WO1997005251A2 - Feline stem cell factor - Google Patents
Feline stem cell factor Download PDFInfo
- Publication number
- WO1997005251A2 WO1997005251A2 PCT/GB1996/001904 GB9601904W WO9705251A2 WO 1997005251 A2 WO1997005251 A2 WO 1997005251A2 GB 9601904 W GB9601904 W GB 9601904W WO 9705251 A2 WO9705251 A2 WO 9705251A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- stem cell
- cell factor
- feline
- fragment
- nucleic acid
- Prior art date
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Classifications
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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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to feline stem cell factor, a nucleic acid sequence encoding feline stem cell factor, a recombinant vector comprising such a nucleic acid sequence, a host cell containing said recombinant vector, a feline stem cell factor polypeptide, antibodies immuno- reactive with said polypeptide and pharmaceutical compositions comprising recombinant feline stem cell factor for use as a prophylactic and/or therapeutic agent and also as an adjuvant in cats.
- SCF Stem cell factor
- Cytokine and anti-cytokine therapies such as the synthesis of inhibitors, soluble cytokine receptors, receptor antagonists or anti-cytokine antibodies are finding increasing application in human medicine (Mire- Sluis, 1993) .
- Cytokine and anti-cytokine therapies such as the synthesis of inhibitors, soluble cytokine receptors, receptor antagonists or anti-cytokine antibodies are finding increasing application in human medicine (Mire- Sluis, 1993) .
- due to a lack of species cross- reactivity of many cytokines and the potential for antibody production against heterologous cytokines (Homes, 1993) there is a need for substantially feline-specific reagents.
- the present invention provides a poiynucleotide fragment, such as a DNA fragment, encoding feline stem cell factor.
- the invention further provides a recombinant feline stem cell factor polypeptide.
- Polynucleotide fragment refers to a chain of nucleotides of any length, such as deoxyribonucleic acid (DNA) sequences and transcription products thereof, (such as RNA) capable of giving rise to feline stem cell factor, or functionally active desivative thereof.
- DNA deoxyribonucleic acid
- RNA transcription products thereof
- the term excludes the whole naturally occurring genome. Thus, this term includes double and single stranded DNA, and RNA sequences derived therefrom.
- the poiynucleotide will be in isolated form, substantially free of biological material with which the whole genome is normally associated in vivo.
- polypeptide refers to a chain or sequence of amino acids displaying a biological activity substantially similar to the biological activity of feline stem cell factor, and does not refer to a specific length of the product as such.
- the polypeptide required can be modified in vivo and/or in vitro. for example by glycosylation, amidation, carboxylation, phosphorylation and/or post-translation cleavage; thus inter alia peptides. oligopeptides proteins and functionally active derivatives thereof are encompassed thereby.
- the poiynucleotide fragment encoding feline stem cell factor can be amplified from feline stem cell factor cDNA obtained by way of reverse transcription of mRNA, which can be recovered from feline embryo cells infected with feline leukaemia virus type A, using polymerase chain reaction (PCR) techniques known in the art. PCR amplification of cDNA can be accomplished using primers designed with respect to conserved regions of stem cell factor coding sequences from a single species or a number of other species.
- An amplified fragment containing the feline stem cell factor is exemplified in Figure 1.
- the DNA fragment of Figure l is shown to encode an open reading frame (ORF) of 825 nucleotides (nucleotides 65-889 inclusive) , corresponding to a protein of 274 amino acids.
- ORF open reading frame
- the fragment shown in Figure l shows the longer isoform of feline stem cell factor.
- a predicted shorter mature isoform can have a deletion of 84 nucleotides, (nucleotides 588 to 671 inclusive, shown underlined) based on information derived from other species and disclosed by McNiece and Shieh (1994) .
- feline stem cell factor protein (249 amino acids) is a transmembrane protein cleavable in vivo between residues Ala 164/Ala 165 and/or Ala 165/Ser 166, of the mature protein, resulting in the release of soluble feline stem cell factor (McNiece and Shieh, 1994) .
- feline stem cell factor cDNA Comparison of the full-length feline stem cell factor cDNA to published sequences in other species shows, similarity, for example identities to canine (Shull et al, 1992), bovine (Zhou et al, 1994), porcine (Zhang and Anthony, 1994) human (Martin et al, 1990), murine (Anderson et al. 1990) and chicken (Zhou et al. 1993) stem cell factor of 95%, 93%, 95%, 92%, 87% and 71% respectively.
- the conceptual amino acid sequence of the feline stem cell factor protein shows an amino acid identity with canine, porcine bovine, human, murine and chicken homologues of 92%, 92%, 91%, 88%, 80% and 53% respectively (using the GCG DNA computer analysis package, and the programs Translate, Compare and Best Fit) .
- the conceptual polypeptide sequence of the longer isoform of feline stem cell factor is shown in Figure 2.
- the first 25 amino acids encode a predicted signal peptide which is cleaved to produce the mature protein; hence the N-terminus of the mature feline stem cell factor of Figure 2 begins KGLCR.
- the present invention includes polypeptide fragments having at least 80%, particularly at least 90% and especially at least 95% similarity with the fragment exemplified in Figure l.
- the present invention includes polypeptide sequences having at least 80%, particularly at least 90% and especially at least 95% similarity with the polypeptide exemplified in Figure 2.
- Similarity refers to both identical and conservative replacement of nucleotides or amino acids, provided that the functionality of feline stem cell factor DNA and/or polypeptide is substantially unimpaired.
- feline stem cell factor polypeptide embraced herein, natural variations can exist between individuals or between species within the felis genus. These variations may be demonstrated by (an) amino acid difference(s) in the overall sequence or by deletions, substitutions, insertions, inversions or additions of (an) amino acid(s) in said sequence. All such derivatives are included within the scope of this invention provided the derivatives produce active feline stem cell factor. For example, for the purpose of the present invention conservative replacements may be made between amino acids, within the following groups:
- the degeneracy of the genetic code permits substitution of bases in a codon resulting in a different codon capable of coding for the same amino acid, e.g. the codon for the amino acid glutamic acid is both GAT and GAA. Consequently, it is clear that for the expression of a polypeptide with the amino acid sequence shown in Figure 2 or fragment thereof, use can be made of a derivative nucleic acid sequence with such an alternative codon composition different from the nucleic acid sequence shown in said Figure 1.
