data are then subjected to Scatchard analysis after which observations and conclusions regarding material activity can be drawn.
  • RF60 radializing factor
  • a receptor assay may be performed and utilized, in the instance where the cellular binding ability of the assayed material may serve as a distinguishing characteristic.
  • An assay useful and contemplated in accordance with the present invention is known as a "cis/trans" assay. Briefly, this assay employs two genetic constructs, one of which is typically a plasmid that continually expresses a particular receptor of interest when transfected into an appropriate cell line, and the second of which is a plasmid that expresses a reporter such as lucif erase, under the control of a receptor/ligand complex.
  • one of the plasmids would be a construct that results in expression of the receptor in the chosen cell line, while the second plasmid would possess a promoter linked to the luciferase gene in which the response element to the particular receptor is inserted.
  • the compound under test is an agonist for the receptor
  • the ligand will complex with the receptor, and the resulting complex will bind the response element and initiate transcription of the luciferase gene.
  • the resulting chemiluminescence is then measured photometrically, and dose response curves are obtained and compared to those of known ligands.
  • the foregoing protocol is described in detail in U.S. Patent No. 4,981,784 and PCT International Publication No. WO 88/03168, for which purpose the artisan is referred.
  • kits suitable for use by a medical specialist may be prepared to determine the presence or absence of predetermined radializing factor (RF60) activity or predetermined radializing factor (RF60) activity capability in suspected target cells.
  • RF60 predetermined radializing factor
  • RF60 predetermined radializing factor
  • one class of such kits will contain at least the labeled radializing factor (RF60) or its binding partner, for instance an antibody specific thereto, and directions, of course, depending upon the method selected, e.g., "competitive,” “sandwich, " “DASP” and the like.
  • the kits may also contain peripheral reagents such as buffers, stabilizers, etc.
  • an assay system for screening potential drugs effective to modulate the activity of the radializing factor (RF60) may be prepared.
  • the radializing factor (RF60) may be introduced into a test system, and the prospective drug may also be introduced into the resulting cell culture, and the culture thereafter examined to observe any changes in the radializing factor (RF60) activity of the cells, due either to the addition of the prospective drug alone, or due to the effect of added quantities of the known radializing factor (RF60).
  • Partial purification/enrichment of the differentiation protein 10 liters of GCB6 conditioned medium were loaded onto an EconocolumnTM (BioRad) packed with 40mls heparin agarose affigel matrix (BioRad) through a Pharmacia FPLC system with P-500 pump at a flow rate of 5 ml/minute. Partially purified bioactivity was obtained by elution from this column with 0.4M KC1 and the activity pool loaded onto an EconocolumnTM packed with ml wheat germ agglutinin matrix (Vector Laboratories Inc, Burlingame, CA).
  • Bioactivity was eluted from this using a gradient of N-acetyl-glucosamine and loaded onto an EconocolumnTM packed with 5 ml MonoQ Sepaharose matrix (Pharmacia).
  • a final pool of partially purified glial differentiation protein was obtained by elution with 0.4M KC1.
  • Total protein determinations were taken for whole conditioned medium, and for the heparin agarose and mono Q bioactive pools and these were screened on normal and reeler astrocytes as described above at serial dilutions 10 ' to lO- 12 ng/ml total protein. After 48 hours cells were fixed, immunostained with anti-GFAP (anti-glial fibrillary acidic protein) and scored for cell phenotype as described above.
  • anti-GFAP anti-glial fibrillary acidic protein
  • Diluted samples were assayed for bioactivity by screening on cultured astrocytes as described above. After 48 hours cells were fixed, immunostained with anti-GFAP (anti-glial fibrillary acidic protein) and scored for cell phenotype as described above.
  • anti-GFAP anti-glial fibrillary acidic protein
  • GCB6 conditioned medium was fractionated using three affinity matrices in sequence: heparin agarose, wheat germ agglutinin and monoQ sepharose, each of which had been found separately in preliminary assays to bind, and permit enrichment of, the glial differentiation protein (data not shown).
  • affinity matrices in sequence: heparin agarose, wheat germ agglutinin and monoQ sepharose, each of which had been found separately in preliminary assays to bind, and permit enrichment of, the glial differentiation protein (data not shown).
  • FIGURE 2 The number of different proteins in the final pool, i.e. pooled eluent from the monoQ sepharose column, were determined by SDS-PAGE analysis. A sample of this pool was loaded onto a denaturing acrylamide gel and silver staining was used to permit visualization of the distribution, number and separation of prominent protein bands present.
