WO1991000100A2 - Toxin uses - Google Patents
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- WO1991000100A2 WO1991000100A2 PCT/GB1990/000992 GB9000992W WO9100100A2 WO 1991000100 A2 WO1991000100 A2 WO 1991000100A2 GB 9000992 W GB9000992 W GB 9000992W WO 9100100 A2 WO9100100 A2 WO 9100100A2
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- toxin
- cells
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- multocida
- fragment
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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
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/285—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pasteurellaceae (F), e.g. Haemophilus influenza
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/6415—Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
- A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
- A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
- A61K47/6817—Toxins
- A61K47/6829—Bacterial toxins, e.g. diphteria toxins or Pseudomonas exotoxin A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
Definitions
- This invention relates to novel uses for the Pasteurella multocida toxin.
- P multocida toxin has been identified as a causative agent of atrophic rhinitis, a disease of growing pigs which results in twisting of the snout and atrophy or loss of the nasal turbinate bones.
- Intraperitoneal injection of crude preparations of the toxin has been shown to produce turbinate atrophy (Rutter & Mackenzie, 1984).
- Nasal lesions produced by the toxin included atrophy of mucosal glands, epithelial hyperplasia, osteolysis and proliferation of mesenchymal cells. Other degenerative, obstructive and hyperplastic lesions were observed in the liver, ureter and bladder.
- the toxin has since been purified and it has been demonstrated (Chanter et al , 1986b) that the purified toxin could reproduce all the effects noted above. (For a review see Chanter and Rutter, 1989). In 1987, the Institute for Animal Health reported the cloning of the toxin gene (J M Rutter, in "Virulence Mechanisms of Bacterial Pathogens” Ed J A Roth, p 234). WO 89/09617 (published October 1989) discloses the use of the toxin and analogues thereof, produced by recombinant techniques, in vaccines against atrophic rhinitis.
- the toxin causes marked proliferation of cells in culture.
- the mode of action was not known (Chanter et al 1986b) and, in view of the complex situation in vivo, the proliferative effects might have been secondary effects or even a repair response to damaged tissue.
- the toxin is cytotoxic for embryonic bovine lung cells in culture (Rutter and Luther, 1984) and all other in vitro studies had also shown only cytotoxic effects of toxin.
- recent papers (Elling et al 1988, Cheville et al 1988) have been entirely in the context of the toxin being necrotic and have noted the similarity between the P multocida toxin and other necrotic bacterial toxins.
- One aspect of the invention provides a non-immunogenic pharmaceutically acceptable composition comprising the P mul tocida toxin or a fragment or variant thereof and one or more carriers or diluents.
- the composition is suitable for topical administration to the skin or the cornea.
- non-immunogenic we mean that the compositions are not deliberately immunogenic, in other words no component is included in the composition in order to provoke or to help provoke an immune response to the composition.
- the compositions are not vaccines and are not intended to be used for raising immune sera.
- Compositions of the invention may, nevertheless, have an incidental degree of immunogenicity if this does not compromise their effective use.
- P multocida toxin we mean the toxin produced by a toxigenic strain of P multocida such as strain LFB3. To avoid any possible ambiguity, we have deposited our strain of P mul tocida with the NCIMB, Aberdeen under the terms of the Budapest Treaty (Accession No NCIMB 40158, deposited 26th June 1989). A toxigenic strain (45/78) is also available from the National Collection of Type Cultures, London, as NCTC 12178.
- the toxin produced by P mul tocida (in the following occasionally abbreviated to PMT) which, as noted above, is generally believed to be the causative agent of porcine atrophic rhinitis, has in the prior literature been variously termed "dermonecrotic toxin", “osteolytic toxin”, “turbinate atrophy toxin” and “heat labile exotoxin”, but it would appear to be the same toxin as the amino acid composition, iso-electric point and biological activities of the variously termed toxins show basic similarities, although minor variations in the properties of toxins isolated from different strains of P multocida appear to exist.
- the estimated amino acid composition of PMT (as deduced from the DNA sequence) is as follows:
- Ala is found 76 times - 5.91%
- Trp is found 18 times - 1.40%
- the total number of amino acid residues is 1285, and the full-length toxin has a molecular weight of 146.5 kd.
- the toxin gene will preferably have the DNA sequence of Figure 6 below or a DNA sequence which encodes the same amino acid sequence and the toxin molecule will preferably have a corresponding amino acid sequence.