- feline stem cell factor poiynucleotide fragment of the present invention is preferably linked to regulatory control sequences.
- control sequences may comprise promoters, operators, inducers, ribosome binding sites, terminators etc.
- Suitable control sequences for a given host may be selected by those of ordinary skill in the art.
- a feline stem cell factor control sequence may be employed in a mammalian host.
- a poiynucleotide fragment according to the present invention can be ligated to various expression controlling DNA sequences, resulting in a so called recombinant DNA molecule.
- the present invention also includes an expression vector containing the expressible nucleic acid molecule.
- Said recombinant nucleic acid molecule can then be used for transformation of a suitable host.
- Such expression vectors are preferably hybrid DNA molecules derived from, for example plasmids, or from nucleic acid sequences derived from bacteriophages or viruses and are termed vector molecules.
- a specific bacterial expression vector pGEX-4T-l (Pharmacia Limited.) has been used for feline stem cell factor production.
- the pGEX-4T-l vector results in production of a fusion protein with glutathione S- transferase (GST) , being expressed.
- GST glutathione S- transferase
- GST or other small N-terminally fused proteins facilitates purification by affinity chromatography and the mature feline stem cell factor may then be cleaved from GST using thrombin, thereby releasing an 18kD polypeptide corresponding to the soluble form of feline stem cell factor.
- the glutathione S-transferase (GST) gene fusion system allows expression, purification and, if required, detection of fusion proteins produced in E. coli .
- the pGEX plasmid vectors are designed for expression of a fusion protein with Schistosoma japonicum GST.
- the resulting fusion proteins are purified from bacterial lysates by affinity chromatography using Glutathione Sepharose 4B.
- the polypeptide of interest can be cleaved from the GST moiety using a site specific protease (thrombin) with a cleavage site upstream of the cloning site on the plasmid. On cleavage the GST molecule remains bound to the Sepharose matrix and the polypeptide of interest can be purified from the GST. (Pharmacia Biotech, GST Gene Fusion System, Second Edition of Manual) .
- the present invention also relates to a transformed cell containing the nucleic acid molecule in expressible form.
- Transformation refers to the introduction of a heterologous nucleic acid sequence into a host cell, irrespective of the method used, for example direct uptake, transfection or transduction.
- the heterologous poiynucleotide fragment may be maintained through autonomous replication or alternatively, may be integrated into the host genome.
- the recombinant nucleic acid molecules preferably are provided with appropriate control sequences compatible with the designated host which can regulate the expression of the inserted poiynucleotide fragment e.g. tetracylcine responsive promoter, thymidine kinase promoter and SV-40 promoter.
- Suitable hosts used for the expression of recombinant nucleic acid molecules can be prokaryotic or eukaryotic in origin.
- the most widely used hosts for expression of recombinant nucleic acid molecules may be selected from bacteria, yeast, insect cells, and mammalian cells.
- the cloning and expression of recombinant feline stem cell factor also facilitates in producing reagents for the production of, for example, probes for in situ expression studies, production of anti-feline stem cell factor antibodies (particularly monoclonal antibodies) and evaluation of in vitro and in vivo biological activity of recombinant feline stem cell factor.
- the antibodies may be employed in diagnostic tests for feline stem cell factor.
- the present invention further provides recombinant feline stem cell factor for the manufacture of reagents for use as prophylactic and/or therapeutic agents and also as -lo ⁇ an adjuvant in cats.
- the invention provides pharmaceutical compositions comprising the recombinant feline stem cell factor together with a pharmaceutically acceptable carrier therefor.
- feline disorders such as disease states attribute to feline stem cell factor deficiencies and/or over abundance and physiological or immunological abnormalities, potentially benefit from feline stem cell factor therapy and/or prophylactic treatment.
- Such disorders include cancer, endotoxaemia, parasitic and bacterial infections, wound therapy, autoimmune and inflammatory diseases and allergies.
- recombinant feline stem cell factor may be used in the treatment of anaemia, leukopaenia, immunosuppression associated with retroviral or non- retroviral agents, chronic infections of bacterial, viral or parasitic origin, and hypo-pigmentation and other skin disorders and for the manipulation of fertility.
- Feline stem cell factor may be used alone, or in combination with other cytokines and drugs, including erythropoietin, interleukins such as IL3, IL4, IL6 and IL12, granulocyte/ macrophage colony-stimulating factor (GM-CSF) and/or granulocyte colony-stimulating factor (G-CSF) .
- cytokines and drugs including erythropoietin, interleukins such as IL3, IL4, IL6 and IL12, granulocyte/ macrophage colony-stimulating factor (GM-CSF) and/or granulocyte colony-stimulating factor (G-CSF) .
- a feline stem cell factor of the present invention may find further utility as a research and/or diagnostic tool.
- RNAgents Trademark
- Total RNA isolation system supplied by Promega Corp.
- Superscript Trademark
- oligonucleotides Two oligonucleotides (see below) were designed against interspecies conserved non-coding regions of stem cell factor mRNA.
- oligonucleotides were then used as primers in a polymerase chain reaction (PCR) experiment, to amplify the feline stem cell factor cDNA.
- the PCR was performed essentially as described by Saiki et al (1987) .
- 10 ⁇ l of lOOng/ ⁇ l template cDNA from the reverse transcribed mRNA was added to a 40 ⁇ l reaction mixture containing 200 ⁇ M of dATP, dCTP, dGTP, dTTP, 50pmol of both primers (I) and (II) , 1 unit of Taq DNA Polymerase and 5 ⁇ l of lOx reaction buffer.
- the reaction buffer contained lOOmM Tris-HCI, 500mM potassium chloride, 0.01 per cent (w/v) gelatin and 1.5mM magnesium chloride, ultrapure water, TE (pH ⁇ .O).
- the solution was overlaid with two drops of mineral oil to prevent evaporation.