  • the final pool was found to contain a number of abundant protein bands but these were well separated visually on the gel (FIGURE la).
  • FOGURE la By running a second lane of the protein on the same gel and leaving this unfixed/unstained, then comparing this gel piece to its silver stained equivalent, it was possible to excise, from the unfixed gel piece, slices of gel representing the approximate position of each of 20 abundant bands.
  • the protein band in each gel slice was eluted and renatured and each sample was screened for bioactivity (defined as the capacity to induce expression of a radial glial phenotype by cultured astrocytes (Hunter and Hatten, 1995)).
  • Bioactivity was found to be restricted to a region of three bands on the gel located around the 60kD molecular weight range (FIGURE lb). This correlates with the previous report that the bioactivity elutes from a size exclusion column in the 50-60kD molecular weight range (Hunter and Hatten, 1995). Allowing for the possibility that bioactivity may have diffused into neighboring bands, and considering the fact that the gel pieces were essentially excised 'blind' from an unmarked gel, it seems therefore that the radial glial differentiation signal is represented by a single gel band in the approximate molecular weight region of 60kD. According to its function and approximate molecular weight, this was termed radializing factor 60 (RF60), which occurs as a single protein band on SDS-PAGE analysis.
  • RF60 radializing factor 60
  • radial glia differentiate poorly, as evidenced by the extension of only short, truncated processes; the radial scaffold is maintained for a shorter time than normal, and radial glia transform prematurely into astrocytes.
  • reeler astroglia show only impaired differentiation in response to the protein. This suggests that an intrinsic defect in glial differentiation contributes to the phenotype of abnormal cortical lamination seen in reeler mouse.
  • the murine autosomal recessive mutation reeler broadly affects cellular organization throughout the central nervous system, including in the cerebral and cerebellar cortices.
  • the reeler phenotype is evident at E14 as a failure in the formation of the cortical plate. Subsequently, patterning of neurons during migration and laminar formation are disturbed, thus leading to an inversion of the normal cortical layers (Falconer, 1951; Goffmet, 1979; Caviness, 1982; Pinto-Lord et al., 1982; Caviness et al., 1988).
  • Molecular cloning of the reeler gene has revealed that the gene encodes a 388kD extracellular protein, reelin.
  • reelin is expressed in Cajal Retzius neurons, cells which are localized to the outermost layer, layer 1, and form a postmitotic, fully differentiated neuronal population well before the bulk of cortical neurogenesis occurs.
  • the early differentiation of Cajal Retzius cells, together with their expression of the reelin protein, indicates a critical role for these early neurons in cortical patterning (Miao et al., 1994; D'Arcangelo et al., 1995).
  • the reeler mouse provides therefore a genetic model for the study of cortical patterning, including the development of the glial scaffold and its role in corticogenesis.
  • the reeler system can be used to elucidate the molecular events which regulate radial glial cell development and the role of the radial glial differentiation signal, radializing factor (RF60) in this.
  • the progression of radial glia and astrocyte phenotype during the perinatal period in reeler and normal cortex can be examined and the effect of the radial glial differentiation factor on reeler and normal astroglia can thus be compared.
  • These analyses aim to determine whether or not the developmental program of radial glial cell development in reeler cortex is normal, whether these cells might directly cause the reeler cortical phenotype, and ultimately, to reveal how the glial differentiation factor functions in the developing forebrain.
  • rl/rl and 4-/rl mice were acquired from matings of rl/rl or +/rl females with +/rl males (Jackson Laboratories, Bar Harbor, ME). Day of plug detection was determined as embryonic day 0 (E0) and day of birth as postnatal day 0 (P0). Pups of stage E14, P0 and P4 were sacrificed and the forebrains removed in ice cold CMF-PBS (calcium-magnesium free buffered saline; GIBCO BRL, Grand Island, NY), fixed overnight in 2% paraformaldehyde in PBS (phosphate buffered saline; reconstituted from tablets (Sigma Chemical Co., St. Louis, MO)), washed in PBS for 3 hours then embedded in agarose (3% low EEO agarose (Sigma) in dH 2 0). Brains were cut into lOO ⁇ m coronal
  • Vibratome sections Two sections from each brain were counterstained with cresyl violet then examined wet and unmounted under light microscopy and phenotyped as normal or reeler according to presence or absence of a cortical plate, respectively.
  • Immunostaining for RC2 and GFAP " glial fibrillary acidic protein) distribution in vivo. Cortical sections were permeablized/preblocked (1 % Tween20 (BioRad, Hercules, CA), 20% normal goat serum (GIBCO) in PBS, 90 minutes) then incubated in either monoclonal mouse-anti-RC2 (tissue culture supernatant, cell line a generous gift of Dr. M.