- gene we mean the nucleotide sequence together with its regulatory sequences.
- variants is intended to include (but not necessarily to be restricted to) minor variations in amino acid residues (such as molecules lacking one or a few residues, having conservative substitutions or minor insertions of residues, or having minor variations of amino acid structure) which do not reduce to less than 10% (preferably no less than 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%) the ability of the compound to induce proliferation of 3T3 cells, or DNA synthesis in 3T3 cells, or inhibit cAMP production in 3T3 cells in the assays described below or to potentiate effects on the proliferation or DNA synthesis or cAMP production of another agent, for example a growth factor (eg EGF).
- variants may have 80%, preferably 85%, 90%, 95% or 99% homology with the P multocida toxin amino acid sequence depicted in Figure 6.
- Conservative substitutions are those where one or more amino acids are substituted for others having similar properties such that one skilled in the art of polypeptide chemistry would expect at least the secondary structure, and preferably the tertiary structure, of the polypeptide to be substantially unchanged.
- typical such substitutions include alanine or valine for glycine, arginine or asparagine for glutamine, serine for asparagine and histidine for lysine.
- Variants may alternatively, or as well, lack up to ten (preferably only one or two) amino acid residues in comparison with P multocida toxin; preferably any such omissions occur at the carboxy terminal of the polypeptide.
- up to ten, but preferably only one or two, amino acids may be added, again at the carboxy terminal for preference.
- “Fragments” of the toxin are those which display useful cell proliferative properties as defined above in relation to variants of the toxin and which have (or which comprise regions having) at least 50% homology, preferably 60%, 70%, 80%, 90%, 95% or 99% homology, with the region of the toxin sequence to which they are most similar.
- the fragments preferably have a molecular weight of at least 10000.
- Useful fragments may be selected by reference to the characteristics detailed below. Particular fragments include those of approximate molecular weight 23000, 64000, 74000, 87000 and 138000, obtainable by the methods of Nakai and Kume (Nakai & Kume 1987a, 1987b and Kume & Nakai 1985).
- toxin is used in this specification to include fragments and variants thereof.
- the toxin used in the compositions and methods of the invention may be isolated and purified by known techniques, using a toxigenic strain of P multocida .
- the Rutter & Mackenzie (1984), Chanter et al (1986b) and Chanter & Rutter (1989) articles are all incorporated herein by reference for this purpose.
- the toxin may be prepared by recombinant DNA techniques, as are known in the art and as are explained in more detail below and in WO 89/09617 which is incorporated herein by reference.
- Variants and fragments may be made by the expression of mutant genes, obtained by site-directed mutagenesis in known ways.
- Fragments may be obtained by proteolytic (eg enzymatic) degradation of recombinant-produced or naturally occurring material (as is disclosed by the Nakai and Kume references) or by peptide synthesis, using the general method of Marglin and Merrifield (Ann. Rev. Biochem., 39 , 841-866, 1970) or by the Fmoc-polyamide method of Atherton, Sheppard and their co-workers and by subsequent refinements of these approaches.
- proteolytic eg enzymatic degradation of recombinant-produced or naturally occurring material (as is disclosed by the Nakai and Kume references) or by peptide synthesis, using the general method of Marglin and Merrifield (Ann. Rev. Biochem., 39 , 841-866, 1970) or by the Fmoc-polyamide method of Atherton, Sheppard and their co-workers and by subsequent refinements of these approaches.
- the nucleotide sequence may be derived from a P multocida genome by screening for genomic sequences hybridizing to a DNA probe prepared on the basis of the full or partial amino acid sequence of the toxin in accordance with established procedures or by establishing a toxin gene library and screening for toxin-producing clones by means of a toxin-specific antibody (for a more detailed description of this procedure, see WO 89/09617, Example 4) or using an overlay technique as is explained in more detail below.
- the gene may be prepared more directly by isolating a 15kb HpaII fragment as is described in Lax & Chanter (1990) J Gen Microbiol 136, 81-87, which is incorporated herein by reference.
- the nucleotide sequence may also be derived from a bacteriophage infectious for P mul tocida , ie one which has been transferred from one bacterial strain which originally carried the sequence to another strain which did not originally carry the sequence by bacteriophage transfection.
- the nucleotide sequence may be derived from a plasmid or other genetic element transferred from one strain to another by conjugation, transformation or the like.