- amplification and analysis of the products were carried out. Thirty five cycles of amplification were performed using a Perkin Elmer Cetus thermal cycler. Each cycle consisted of 1 min. at 95°C to denature the DNA, 1 min. at 50°C to anneal the primers to the template and l min. at 72°C for primer extension. After the last cycle a further incubation for lOmins. at 72°C was performed to allow extension of any partially completed product. On completion of the amplification, lO ⁇ l of the reaction mixture was electrophoresed through a 1.5 per cent agarose gel. The DNA was visualised by staining with ethidium bromide and exposure to ultraviolet light (320nm) .
- the Blue-Script (Trademark) derived vector, containing the cloned feline stem cell factor DNA was prepared and purified. Double stranded DNA sequencing was carried out on this DNA using the T7 polymerase, "Sequenase"
- the open reading frame (ORF) encoding the feline stem cell factor was excised from the Blue-script (TRADE MARK) derived vector and sub-cloned into the plasmid vector pGEX- 4T-1 (Pharmacia Ltd) .
- a liquid culture of the chosen clone was grown as described above and the bacteria were collected by centrifugation and resuspended in lx phosphate buffered saline (PBS) and lysed by sonication. The insoluble cell debris was removed by centrifugation and the supernatant was mixed with a 50% (w/v) slurry of Glutathione Sepharose 4B. The resulting mixture was incubated at 20°C for five minutes and then the Sepharose beads were pelleted by brief centrifugation. The supernatant was removed and the beads were resuspended in PBS and again pelleted, this washing step was repeated twice.
- PBS lx phosphate buffered saline
- Sepharose bead pellet with bound fusion protein were mixed with a thrombin solution at 10 cleavage units per mg of protein in PBS.
- the mixture was incubated with gentle mixing for 16h at 20°C the beads were pelleted from the mixture by centrifugation and the supernatant containing the cleaved recombinant protein was stored for assay.
- the murine mast cell line MC/9 and human crythroleukaemia cell line TF-1 show cell proliferation in response to stem cell factor. Both cell lines have been used to assay the biological activity of feline stem cell factor as assessed by the incorporation of H 3 - thymidine (according to methodology essentially as described in Current Protocols in Immunology (1992) . Measurement of human and murine stem cell factor (c-kit ligands) Unit 6.17.1. John Wiley and Sons, Inc. USA).
- TF-1 cells show greater sensitivity to feline stem cell factor with an ED 50 of approximately 5ng/ml cf. 40ng/ml for MC/9 cells; in addition these cells are easier to maintain in culture and have less tendency to become factor independent. Thus TF-1 cells are the preferred cells for assaying feline stem cell factor bioactivity.
- TF-1 cells were maintained in RPMI-1640 medium supplemented with 5% FCS, glutamine, penicillin/streptomycin and 2ng/ml human granulocyte- acrophage colony stimulating factor. Cells were taken 2-3 days after passaging and washed three times in RPMI-1640 medium. The washed cells were counted, cell viability assessed by trypan blue exclusion and cells were resuspended to a final concentration of l x io 5 cells/ml in RPMI-1640 medium containing 5% FCS, glutamine and penicillin/streptomycin. Recombinant feline stem cell factor was serially diluted in lOO ⁇ l volume in triplicate in 96 well microtitre plates. Recombinant murine stem cell factor was used as a positive control.
- MC/9 cells were maintained in MC/9-medium. Before assay the cells were washed and resuspended in growth medium without concanavalin-A activated supernatant. The recombinant purified feline stem cell factor was diluted in medium in microtiter plates and 5 x IO 3 cells were added to each well. The thymidine incorporation was then measured by scintillation counting.
- Figure 4 shows the biological activity of recombinant feline stem cell factor as determined by MC/9 cell proliferation measured by 3 H Thymidine incorporation. As can be seen thymidine incorporation as a result of feline stem cell factor was significantly above background levels. especially at concentrations greater than about 40 mg feline stem cell factor per ml.
- G-CSF human granulocyte colony stimulating factor
- PHA-CM feline phytohaemagglutinin spleen cell conditioned medium
- Bone marrow was collected from the femur or humerus of SPF (Specified Pathogen Free) cats immediately post mortem into 25ml IMDM (Iscove's Modified Dulbecco's Medium Supplied by Life Technolgies) containing 100 iu/ml heparin. Low density bone marrow mononuciear cells were isolated by centrifugation over a Ficoll-Ditrizoate gradient. Cells were washed in IMDM and counted.
- IMDM Iscove's Modified Dulbecco's Medium Supplied by Life Technolgies
- the cells were suspended at a concentration of 5 x IO 4 cells/ml in a mixture containing 0.66ml batch-tested fetal calf serum, 0.033ml batch-tested bovine serum albumin and IMDM (with added penicillin/streptomycin and glutamine) to a total volume of 2.97 ml; 0.33 ml agar was added at boiling point to the cell suspension.
- Growth factors were included (as single factors or a combination) at the following concentrations: human granulocyte colony stimulating factor, 20ng/ml; feline stem cell factor (fSCF) , lOOng/ml; feline phytohaemagglutinin spleen cell conditioned medium, 5%.
- Control cultures were set up omitting any growth factors.
- the resultant mix was then transferred in 1 ml aliquots to three 35 x 10 mm petri dishes, allowed to set and incubated in a humidified incubator at 37°C, 5% C02. Colonies (over 50 cells) and clusters (5-50 cells) were counted on day 8 under a Leitz Labovert inverted microscope.
- Feline stem cell factor has a synergistic effect on granulocyte-macrophage colony formation in soft agar in combination with human granulocyte colony stimulating factor or feline phytohaemagglutin spleen cell conditioned medium. Feline stem cell factor additionally promotes cluster formation alone; this activity is enhanced by the addition of either human granulocyte colony stimulating factor or feline phytohaemagglutinin spleen cell conditioned medium.
- Canine stem cell factor supports the survival of hematopoietic progenitors in long term canine marrow culture. Exp. He atol. 20, 1118-1124. McNiece IK and SHeih J-H. (1994). Stem Cell Factor. ppl77-180 in Guide Book to Cytokines and Their Receptors.