  • GFAP glial fibrillary acidic protein
  • bound secondary antibody was detected by bromochloroindoyl phosphate/nitro blue tetrazolium development as described (Towbin et al., 1979). Screening size fractionated protein on reeler and normal astrocytes. Astrocytes were purified from forebrains of PO +/rl and rl/rl pups.
  • GCB6 cerebellar cell line (Gao and Hatten, 1994) as follows: GCB6 cells were plated into Integrid TM culture plates (Falcon) at 1/10 dilution in DMEMF12 culture medium (GIBCO) supplemented with 10% fetal bovine serum (FBS; GIBCO). On reaching confluence (5-7 days) GCB6 cells were transferred to a minimal volume of serum free DMEMF12. This medium was harvested and replaced daily; harvested medium was filtered through a 0.22mm membrane filter to remove cell debris, then stored at 4°C.
  • GIBCO DMEMF12 culture medium
  • FBS fetal bovine serum
  • 5mls of conditioned medium was fractionated on a HiLoad Superdex 16/60 size exclusion column (Pharmacia) as previously described (Hunter and Hatten, 1995). Fractions eluted from the sizing column were screened on normal and reeler astrocytes; after 48 hours cells were fixed in 2% paraformaldehyde, and immunostained for GFAP (glial fibrillary acidic protein) expression as previously described (Hunter and Hatten, 1995). Approximately 200 cells on each coverslip were scored according to phenotype as 'normal' or 'radial' , and the percentage of radial cells on each coverslip was calculated. 1 unit of bioactivity was defined as the amount of protein pool required in each case to effect 'conversion' (switch from epithelioid to radial phenotype) of 25% of astrocytes.
  • Radializing factor (RF60) was prepared and purified as described in Example 1.
  • the radial glial scaffold develops poorly and disappears prematurely in reeler cortex
  • the architecture and organization of the radial glial scaffold was examined in coronal sections of reeler and normal cortex of developmental stages E14, P0 and P4. These stages represent the early, middle and late phases of the radial glia-astrocyte developmental pathway.
  • Radial glia marked by expression of the RC2 antigen (Misson et al., 1988) are fully elaborated in the cortical wall at E14 and persist throughout the late embryonic period; around the time of birth they begin to transform into astrocytes, as evidenced by expression of glial intermediate filament protein, glial fibrillary acidic protein (GFAP) (Dahl and Bignami, 1973; Levitt and Rakic, 1980).
  • GFAP glial fibrillary acidic protein
  • Transformation is complete by the end of the first postnatal week (Misson et al. , 1991) at which time highly elongated, RC2-positive cells have disappeared and stellate GFAP-positive astrocytes are prevalent.
  • the switch from RC2 expression to GFAP expression in the perinatal period occurs concomitantly with the establishment of the cortical layers, suggesting that the transformation of radial glial into astrocytes follows changing roles for astroglial cells in corticogenesis.
  • E14 cortical sections were immunostained with RC2 antibody while P0 and P4 sections were immunostained with anti-GFAP.
  • Radial glia in the E14 reeler cortex were organized in an approximately radial manner across the thickness of the cortical strom.
  • the radial scaffold which they constituted was sparse and the fibers were loosely packed. Instead of projecting in the radial plane, fibers projected in all directions within the cortical wall. These fibers were poorly differentiated, extending shorter processes that often failed to form endfeet along the pial surface (FIGURE 3B).
  • the forms of radial glia in normal E14 cortex recognized by RC2 antibody were as reported in classical studies (reviewed in Misson et al., 1991).
  • the radial glial scaffold was present but the cell processes were thick and fibrous, the whole scaffold having a coarse appearance.
  • the most striking feature of the PO reeler cortex was the abundance of mature astrocytic forms present. These cells were located directly beneath the pial surface, being highly abundant and forming a dense network of stellar cell forms (FIGURE 3D).
  • astroglial phenotype in normal P4 cortex was reminiscent of that classically described as transitional astroglia, cells which bear features of both radial glia and astrocytes, having long radial glia-like cell processes oriented perpendicular to the pial surface, as well as shorter, astrocytic processes close to the cell body (FIGURE 3E).
  • the abundance of these cell forms is indicative of the fact that the transformation of radial glia into astrocytes is still in progress within the cortical wall.