- nucleotide sequence coding for the toxin may be a synthetic sequence, that is, one prepared according to standard procedures, eg as described in Matthes et al (1984).
- nucleotide sequence may be a mixed genomic and synthetic or mixed cDNA and synthetic sequence prepared by ligating DNA fragments of genomic, cDNA or synthetic origin (as appropriate) which DNA fragments each contain part of the nucleotide sequence encoding the toxin, in accordance with established methods.
- the DNA fragment may be one which has been modified by substitution, addition, insertion or deletion of one or more nucleotides in the sequence with the purpose of establishing a sequence which, when expressed, results in the production of a useful toxin or toxin variant.
- the invention relates to a DNA fragment which comprises a nucleotide sequence substantially as shown in Fig 6 (a)-(j) or a modification thereof as indicated above.
- the sequence coding for the full-length toxin starts at position 219 (or 213) of the sequence shown in the figure, while the end of the sequence is at position 4073.
- the DNA sequence shown in Fig 6 (a)-(j) has been established by well-known methods.
- the DNA fragment of the invention may further comprise a nucleotide sequence encoding another polypeptide fused to the nucleotide sequence encoding the toxin with the purpose of producing a fused polypeptide, as explained above.
- a further purpose of preparing a fused polypeptide may be to facilitate purification of the toxin.
- the fused sequence may be inserted into an appropriate vector which is transformed to a suitable host microorganism which is grown under conditions ensuring expression of the fused sequence after which the fused polypeptide is recovered from the culture by subjecting the fused polypeptide to affinity chromatography involving an antibody or any other ligand reacting with the second polypeptide.
- the second polypeptide may then be removed, for instance by suitable proteolytic cleavage followed by separation of the two polypeptides.
- the toxin may involve: a) isolating a nucleotide sequence coding for the P multocida toxin, b) inserting said sequence, optionally in suitably modified form in an expression vector, c) transforming a suitable host microorganism with the vector produced in step b), d) cultivating the microorganism produced in step c) under suitable conditions for expressing the toxin, e) harvesting the toxin from the culture, and f) optionally subjecting the toxin to posttranslational modifications to produce a (further) variant.
- the nucleotide sequence may for instance be isolated by establishing a P mul tocida gene library and screening for toxin-positive clones in accordance with established methods as indicated above as well as described in detail below.
- the modification of the sequence optionally carried out may be performed before or after the sequence has been inserted in the vector.
- the modification may comprise substitution, addition, insertion or deletion of one or more nucleotides in the sequence or a combination thereof, as explained above.
- step c) of the method may be carried out by standard procedures, such as disclosed in Maniatis et al (1982).
- step d) of the method may be carried out in a culture medium conventionally used for fermentation purposes, eg Luria Broth medium, and under conditions with respect to pH, temperature, aeration, etc suited to the type of microorganism in question, eg as disclosed in Maniatis et al (1982).
- a culture medium conventionally used for fermentation purposes
- the harvesting of the toxin may proceed by well-known methods such as by precipitation, gel filtration, ion exchange or HPLC reverse phase chromatography or immunoaffinity chromatography.
- compositions of the invention also comprise an acceptable carrier or vehicle.
- vehicle may be any vehicle usually employed in the preparation of pharmaceutical compositions, for example a diluent such as isotonic saline or suspending agent.
- the composition may be prepared by mixing an effective amount of the toxin with the vehicle in an amount resulting in the desired concentration of the toxin in the preparation.
- the mitogenic properties of the toxin mean that it may be put to many uses.
- the toxin may be used to aid wound healing and the invention therefore also provides a method of promoting wound healing in a mammal, for example healing skin ulcers, burns, severe cuts or abrasions, facial incisions resulting from cosmetic surgery, or cuts or burns on the cornea.
- the toxin may also be used to accelerate growth of bone marrow following anti-cancer chemotherapy or irradiation. The marrow may be removed from the patient, exposed to the toxin and then returned to the patient.
- marrow with an increased growth rate can be returned to the patient without there being any extracellular toxin and, in at least some cases, without any detectable intracellular toxin.
- a further aspect of the invention includes cells, for example bone marrow cells (specifically, stem cells) which have altered characteristics following exposure to the toxin.
- the "altered characteristics" may include an increased rate of division and/or DNA synthesis in relation to a control sample which was not exposed to the toxin, or in relation generally to what would have been expected.