Abstract
The present invention provides a polynucleotide fragment encoding feline stem cell factor, a recombinant vector comprising such a polynucleotide fragment, a host cell containing said polynucleotide fragment, a recombimant vector, a recombinant feline stem cell factor polypeptide, and pharmaceutical compositions comprising recombinant feline stem cell factor for use as a prophylactic and/or therapeutic agent.
Description
Feline Stem Cell Factor
Field of Invention
The present invention relates to feline stem cell factor, a nucleic acid sequence encoding feline stem cell factor, a recombinant vector comprising such a nucleic acid sequence, a host cell containing said recombinant vector, a feline stem cell factor polypeptide, antibodies immuno- reactive with said polypeptide and pharmaceutical compositions comprising recombinant feline stem cell factor for use as a prophylactic and/or therapeutic agent and also as an adjuvant in cats.
Background of the Invention
Stem cell factor (SCF) is a pleiotropic (multiple effect) cytokine involved in the regulation of haemopoietic reproductive germ cells and pigment cells. SCF has a direct action on myeloid and lymphoid cell development. In addition, SCF acts in synergy with other growth factors such as interleukin 1, 3, 6, 7 , 11 and/or erythropoietin, to stimulate the production of myeloid, lymphoid, megakaryocytic or erythroid cells. (McNiece and Shieh, 1994) .
Cytokine and anti-cytokine therapies such as the synthesis of inhibitors, soluble cytokine receptors, receptor antagonists or anti-cytokine antibodies are finding increasing application in human medicine (Mire- Sluis, 1993) . However, due to a lack of species cross-
reactivity of many cytokines and the potential for antibody production against heterologous cytokines (Homes, 1993), there is a need for substantially feline-specific reagents.
Summary of the Invention
The present invention provides a poiynucleotide fragment, such as a DNA fragment, encoding feline stem cell factor. The invention further provides a recombinant feline stem cell factor polypeptide.
"Poiynucleotide fragment" as used herein refers to a chain of nucleotides of any length, such as deoxyribonucleic acid (DNA) sequences and transcription products thereof, (such as RNA) capable of giving rise to feline stem cell factor, or functionally active desivative thereof. The term excludes the whole naturally occurring genome. Thus, this term includes double and single stranded DNA, and RNA sequences derived therefrom.
Generally the poiynucleotide will be in isolated form, substantially free of biological material with which the whole genome is normally associated in vivo.
In general, the term "polypeptide" refers to a chain or sequence of amino acids displaying a biological activity substantially similar to the biological activity of feline stem cell factor, and does not refer to a specific length of the product as such. The polypeptide required can be modified in vivo and/or in vitro. for example by glycosylation, amidation, carboxylation, phosphorylation and/or post-translation cleavage; thus inter alia peptides.
oligopeptides proteins and functionally active derivatives thereof are encompassed thereby.
The poiynucleotide fragment encoding feline stem cell factor can be amplified from feline stem cell factor cDNA obtained by way of reverse transcription of mRNA, which can be recovered from feline embryo cells infected with feline leukaemia virus type A, using polymerase chain reaction (PCR) techniques known in the art. PCR amplification of cDNA can be accomplished using primers designed with respect to conserved regions of stem cell factor coding sequences from a single species or a number of other species. An amplified fragment containing the feline stem cell factor is exemplified in Figure 1.
The DNA fragment of Figure l is shown to encode an open reading frame (ORF) of 825 nucleotides (nucleotides 65-889 inclusive) , corresponding to a protein of 274 amino acids. The fragment shown in Figure l shows the longer isoform of feline stem cell factor. A predicted shorter mature isoform can have a deletion of 84 nucleotides, (nucleotides 588 to 671 inclusive, shown underlined) based on information derived from other species and disclosed by McNiece and Shieh (1994) .
The full length feline stem cell factor protein (249 amino acids) is a transmembrane protein cleavable in vivo between residues Ala 164/Ala 165 and/or Ala 165/Ser 166, of the mature protein, resulting in the release of soluble feline stem cell factor (McNiece and Shieh, 1994) .
Comparison of the full-length feline stem cell factor
cDNA to published sequences in other species shows, similarity, for example identities to canine (Shull et al, 1992), bovine (Zhou et al, 1994), porcine (Zhang and Anthony, 1994) human (Martin et al, 1990), murine (Anderson et al. 1990) and chicken (Zhou et al. 1993) stem cell factor of 95%, 93%, 95%, 92%, 87% and 71% respectively. The conceptual amino acid sequence of the feline stem cell factor protein shows an amino acid identity with canine, porcine bovine, human, murine and chicken homologues of 92%, 92%, 91%, 88%, 80% and 53% respectively (using the GCG DNA computer analysis package, and the programs Translate, Compare and Best Fit) .
The conceptual polypeptide sequence of the longer isoform of feline stem cell factor is shown in Figure 2. The first 25 amino acids encode a predicted signal peptide which is cleaved to produce the mature protein; hence the N-terminus of the mature feline stem cell factor of Figure 2 begins KGLCR.
The present invention includes polypeptide fragments having at least 80%, particularly at least 90% and especially at least 95% similarity with the fragment exemplified in Figure l. The present invention includes polypeptide sequences having at least 80%, particularly at least 90% and especially at least 95% similarity with the polypeptide exemplified in Figure 2. "Similarity" refers to both identical and conservative replacement of nucleotides or amino acids, provided that the functionality of feline stem cell factor DNA and/or polypeptide is
substantially unimpaired.
The skilled man will appreciate that it is possible to genetically manipulate the gene or derivatives thereof, for example to clone the gene by recombinant DNA techniques generally known in the art and to express the polypeptide encoded thereby in vitro or in vivo. DNA fragments having the nucleotide sequence depicted in Figure 1 or derivatives thereof are preferably used for the expression of feline stem cell factor.