  • the astroglial forms present are of more immature forms than those in P4 reeler animals. Therefore, transformation of radial glia into astrocytes, normally not concluded until the end of the second postnatal week (reviewed in Misson et al., 1991), seems to be completed ahead of schedule in the reeler cortex.
  • GFAP was present in reeler cortex at the earliest developmental stage screened, El 4, a time when the radial glial scaffold is most well differentiated, and transformation has not yet been initiated. GFAP levels then remained constant throughout the embryonic period but there was a marked increase in at the time of birth, and very high levels of GFAP were seen in the P21 reeler cortex. Overall, in reeler cortex levels of GFAP were substantial throughout the embryonic and postnatal periods.
  • GFAP was absent from normal cortex during embryogenesis, being first detectable at PO, a time more in keeping with its observed appearance in immunostained sections, as shown here and previously (reviewed in Misson et al., 1991). Furthermore, even at PO GFAP levels were still low in normal cortex, particularly when compared to levels in reeler cortex at this time. Indeed of all stages of normal cortex screened, only the most mature, that harvested from P21 cortex, contained substantial amounts of GFAP, and again this was markedly lower than levels in P21 reeler cortex (FIGURE 4). Indeed, GFAP levels in PO reeler cortex were comparable to those in the mature P21 normal cortex (FIGURE 4).
  • GFAP the characteristic astrocyte marker
  • This enhanced expression of GFAP confirms and supports the immunohistochemical finding that in reeler cortex there is premature transformation of radial glia into mature astrocytes and further shows that astrocytes in mature reeler cortex express levels of GFAP which are in excess of those seen in normal cortex.
  • Reeler astroglia have an autonomous defect in their ability to respond to RF60
  • Partially purified RF60 was prepared as previously described by elution from a size exclusion column (Hunter and Hatten, 1995).
  • reeler astrocytes Only a small percentage of reeler astrocytes were induced to express a radial phenotype, the peak response being only 20% compared to almost 70% in the case of normal astrocytes; instead, the majority of cells remained epithelioid in form (FIGURE 2A).
  • This suggested reeler astrocytes may have a reduced ability to respond to RF60.
  • reeler astrocytes have a shifted dose-response for RF60, i.e. require higher levels than normal astrocytes to exhibit the same degree of phenotype change.
  • GCB6 conditioned medium was progressively purified by fractionation on three sequential affinity matrices: heparin agarose, wheat germ agglutinin and monoQ sepharose, steps which progressively enriched RF60.
  • the bioactivity pool from each column was screened in serial dilution on reeler and normal astrocytes. After each step the protein pool became more potent in inducing radial differentiation of normal astrocytes (FIGURE 2A, 2B, 2C). However as in the size fractionation screen, overall, very few reeler astrocytes were induced to differentiate.
  • radial glial differentiation and transformation into astrocytes is bidirectional in the mammalian forebrain is bidirectional, with the availability of a diffusible factor present in embryonic but not adult brain, acting to regulate the form and function of astroglial cells (Hunter and Hatten, 1995).
  • this factor was purified by biochemical methods, using a previously described bioassay, and defined as a single bioactivity, termed RF60.
  • the findings presented here reveal that there are intrinsic defects in the program of development of reeler radial glia development and transformation in reeler forebrain which involves the function of RF60.
  • RF60 is present in embryonic reeler cortex, reeler astroglia are unresponsive to this; the protein is ineffectual in inducing a radial glia phenotype in cultured reeler astrocytes; even saturating levels of RF60 are ineffective in inducing a radial phenotype in these cells, suggesting there is a glia-autonomous defect in reeler cortex which involves a failure of RF60 function, either in terms of expression of receptors for this protein, or in its signalling pathway.
  • EXAMPLE 3 To ascertain the effect/ impact of radializing factor (RF60) in vivo, stab wounds in the adult mouse cortex were studied both with and without the administration of radializing factor (RF60), infused into the would using as osmotic micropump system. The form of the astrocytes around the wound were examined at two, five and seven days after the wounding/infusion.
  • RF60 radializing factor
  • mice were anaesthetized with Nembural according to body weight; during surgery deep anaesthesia was maintained by administering the vaporous anaesthetic AerraneTM. An incision was made through the skin and muscle over the forebrain. Using a stereotactic device a fine needle was driven through the skull and into the wall of the left cortical hemisphere. A catheter was inserted into this hole and then the catheter was connected via fine tubing to an AlzetTM osmotic micropump, which had been pre-filled with a partially purified fraction of RF60 in saline. The pump itself was inserted under the skin of the back of the mouse.