- the toxin may also be used in cell fermentations, particularly eukaryotic cell fermentations (including hybridomas), to increase the yield of cells or their products, including viruses or expression products of recombinant nucleotides, or to accelerate the fermentation.
- a further embodiment of the invention therefore provides a cell fermentation medium comprising the P multocida toxin and being suitable for fermenting cells other than P multocida.
- the cells which are most likely to proliferate when exposed to the toxin are those whose growth characteristics are influenced by protein kinase C.
- the person skilled in the art will readily be able to determine whether any given cell type responds to the toxin in a desirable way.
- the toxin will function in the absence of serum, although small amounts of serum may still be desirable, for example to encourage adhesion of cells to a support.
- the amount of serum can be reduced from, say, 10%, to about 2% or less, for example 1%, 0.5% or 0.1%. This reduces the costs of the medium and frequently aids subsequent purification of the fermentation product.
- the toxin has been found to inhibit cAMP production and may therefore be used to treat E coli-induced diarrhoea, cholera, psoriasis or other conditions characterised by excessive cAMP production, for example the overproduction of some hormones.
- the proliferative effects of the toxin may also be used to stimulate specific cell types within the body, a reverse of the so-called "magic bullet".
- the toxin should preferably be targeted to the specific cell type required.
- a further embodiment of the invention therefore provides a conjugate of P multocida toxin or a fragment thereof and means to bind to a specific cell type.
- conjugate is used to cover molecules which have been made by merely joining the toxin or fragment thereof to a binding means (for example by methods taught by O'Sullivan et al (1979) Anal Biochem 100 , 100-108) and also molecules in which the toxin and the binding means have been synthesized integrally by chemical peptide synthesis techniques or recombinant DNA techniques (ie by using fused DNA sequences to express a single polypeptide product).
- the "means to bind to a specific cell type” may be any molecule which binds to a specific cell type in the body. Such a molecule may be an antibody, a naturally occurring molecule or fragment thereof or a synthetic peptide.
- the cell-binding portion may direct the conjugate to the bone-marrow or the gut epithelium, these being examples of rapidly-dividing tissues which may be damaged by anti-cancer chemotherapy or irradiation and whose growth one might wish to encourage.
- the cell-binding portion may be an antibody directed against a component of the gut pili (for example adhesins or colonisation factor antigens).
- the cell-binding portion of the conjugate may be low density lipoprotein or a cell-binding portion of human plasma fibronectin, such as the 11.5 kDalton polypeptide described by Ruoslahti & Pierschbacher in WO 84/00540, the Arg-Gly-Asp-Ser minimal cell-binding part thereof or any intermediate peptide between those two peptides.
- a nucleotide sequence coding for such a peptide is fused to a nucleotide sequence coding for the P multocida toxin or part thereof (either upstream or downstream thereof) in a known manner, incorporated in an expression vector in a suitable host and used to produce a hybrid polypeptide.
- the conjugate is preferably one which will be internalised.
- the toxin or a conjugate of whole toxin or an active part of the toxin with a specific binding compound (eg protein) for osteoclasts or osteoblasts can be used to modulate the interaction between these cell types by affecting (increasing or reducing) the production of growth factors, cytokines or paracrine substances by which osteoblasts regulate the function of osteoclasts.
- a specific binding compound eg protein
- osteoclasts or osteoblasts for example a monoclonal antibody specific to the osteoclast or osteoblast
- Monoclonal antibody 23C6 which is specific for the vitronectin receptor of osteoclasts is an example of a binding compound [M Horton et al "Monoclonal antibodies to osteoclastomas - definition of osteoclast-specific antigens" (1985) Cancer Res, 45, 5663-5669].
- the toxin or a conjugate of whole toxin or an active part of the toxin with a specific binding protein for lymphocytes, can be used to reduce the production of pharmacologically active lymphokines, cytokines or paracrine substances from these cells especially when they are over stimulated.
- the toxin conjugates may be used to treat conditions where there is excessive lymphocyte stimulation, such as that following transplantation which may cause some lymphoid malignancies, that which occurs in the incubation phase of Acquired Immune Deficiency Syndrome and may be required for the development of disease, and that which occurs during acute infections such as those involving the staphylococcal enterotoxins.
- the modulation of production and secretion of pharmacologically active substances from stimulated lymphocytes can reduce the consequences of stimulation of these cells.