It will be understood that for the particular feline stem cell factor polypeptide embraced herein, natural variations can exist between individuals or between species within the felis genus. These variations may be demonstrated by (an) amino acid difference(s) in the overall sequence or by deletions, substitutions, insertions, inversions or additions of (an) amino acid(s) in said sequence. All such derivatives are included within the scope of this invention provided the derivatives produce active feline stem cell factor. For example, for the purpose of the present invention conservative replacements may be made between amino acids, within the following groups:
(i) Alanine, serine and threonine (ii) Glutamic acid and aspartic acid (iii) Arginine and Lysine; (iv) Asparagine and Glutamine: (v) Isoleucine, leucine, and valine; (vi) Phenylalanine, Tyrosine and Tryptophan.
Moreover, recombinant DNA technology may be used to prepare nucleic acid sequences encoding these various derivatives outlined above.
As is well known in the art, the degeneracy of the genetic code permits substitution of bases in a codon resulting in a different codon capable of coding for the same amino acid, e.g. the codon for the amino acid glutamic acid is both GAT and GAA. Consequently, it is clear that for the expression of a polypeptide with the amino acid sequence shown in Figure 2 or fragment thereof, use can be made of a derivative nucleic acid sequence with such an alternative codon composition different from the nucleic acid sequence shown in said Figure 1.
Furthermore, functionally active derivatives derived from the feline stem cell factor polypeptide or from the amino acid sequence depicted in Figure 2 which still display feline stem cell factor properties, or derivatives from the nucleotide sequence encoding the feline stem cell factor polypeptide or derived from the nucleotide sequence depicted in Figure l encoding fragments of said feline stem cell factor polypeptides are also included in the present invention.
All such modifications mentioned above resulting in such derivatives of the feline stem cell factor polypeptide or gene are covered by the present invention so long as the characteristic feline stem cell factor biological properties remain substantially unaffected.
The feline stem cell factor poiynucleotide fragment of the present invention is preferably linked to regulatory control sequences. Such control sequences may comprise promoters, operators, inducers, ribosome binding sites, terminators etc. Suitable control sequences for a given host may be selected by those of ordinary skill in the art. In particular, a feline stem cell factor control sequence may be employed in a mammalian host.
A poiynucleotide fragment according to the present invention can be ligated to various expression controlling DNA sequences, resulting in a so called recombinant DNA molecule. Thus the present invention also includes an expression vector containing the expressible nucleic acid molecule. Said recombinant nucleic acid molecule can then be used for transformation of a suitable host. Such expression vectors are preferably hybrid DNA molecules derived from, for example plasmids, or from nucleic acid sequences derived from bacteriophages or viruses and are termed vector molecules.
Specific vectors which can be used to clone nucleic acid sequences according to the invention are known in the art (e.g. Rodriguez, R.L. and D.T. Denhardt, edit.. Vectors: A survey of molecular cloning vectors and their uses, Butterworths, 1988) .
A specific bacterial expression vector pGEX-4T-l (Pharmacia Limited.) has been used for feline stem cell factor production. The pGEX-4T-l vector results in production of a fusion protein with glutathione S-
transferase (GST) , being expressed. The use of GST or other small N-terminally fused proteins facilitates purification by affinity chromatography and the mature feline stem cell factor may then be cleaved from GST using thrombin, thereby releasing an 18kD polypeptide corresponding to the soluble form of feline stem cell factor.
The glutathione S-transferase (GST) gene fusion system allows expression, purification and, if required, detection of fusion proteins produced in E. coli . The pGEX plasmid vectors are designed for expression of a fusion protein with Schistosoma japonicum GST. The resulting fusion proteins are purified from bacterial lysates by affinity chromatography using Glutathione Sepharose 4B. The polypeptide of interest can be cleaved from the GST moiety using a site specific protease (thrombin) with a cleavage site upstream of the cloning site on the plasmid. On cleavage the GST molecule remains bound to the Sepharose matrix and the polypeptide of interest can be purified from the GST. (Pharmacia Biotech, GST Gene Fusion System, Second Edition of Manual) .
The methods used in the construction of a recombinant nucleic acid molecule according to the invention are known to the skilled addressee and are inter alia set forth in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1989) .
The present invention also relates to a transformed cell containing the nucleic acid molecule in expressible
form. "Transformation", as used herein, refers to the introduction of a heterologous nucleic acid sequence into a host cell, irrespective of the method used, for example direct uptake, transfection or transduction. The heterologous poiynucleotide fragment may be maintained through autonomous replication or alternatively, may be integrated into the host genome. The recombinant nucleic acid molecules preferably are provided with appropriate control sequences compatible with the designated host which can regulate the expression of the inserted poiynucleotide fragment e.g. tetracylcine responsive promoter, thymidine kinase promoter and SV-40 promoter.
Suitable hosts used for the expression of recombinant nucleic acid molecules can be prokaryotic or eukaryotic in origin. The most widely used hosts for expression of recombinant nucleic acid molecules may be selected from bacteria, yeast, insect cells, and mammalian cells.
The cloning and expression of recombinant feline stem cell factor also facilitates in producing reagents for the production of, for example, probes for in situ expression studies, production of anti-feline stem cell factor antibodies (particularly monoclonal antibodies) and evaluation of in vitro and in vivo biological activity of recombinant feline stem cell factor. The antibodies may be employed in diagnostic tests for feline stem cell factor.
The present invention further provides recombinant feline stem cell factor for the manufacture of reagents for use as prophylactic and/or therapeutic agents and also as
-lo¬ an adjuvant in cats. In particular the invention provides pharmaceutical compositions comprising the recombinant feline stem cell factor together with a pharmaceutically acceptable carrier therefor.
A wide range of feline disorders, such as disease states attribute to feline stem cell factor deficiencies and/or over abundance and physiological or immunological abnormalities, potentially benefit from feline stem cell factor therapy and/or prophylactic treatment. Such disorders include cancer, endotoxaemia, parasitic and bacterial infections, wound therapy, autoimmune and inflammatory diseases and allergies. In particular, it is believed that recombinant feline stem cell factor may be used in the treatment of anaemia, leukopaenia, immunosuppression associated with retroviral or non- retroviral agents, chronic infections of bacterial, viral or parasitic origin, and hypo-pigmentation and other skin disorders and for the manipulation of fertility. Feline stem cell factor may be used alone, or in combination with other cytokines and drugs, including erythropoietin, interleukins such as IL3, IL4, IL6 and IL12, granulocyte/ macrophage colony-stimulating factor (GM-CSF) and/or granulocyte colony-stimulating factor (G-CSF) .