  • anti-GFAP antibody recognized stellate cells with the characteristic appearance of mature astrocytes. GFAP levels appeared to be constant across the cortical mass in these cases, i.e., no regional variations. RC2 antibody did not detect antigen at any time in these animals, indicating that this embryonic marker is not normally expressed in adult cortex.
  • astrocytes have a characteristic stellar appearance, and express GFAP, but not RC2, the latter being an embryonic glial marker. Following stab injury of the cortex, astrocytes usually assume a reactive phenotype, becoming even more stellar and increasing their expression of GFAP. This reactive cell phenotyped is seen at two days following injury and is still in evidence even seven days after injury. These reactive astrocytes do not express RC2 at any time.
  • RF60 protein is infused into the wound site following injury, a very different pattern is observed.
  • reactive astrocytes are not seen at the wound site. Rather, GFAP levels are reduced, being even lower than in unstabbed cortical areas; however, a number of astrocytes are recognized by RC2 antibody and a number of these cells have a "radialized” phenotype. Five days after wounding/infusion these cortices look very similar to those wounded but not infused with RF60 protein.
  • Neocortical histogenesis in normal and reeler mice a developmental study based on [ 3 H] thymidine autoradiography. Dev. Brain Res. 4:293-302
  • Radial glial cell transformation is bidirectional: regulation by a diffusible factor in embryonic forebrain. Proc. Natl. Acad. Sci.92:2061-2065.
  • Rakic P. (1972). Mode of cell migration to the superficial layers of fetal monkey cortex. J. Comp. Neurol. 145:61-84.

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Abstract

On a identifié, isolé, purifié et caractérisé des modulateurs de différenciation et de transformation radiale glie-astrocyte comprenant un facteur de différenciation radiale gliale sécrété. Ce facteur radial, appelé RF60 aux fins de l'invention, a un poids moléculaire apparent d'environ 60 kD et tient lieu de signal radial de maintenance gliale, avec possibilité de régulation par la protéine du phénotype de la glie corticale. On peut aussi utiliser RF60 pour inverser le phénomène de cicatrisation gliale intervenant en cours de traumatisme du système nerveux central, et revenir ainsi au stade précédant l'événement traumatique. D'où la possibilité d'utiliser RF60 pour induire dans les cellules le retour à la forme embryonnaire et la régénérescence normale, de manière à traiter les cas de lésion ou de chirurgie cérébrale ou spinale, les troubles ou maladies neurologiques avec diminution de l'activité normale du cerveau ou du système nerveux central (par exemple, démence, maladie d'Alzheimer, sclérose en plaques) et les situations où la régénérescence du tissu du système nerveux central joue un rôle thérapeutique, par exemple dans les cas suivants: chirurgie, traumatisme, ictus dû à des phénomènes ischémiques ou hémorragiques, infections bactériennes et virales, néoplasmes et autres.
PCT/US1997/023818 1996-12-31 1997-12-31 Modulateurs de differenciation et de transformation radiale glie-astrocyte, et utilisations diagnostiques et therapeutiques WO1998029547A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002043654A2 (fr) * 2000-12-01 2002-06-06 Milos Pekny Composition pharmaceutique et methode de traitement d'une lesion cerebrale, d'une lesion de la moelle epiniere, d'un accident vasculaire cerebral, d'une maladie neurodegenerative et d'autres etats pathologiques

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EP0503297A1 (fr) * 1991-02-14 1992-09-16 Takeda Chemical Industries, Ltd. Facteur d'activation des cellules gliales et sa production
WO1996018103A1 (fr) * 1994-12-09 1996-06-13 The Scripps Research Institute Inhibition de la proliferation de cellules gliales

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Publication number Priority date Publication date Assignee Title
EP0503297A1 (fr) * 1991-02-14 1992-09-16 Takeda Chemical Industries, Ltd. Facteur d'activation des cellules gliales et sa production
WO1996018103A1 (fr) * 1994-12-09 1996-06-13 The Scripps Research Institute Inhibition de la proliferation de cellules gliales

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002043654A2 (fr) * 2000-12-01 2002-06-06 Milos Pekny Composition pharmaceutique et methode de traitement d'une lesion cerebrale, d'une lesion de la moelle epiniere, d'un accident vasculaire cerebral, d'une maladie neurodegenerative et d'autres etats pathologiques
WO2002043654A3 (fr) * 2000-12-01 2002-09-06 Milos Pekny Composition pharmaceutique et methode de traitement d'une lesion cerebrale, d'une lesion de la moelle epiniere, d'un accident vasculaire cerebral, d'une maladie neurodegenerative et d'autres etats pathologiques

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