- the P mul tocida toxin itself contains at least one cell binding portion. Many drugs need to be directed to specific cells on which they are intended to act, an example being cytotoxic drugs for tumour chemotherapy.
- the toxin or a cell-binding portion thereof may be used as the delivery vehicle.
- a further embodiment of the invention therefore provides a conjugate of P mul tocida toxin or a cell-binding variant or portion thereof and a pharmacologically active compound. Preferably, the variant or portion is not also mitogenic.
- conjugate is used in the same sense as above, and encompasses chemically-joined fragments and fused hybrids produced by automated polypeptide synthesis or expression of fused nucleotides.
- the pharmacologically active agent which is conjugated to the toxin or portion thereof may be any cytotoxic or antiviral agent, for example vincristine, vinblastine or methotrexate.
- Cell-binding portions of the toxin may be readily identified by radio-labelling a candidate portion and detecting its location on the surface of cells, or by exposing the cells to the candidate portion and then adding a labelled antibody to the portion.
- the methods of WO 84/00540 or Yamada (1983) may be used.
- Target cells include the bladder, liver and spleen.
- At least some of the various effects of the toxin and conjugates of all or part thereof may be potentiated by one or more growth factors, such as EGF, PDGF, FGFb or insulin.
- growth factors such as EGF, PDGF, FGFb or insulin.
- Figure 1 is a restriction map of the recombinant plasmids pAJL12 and pAJL13.
- the arrows indicate the direction of readthrough from the tetracycline gene of pAT153.
- the insert in pAJL12 was not cut by HpaII , KpoI , PvuI , PvuII , SalI or SstI .
- the BamEI site in parentheses was lost upon ligation of the vector BamHI site to the insert SauIIIA site. Two HindIII sites between the HindIII sites at the right hand end of the insert were not mapped.
- Figure 2 is a representation of an agarose gel of chromosomal DNA from P multocida LFB3; uncut (lane 1) or cut with HpaI (lane 2) or HpaII (lane 3) (cross-hatching representing dark area and open boxes representing light bands) and, alongside, a Southern blot of the gel probed with the large HpaII fragment of pALJ12 (lanes 4-6 as lanes 1-3).
- Figure 3 is a SDS PAGE of purified toxin from P multocida LFB3 (lane 1) and E coli TOX1 (lane 2).
- Figure 4 shows the result of crossed immunoelectrophoresis of purified recombinant toxin reacted against gnotobiotic pig antiserum to purified toxin from P multocida LFB3.
- Figure 5 shows an immunoblot probed with gnotobiotic pig serum to toxin purified from P multocida LFB3: Lane 1 purified toxin from P mul tocida LFB3, Lane 2 purified toxin from E coli TOX1, Lane 3 whole cell lysate of a recombinant which reacted with the HpaI fragment of the insert in pALJ12, Lane 4 whole cell lysate from E coli HB101 containing pAT153, Lanes 5-8 as Lanes 1-4 without antibody to toxin.
- Figure 6 shows DNA sequence data for the PMT gene, taken from WO 89/09617.
- Our sequence which is considered to be the more authoritative, differs therefrom in that at their position 460 they have an A whereas we have a C; at 461 they have C and we have T; at 2541, C, G; at 2542, G, C; and at 110, outside the coding region, we have an extra C which therefore alters the numbering by one residue.
- Figure 7 shows the PMT amino acid sequence data, deduced from the nucleotide sequence of Figure 6.
- P mul tocida strain LFB3 is a toxigenic isolate from a pig with atrophic rhinitis (Rutter, 1983) and E coli HB101 and HB101 harbouring pAT153 were obtained from Dr J G Williams, Imperial Cancer Research Fund, London. All bacteria were stored as cell suspensions at -70°C in 12% (v/v) glycerol. P multocida were grown in Bacto tryptose broth (Jones and Matthews, 1975) at 37°C with agitation. E coli strains were grown on LB agar or in L broth (Maniatis et al , 1982).
- P multocida DNA was isolated by a modification of the method of Saito and Miura (1963). Cells were collected by centrifugation and washed in an aqueous solution of 0.15M NaCl, 0.1M EDTA, pH8.0 and were resuspended in the same buffer at one tenth the original volume of culture. Lysozyme (1mg/ml) was added, and the mixture was incubated at 37°C for 30 minutes and was then rapidly immersed in a dry ice/acetone bath.