A feline stem cell factor of the present invention may find further utility as a research and/or diagnostic tool.
Detailed Description of the Invention
An embodiment of the invention will now be described
by way of example only.
Example 1
Molecular cloning of the feline stem cell factor mRNA was isolated from feline leukaemia virus infected feline embryo cells using the RNAgents (Trademark) Total RNA isolation system (supplied by Promega Corp.) 5μg of the mRNA was converted to cDNA using the Superscript (Trademark) system (supplied by Life Technologies) .
Two oligonucleotides (see below) were designed against interspecies conserved non-coding regions of stem cell factor mRNA.
Upstream primer (I):- 5'- CCAGAACAGCTAAACGGAGT-3' Downstream primer (II):- 5'- ATGAAGCAAACATGAACTGT-3'
These oligonucleotides were then used as primers in a polymerase chain reaction (PCR) experiment, to amplify the feline stem cell factor cDNA.
The PCR was performed essentially as described by Saiki et al (1987) . 10 μl of lOOng/μl template cDNA from the reverse transcribed mRNA was added to a 40μl reaction mixture containing 200μM of dATP, dCTP, dGTP, dTTP, 50pmol of both primers (I) and (II) , 1 unit of Taq DNA Polymerase and 5μl of lOx reaction buffer. The reaction buffer contained lOOmM Tris-HCI, 500mM potassium chloride, 0.01 per cent (w/v) gelatin and 1.5mM magnesium chloride, ultrapure water, TE (pHδ.O). The solution was overlaid
with two drops of mineral oil to prevent evaporation. To eliminate the possibility of false positives from the contamination of genomic samples with preparation of cDNA, amplification and analysis of the products were carried out. Thirty five cycles of amplification were performed using a Perkin Elmer Cetus thermal cycler. Each cycle consisted of 1 min. at 95°C to denature the DNA, 1 min. at 50°C to anneal the primers to the template and l min. at 72°C for primer extension. After the last cycle a further incubation for lOmins. at 72°C was performed to allow extension of any partially completed product. On completion of the amplification, lOμl of the reaction mixture was electrophoresed through a 1.5 per cent agarose gel. The DNA was visualised by staining with ethidium bromide and exposure to ultraviolet light (320nm) .
An amplified reaction product of approximately 950 bp was observed and the DNA purified and cloned into the phagemid vector Blue-Script (Trademark) sk (+) (supplied by Stratagene) .
Example 2
Seguencing of the cloned feline stem cell factor cDNA
The Blue-Script (Trademark) derived vector, containing the cloned feline stem cell factor DNA was prepared and purified. Double stranded DNA sequencing was carried out on this DNA using the T7 polymerase, "Sequenase"
(Trademark) kit (supplied by USB Corp.) as per the
manufacturers instructions.
The DNA sequence obtained is shown in Figure 1.
A translation of the DNA sequence into its corresponding amino acid sequence (one-letter code) is shown in Figure 2.
Example 3
Expression and purification of recombinant feline stem cell factor in Escherichia coli
The open reading frame (ORF) encoding the feline stem cell factor was excised from the Blue-script (TRADE MARK) derived vector and sub-cloned into the plasmid vector pGEX- 4T-1 (Pharmacia Ltd) .
Two ml cultures of E. coli transformed with various expression constructions were grown with shaking at 37°C to late log phase (O . D . f^^ of 0.7) and induced by the addition of IPTG to 0.1 mM. Induced cultures were then incubated for a further one hour after which the bacteria were collected by centrifugation. The bacterial pellet was lysed by boiling in SDS-PAGE sample buffer and the protein profile of the induced bacteria was analysed on a 12% acrylamide gel (Laemmli, 1970) followed by staining with coomasie brilliant blue dye.
An overexpressed band corresponding to overexpressed feline stem cell factor fusion protein was observed.
A clone which was shown to overexpress feline stem cell factor fusion protein by gel electrophoresis, was used
to prepare sufficient quantities of purified feline stem cell factor.
A liquid culture of the chosen clone, was grown as described above and the bacteria were collected by centrifugation and resuspended in lx phosphate buffered saline (PBS) and lysed by sonication. The insoluble cell debris was removed by centrifugation and the supernatant was mixed with a 50% (w/v) slurry of Glutathione Sepharose 4B. The resulting mixture was incubated at 20°C for five minutes and then the Sepharose beads were pelleted by brief centrifugation. The supernatant was removed and the beads were resuspended in PBS and again pelleted, this washing step was repeated twice. For thrombin cleavage the Sepharose bead pellet with bound fusion protein were mixed with a thrombin solution at 10 cleavage units per mg of protein in PBS. The mixture was incubated with gentle mixing for 16h at 20°C the beads were pelleted from the mixture by centrifugation and the supernatant containing the cleaved recombinant protein was stored for assay.
Example 4
Cell proliferation assay for feline stem cell factor activity
Demonstration of feline stem cell factor bioactivity.
A. Cell Proliferation Assays
The murine mast cell line MC/9 and human crythroleukaemia cell line TF-1 show cell proliferation in
response to stem cell factor. Both cell lines have been used to assay the biological activity of feline stem cell factor as assessed by the incorporation of H3 - thymidine (according to methodology essentially as described in Current Protocols in Immunology (1992) . Measurement of human and murine stem cell factor (c-kit ligands) Unit 6.17.1. John Wiley and Sons, Inc. USA). TF-1 cells show greater sensitivity to feline stem cell factor with an ED50 of approximately 5ng/ml cf. 40ng/ml for MC/9 cells; in addition these cells are easier to maintain in culture and have less tendency to become factor independent. Thus TF-1 cells are the preferred cells for assaying feline stem cell factor bioactivity.