- the suspension was centrifuged and the aqueous phase was re-extracted with phenol, and after phase separation residual phenol was removed from the aqueous phase by extensive dialysis against an aqueous solution of 50mM Tris-HCl, 10mM EDTA, 10mM NaCl, pH8.
- the preparation was treated with ribonuclease as described by Maniatis et al (1982), and the purified DNA was stored at 4°C.
- Genomic DNA from P multocida was partially digested with SauIIIA to obtain fragments about 10kb.
- the digest was fractionated on a sucrose gradient as described by Maniatis et al (1982). Fractions were selected containing fragments in the size range 7-12kb.
- the restricted DNA was ligated overnight at 15°C to pAT153, previously cut with BamEI and treated with phosphatase. Competent HB101 obtained from BRL were transformed.
- the transformed cells were plated onto L agar containing ampicillin (200 ⁇ g/ml). After incubation at 37°C overnight, the 6500 bacterial colonies obtained were replica plated onto agar containing tetracycline (12 ⁇ g/ml).
- the 2500 Ap r Tc s colonies were tested for toxicity for embryonic bovine lung (EBL) cells by the overlay method (Chanter et al 1986a) and stored at -70°C in 12% glycerol in microtitre trays.
- Plasmids were isolated according to Ish-Horowitz and Burke (1981). Other molecular biological techniques were as described in Maniatis et al (1982).
- DNA sequencing was carried out using an Applied Biosystems 370A Sequencer with the Tag polymerase kit, according to the manufacturer's instructions.
- the recombinant plasmid from TOX2 was digested with HpaII and the 4.9kb fragment was separated on an agarose gel, and extracted using Geneclean (Stratech Scientific, London).
- the DNA was digested with AluI or SauIIIA, and either randomly inserted into M13 (mp10, mp18 or mp19) or fragments were gel purified as described above prior to ligation into M13 (mp10, mp18 or mp19).
- Protein Sequencing Protein sequencing was carried out using an Applied Biosystems 477A Gas Liquid Protein Sequencer with on-line 120A PTH Amino Acid analysis according to the manufacturer's instructions. Cloning the toxin gene. One clone (TOX1) out of the 2500 Ap r Tc s clones with inserts was positive for toxin production. The plasmid from this clone, pAJL12, was purified and transformed into competent E coli cells; 20 transformed bacteria were selected and all were toxigenic.
- the plasmid pAJL12 contained a 10.7kb insert.
- the restriction map ( Figure 1) was unusual since the insert was not cut by HpaII or MspI , and only cut three times by HaeIII. These three enzymes have a four base pair recognition sequence which contains only G and C, which suggested that the G + C ratio of the DNA might be quite low.
- the enzymes DraI and SpeI which have a six base pair recognition sequence containing only A and T, cut the insert at least 9 and 11 times respectively (data not shown). Genomic DNA from P mul tocida was prepared and cut with HpaI and HpaII , and Southern blots of the gels were probed with fragments from the insert.
- Figure 2 shows that most of the DNA was digested by HpaII to fragments smaller than 4kb, but there were discrete bands of higher molecular weight. A band at about 15kb hybridised to the probe. Three other toxigenic P multocida isolates produced a HpaII fragment of similar molecular weight which also reacted with the probe. A band of about 6kb in HpaI digests reacted with the probe.
- the 2500 colonies in the clone bank were probed with the HpaI fragment from the insert in pAJL12, and 12 colonies hybridised to the probe. None produced toxin when tested with the EBL overlay test. Preliminary analysis of the 12 hybridising colonies showed that they contained plasmids of different size, but none contained the whole toxin gene.
- Plasmid pAJL12 was partially digested with SauIIIA, to produce fragments about 5kb, the predicted size of the toxin gene. Fragments of size 5 - 8kb were selected and ligated into pAT153, previously BamEI cut and phosphatased. Recombinants were screened for toxicity by the EBL overlay test, and one (TOX2) contained a 5.0kb insert and produced toxin. The recombinant (pAJL13) was mapped ( Figure 1), and was located at one end of the insert in pAJL12, with a duplication of about 0.1kb of the vector sequence. The insert is in the opposite orientation to pAJL12, which implies that in at least one of the constructs the gene is being read off its own promoter.