TF-1 cells were maintained in RPMI-1640 medium supplemented with 5% FCS, glutamine, penicillin/streptomycin and 2ng/ml human granulocyte- acrophage colony stimulating factor. Cells were taken 2-3 days after passaging and washed three times in RPMI-1640 medium. The washed cells were counted, cell viability assessed by trypan blue exclusion and cells were resuspended to a final concentration of l x io5 cells/ml in RPMI-1640 medium containing 5% FCS, glutamine and penicillin/streptomycin. Recombinant feline stem cell factor was serially diluted in lOOμl volume in triplicate in 96 well microtitre plates. Recombinant murine stem cell factor was used as a positive control.
Demonstration of the specificity of feline stem cell factor activity was achieved by pre-incubating feline stem
cell factor with anti-human stem cell factor neutralising antibody at a concentration of 40μg/ml for 1 hour. To each well of the plate was added lOOμl of washed cells, the plates were then incubated at 37°C in a humidified C02 incubator for 48 hours. Thymidine - H3 (0.5μCi) was added to each well and the plates incubated for 4 hours at 37°C. Cells were harvested using a cell harvester onto filters and the incorporated radioactivity was measured by liquid scintillation counting.
Results of a typical experiment are shown in Figure 3. It can be seen that feline stem cell factor induces proliferation of TF-l cells at concentrations over approximately 2-4 ng/ml and tht its activity can be neutralised by anti-human stem cell factor neutralising antibody.
MC/9 cells were maintained in MC/9-medium. Before assay the cells were washed and resuspended in growth medium without concanavalin-A activated supernatant. The recombinant purified feline stem cell factor was diluted in medium in microtiter plates and 5 x IO3 cells were added to each well. The thymidine incorporation was then measured by scintillation counting.
Figure 4 shows the biological activity of recombinant feline stem cell factor as determined by MC/9 cell proliferation measured by 3H Thymidine incorporation. As can be seen thymidine incorporation as a result of feline stem cell factor was significantly above background levels.
especially at concentrations greater than about 40 mg feline stem cell factor per ml.
B. Colony Forming Assays
In order to assess the ability of feline stem cell factor alone and in combination with other growth factors (human granulocyte colony stimulating factor (G-CSF) or feline phytohaemagglutinin spleen cell conditioned medium (PHA-CM) ) to support growth of feline bone marrow cells in vitro , granulocyte-macrophage-colony forming cell (GM-CFC) assays were performed.
Bone marrow was collected from the femur or humerus of SPF (Specified Pathogen Free) cats immediately post mortem into 25ml IMDM (Iscove's Modified Dulbecco's Medium Supplied by Life Technolgies) containing 100 iu/ml heparin. Low density bone marrow mononuciear cells were isolated by centrifugation over a Ficoll-Ditrizoate gradient. Cells were washed in IMDM and counted. The cells were suspended at a concentration of 5 x IO4 cells/ml in a mixture containing 0.66ml batch-tested fetal calf serum, 0.033ml batch-tested bovine serum albumin and IMDM (with added penicillin/streptomycin and glutamine) to a total volume of 2.97 ml; 0.33 ml agar was added at boiling point to the cell suspension. Growth factors were included (as single factors or a combination) at the following concentrations: human granulocyte colony stimulating factor, 20ng/ml; feline stem cell factor (fSCF) , lOOng/ml; feline phytohaemagglutinin spleen cell conditioned medium, 5%.
Control cultures were set up omitting any growth factors. The resultant mix was then transferred in 1 ml aliquots to three 35 x 10 mm petri dishes, allowed to set and incubated in a humidified incubator at 37°C, 5% C02. Colonies (over 50 cells) and clusters (5-50 cells) were counted on day 8 under a Leitz Labovert inverted microscope.
Results of a typical experiment are shown in Table l below. Feline stem cell factor has a synergistic effect on granulocyte-macrophage colony formation in soft agar in combination with human granulocyte colony stimulating factor or feline phytohaemagglutin spleen cell conditioned medium. Feline stem cell factor additionally promotes cluster formation alone; this activity is enhanced by the addition of either human granulocyte colony stimulating factor or feline phytohaemagglutinin spleen cell conditioned medium.
Table 1
Mean/SD per 100,00 Cells
Growth Factor Colonies Clusters
None 0 18.7/4.6
SCF 0 90.7/4.6
G-CSF 2.7/4.6 56.0/16.0
PHA-CM 0 77.3/30.3
SCF + G-CSF 21.3/9.2 234.7/48.2
SCF + PHA-CM 24.0/8.0 200/48.7
SCF + PHA-CM+GCSF 16.0/8.0 194.7/44.1
REFERENCES
Anderson, D.M. , Lyman, S.D., Baird, A., Wignall J.M. , Eisenman, J., Rauch, C, March, C.J., Boswell, H.S., Gimpel, S.D., Cos an, D. , and Williams, D.E. (1990). Molecular Cloning of Mast Cell Growth Factor, A Hematopoietin That is Active in Both Membrane Bound and Soluble Forms. Cell 63, 235-243. Homes MA. (1993) . An important milestone in feline immunology: feline interleukin 2. Feline Vet.J.. 25, 180. Laemmli UK. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227, 680-685. Martin, F.H. , Suggs, S.V. , Langley, K.E., Lu, H.S. , Ting, J. , Okino, K.H., Morris, C.F., McNiece, I.K., Jacobsen, F.W., Mendiaz, E.A. , and et. al, (1990). Primary structure and functional expression of rat and human stem cell factor DNAs. Cell 63, 203-211. Mire-Sluis A. (1983) . Cytokines and disease. Trends in Biotech.. 11, 74-77. Sambrook, J, Fritsch EF, Mamiatis T. (1989) . Molecular Cloning, 2nd edition. Cold Spring Harbour Press. Shull, R.M. , Suggs, S.V. , Langley, K.E., Okino, K.H. , Jacobsen, F.W. and Martin, F.H. (1992) . Canine stem cell factor (c-kit ligand) supports the survival of hematopoietic progenitors in long term canine marrow culture. Exp. He atol. 20, 1118-1124.
McNiece IK and SHeih J-H. (1994). Stem Cell Factor. ppl77-180 in Guide Book to Cytokines and Their Receptors. Ed NA Nicola. Oxford University Press Inc. New York. Zhang, Z. and Anothony, R.V. (1994). Porcine stem cell factor/c-kit ligand: its molecular cloning and localization within the uterus. Biol. Reprod. SO, 95- 102. Zhou, J.H., Ohtaki, M. , and Sakurai, M. (1993). Sequence of a cDNA encoding chicken stem cell factor. Gene 127, 269-270. Zhou, J.H. , Hikono, H. , Ohtaki, M. , Kubota, T.f and Sakurai, M. (1994). Cloning and characterization of cDNAs encoding two normal isoforms of bovine stem cell factor. Biochim. Biophys. Acta 1223, 148-150.
Claims
1. A poiynucleotide fragment encoding feline stem cell factor.
2. A poiynucleotide fragment according to claim 1 wherein the poiynucleotide fragment is a deoxyribose nucleic acid (DNA) fragment.
3. A poiynucleotide fragment according to either of claims 1 or 2 characterised in that said poiynucleotide fragment encodes a polypeptide having an amino acid sequence shown in Figure 2 or a functionally active derivative thereof.
4. A poiynucleotide fragment according to claim 1 characterised in that it is a poiynucleotide fragment which is substantially the same as the poiynucleotide fragment shown in Figure 1 or a functionally active derivative thereof.
5. A recombinant nucleic acid molecule comprising a poiynucleotide fragment according to any one of claims 1 to
4.
6. A recombinant nucleic acid molecule according to claim 5 characterised in that the recombinant nucleic acid molecule comprises regulatory control sequences operably linked to said poiynucleotide fragment for controlling expression of said poiynucleotide fragment.
7. A recombinant nucleic acid molecule according to either of claims 5 or 6 wherein the recombinant nucleic acid molecule is a plasmid.
8. A recombinant nucleic acid molecule according to either of claims 5 or 6 wherein the recombinant nucleic acid molecule is derived from a viral vector.
9. A prokaryotic or eukaryotic host cell transformed by a poiynucleotide fragment or recombinant molecule according to any preceding claim.
10. A recombinant feline stem cell factor polypeptide or functionally active derivative thereof displaying feline stem cell factor activity.
11. A recombinant feline stem cell factor polypeptide as shown in Figure 2 or functionally active derivative thereof.
12. An antibody immuno-reactive with a polypeptide or fragment according to claim 10 or 11.
13. A poiynucleotide fragment according to any one of claims 1- 4 for use in therapy.
14. A recombinant nucleic acid molecule according to any one of claims 5 to 8 for use in therapy.
15. A recombinant polypeptide or functionally active derivative thereof according to either of claims 10 or 11 for use in therapy.
16. A pharmaceutical composition comprising a poiynucleotide fragment according to any of claims 1 to 4.
17. A pharmaceutical composition comprising a polypeptide or derivative thereof according to claims 10 or 11.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU67457/96A AU6745796A (en) | 1995-08-02 | 1996-08-02 | Feline stem cell factor |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GBGB9515839.0A GB9515839D0 (en) | 1995-08-02 | 1995-08-02 | Feline stem cell factor |
| GB9515839.0 | 1995-08-02 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| WO1997005251A2 true WO1997005251A2 (en) | 1997-02-13 |
| WO1997005251A3 WO1997005251A3 (en) | 1997-04-17 |
Family
ID=10778647
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/GB1996/001904 WO1997005251A2 (en) | 1995-08-02 | 1996-08-02 | Feline stem cell factor |
Country Status (3)
| Country | Link |
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| AU (1) | AU6745796A (en) |
| GB (1) | GB9515839D0 (en) |
| WO (1) | WO1997005251A2 (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1991005795A1 (en) * | 1989-10-16 | 1991-05-02 | Amgen Inc. | Stem cell factor |
| WO1992000376A1 (en) * | 1990-06-25 | 1992-01-09 | Immunex Corporation | Mast cell growth factor |
| WO1992003459A1 (en) * | 1990-08-27 | 1992-03-05 | Sloan-Kettering Institute For Cancer Research | LIGAND FOR THE c-KIT RECEPTOR AND METHODS OF USE THEREOF |
| WO1994009803A1 (en) * | 1992-10-27 | 1994-05-11 | Biotransplant, Inc. | Enhancement of xenograft tolerance and porcine cytokines therefor |
| WO1995017206A1 (en) * | 1993-12-22 | 1995-06-29 | Amgen Inc. | Lyophilized stem cell factor formulations |
-
1995
- 1995-08-02 GB GBGB9515839.0A patent/GB9515839D0/en active Pending
-
1996
- 1996-08-02 AU AU67457/96A patent/AU6745796A/en not_active Abandoned
- 1996-08-02 WO PCT/GB1996/001904 patent/WO1997005251A2/en active Application Filing
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1991005795A1 (en) * | 1989-10-16 | 1991-05-02 | Amgen Inc. | Stem cell factor |
| WO1992000376A1 (en) * | 1990-06-25 | 1992-01-09 | Immunex Corporation | Mast cell growth factor |
| WO1992003459A1 (en) * | 1990-08-27 | 1992-03-05 | Sloan-Kettering Institute For Cancer Research | LIGAND FOR THE c-KIT RECEPTOR AND METHODS OF USE THEREOF |
| WO1994009803A1 (en) * | 1992-10-27 | 1994-05-11 | Biotransplant, Inc. | Enhancement of xenograft tolerance and porcine cytokines therefor |
| WO1995017206A1 (en) * | 1993-12-22 | 1995-06-29 | Amgen Inc. | Lyophilized stem cell factor formulations |
Non-Patent Citations (1)
| Title |
|---|
| DNA SEQUENCE, vol. 6, no. 4, 1996, pages 233-237, XP000647329 DUNHAM, S.P., ET AL . : "THE CLONING AND SEQUENCING OF cDNAs ENCODING TWO ISOFORMS OF FELINE STEM CELL FACTOR" * |
Also Published As
| Publication number | Publication date |
|---|---|
| AU6745796A (en) | 1997-02-26 |
| WO1997005251A3 (en) | 1997-04-17 |
| GB9515839D0 (en) | 1995-10-04 |
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