- Toxin Purification Toxin was purified from a crude extract of the toxigenic recombinants TOX1 or TOX2, grown on L agar containing ampicillin, prepared by the lysis method of Rimler and Brogden (1986). Crude extract, treated with RNase, DNase, benzamidine and phenylmethyl-sulphonylfluoride, was sequentially fractionated by DEAE Sephacel chromatography and preparative polyacrylamide gel electrophoresis (Chanter et al , 1986b). In the final step purified toxin was electroeluted from the polyacrylamide. Quantities of toxin in each fraction were measured using toxicity for EBL cells (Rutter and Luther, 1984).
- toxin purified from recombinant E coli Characterisation of toxin purified from recombinant E coli .
- the homogeneity and molecular weight of the polypeptide(s) in toxin purified from the recombinants were estimated by SDS polyacrylamide gel electrophoresis by the method of Laemmli (1970) and staining of gels with silver as described before (Chanter et al , 1986b).
- the antigenic similarity of the toxin purified from the recombinants with that purified from P mul tocida was determined using antiserum to toxin purified from P multocida that was produced in a gnotobiotic pig (Chanter et al , 1986b) in an enzyme-linked immunosorbent assay, immunoblotting, a cytotoxin neutralisation test (Rutter and Luther, 1984) and by crossed immunoelectrophoresis by the method of Moore (1985).
- microtitre plates (Falcon-Becton Dickenson) were coated overnight with 100 ⁇ l of different concentrations of toxin in carbonate/bicarbonate buffer, pH 9.6. Plates were washed in phosphate buffered saline containing 0.03% Tween 20 (PBS/Tween) and incubated at 37°C for 1 hour with serial dilutions of pig antiserum in PBS/Tween with 1% dehydrated skimmed milk (Marvel, Cadbury Ltd).
- Protein separated by SDS-PAGE were immunoblotted by the method of Towbin et al (1979) using a Transblot apparatus and the recommended protocols of the manufacturer (Biorad). Toxicity was assayed using EBL cells (Rutter and Luther, 1984) and intraperitoneal injection of gnotobiotic pigs (Rutter and Mackenzie, 1984) and was related to protein content assayed by a Coomassie dye binding method (Biorad).
- the toxin purified from recombinants TOX1 and TOX2 had the same high molecular weight as that produced by P multocida LFB3. Comparison of the yield and the efficiency of purification indicated that TOX1 produced approximately five fold as much toxin as TOX2 and ten times as much as P multocida . Like P mul tocida both recombinants produced a faint band above the main band.
- a chequerboard titration of toxin was used to coat the microtitre plate for an ELISA and dilutions of gnotobiotic pig serum against toxin purified from P multocida were made.
- the optimum coating concentration of antigen was 1 ⁇ g/ml of coating buffer for toxin from P multocida or from either toxigenic recombinant. At this concentration the serum gave an identical titre of 10 4 for all toxin preparations. Control serum did not react with any of the toxin preparations.
- the gnotobiotic pig serum had a titre of 10 3 with 10 cytotoxic units of toxin purified from either P multocida or the recombinants.
- PVDF membrane Immobilon from Millipore
- a 1cm square of PVDF membrane was wetted with ethanol then 50% ethanol/H 2 O and finally H 2 O. This was applied to a heating block (at 55°C) covered with aluminium foil.
- the sample was applied in 3 ⁇ 30 ⁇ l aliquots (total of 30 ⁇ g) and dried. The membrane was then extensively washed in
- Partial sequence data are as follows.
- PMT PasteureIIa multocida toxin
- rPMT acts in this manner to stimulate mitogenesis
- quiescent 3T3 cells were incubated with rPMT at 1, 5 or 20 ng/ml for various times.
- the level of ( 3 H ) thymidine incorporation was similar to that induced in the cultures incubated continuously with rPMT.
- Lower concentrations of rPMT required longer preincubation times to induce maximal DNA synthesis after removal of unbound toxin.
- the potent mitogenic effect of rPMT persists after the medium containing it has been removed.
- the cells were preincubated with 5 ng/ml rPMT for various times and then transferred to media in the absence or presence of PMT antiserum.
- the cells were treated with 5 ng/ml rPMT for 1 h, subsequent DNA synthesis was markedly inhibited in cultures transferred to medium containing antiserum.
- the mitogenic effect of the toxin is no longer blocked by the addition of antiserum.
- the rPMT undergoes a time-dependent internalization into a compartment not accessible to external antibodies.
- rPMT mitogenic activity of rPMT in cultures of Swiss 3T3 cells also could be readily shown when cell number (rather than ( %)thymidine incorporation into acid-precipitable material) was monitored over a period of several days either in confluent or subconfluent cells.
- Addition of rPMT at 10 ng/ml to the medium in which confluent and quiescent 3T3 cells were grown (depleted medium) resulted in loss of density-dependent inhibition of growth: after 3 days, the cell number was about twice that of the control.
- rPMT stimulated reinitiation of cell proliferation in a concentration-dependent manner.
- addition of rPMT to subconfluent 3T3 cells resulted in a striking increase in cell proliferation; the final saturation density increased 6-fold.
- cpm ⁇ 10 -3 Numbers expressed as cpm ⁇ 10 -3 , means of two determinations.
- concentrations used were as follows: rPMT (10 ng/ml); insulin (1 ⁇ g/ml); rFGFb (5 ng/ml); EGF (5 ng/ml) and PDGF (10 ng/ml).
- 10% FBS gave incorporations of 139 ⁇ 10 3 cpm with rPMT.
- Human foreskin fibroblasts were used at the 18th passage and rendered quiescent by incubation in 0.5% FBS for 4d.
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Abstract
Description
Claims
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB9125997A GB2252105A (en) | 1989-06-29 | 1991-12-06 | Toxin uses |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB8914984.3 | 1989-06-29 | ||
GB898914984A GB8914984D0 (en) | 1989-06-29 | 1989-06-29 | Nucleotide sequences |
Publications (2)
Publication Number | Publication Date |
---|---|
WO1991000100A2 true WO1991000100A2 (en) | 1991-01-10 |
WO1991000100A3 WO1991000100A3 (en) | 1991-04-04 |
Family
ID=10659288
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB1990/000992 WO1991000100A2 (en) | 1989-06-29 | 1990-06-27 | Toxin uses |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP0482018A1 (en) |
JP (1) | JPH05500657A (en) |
GB (2) | GB8914984D0 (en) |
WO (1) | WO1991000100A2 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1994008011A1 (en) * | 1992-09-30 | 1994-04-14 | Bioteknologisk Institut | Recombinant mutant pasteurella multocida protein and process for preparing the same |
WO1997020579A2 (en) * | 1995-12-05 | 1997-06-12 | Smithkline Beecham Plc | Novel compounds and use |
WO2006010360A2 (en) * | 2004-07-22 | 2006-02-02 | Biotecon Therapeutics Gmbh | Carrier for medicaments for obtaining oral bioavailability |
-
1989
- 1989-06-29 GB GB898914984A patent/GB8914984D0/en active Pending
-
1990
- 1990-06-27 WO PCT/GB1990/000992 patent/WO1991000100A2/en not_active Application Discontinuation
- 1990-06-27 EP EP90909525A patent/EP0482018A1/en not_active Withdrawn
- 1990-06-27 JP JP2508958A patent/JPH05500657A/en active Pending
-
1991
- 1991-12-06 GB GB9125997A patent/GB2252105A/en not_active Withdrawn
Non-Patent Citations (1)
Title |
---|
Proc. Natl. Acad. Sci. USA, vol. 87, January 1990, E. Rozengurt et al.: "Pasteurella multocida toxin: Potent mitogen for cultured fibroblasts", pages 123-127 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1994008011A1 (en) * | 1992-09-30 | 1994-04-14 | Bioteknologisk Institut | Recombinant mutant pasteurella multocida protein and process for preparing the same |
WO1997020579A2 (en) * | 1995-12-05 | 1997-06-12 | Smithkline Beecham Plc | Novel compounds and use |
WO1997020579A3 (en) * | 1995-12-05 | 1997-09-25 | Smithkline Beecham Plc | Novel compounds and use |
WO2006010360A2 (en) * | 2004-07-22 | 2006-02-02 | Biotecon Therapeutics Gmbh | Carrier for medicaments for obtaining oral bioavailability |
WO2006010360A3 (en) * | 2004-07-22 | 2007-12-27 | Biotecon Therapeutics Gmbh | Carrier for medicaments for obtaining oral bioavailability |
Also Published As
Publication number | Publication date |
---|---|
GB9125997D0 (en) | 1992-05-20 |
EP0482018A1 (en) | 1992-04-29 |
GB2252105A (en) | 1992-07-29 |
GB8914984D0 (en) | 1989-08-23 |
JPH05500657A (en) | 1993-02-12 |
WO1991000100A3 (en) | 1991-04-04 |
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