Classifications

• • 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
  • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

• This invention relates to polypeptides derived from fragment C of the tetanus toxin and their use in vaccine compositions.
• Tetanus is a highly infectious, worldwide disease caused by the organism Clostridium tetani. Its spores can remain dormant for indefinite periods in house dust and soil which are the prime sources of infection for open wounds. Contaminated animal faeces are also responsible for the spread of the disease and may further contaminate previously uninfected locales.
• Tetanus infection is always a risk for the wounded and is usually associated with great pain and may even lead to death.
• causative agent was discovered to be a bacterium, great effort was devoted to the development of an effective vaccine.
• the first successful vaccination in human subjects was carried out in 1926, when the vaccine used comprised crude tetanus toxin treated to render it atoxic.
• Tetanus toxin provided the target for the first vaccine as it had already been discovered that symptoms are not due to the bacterium infecting host cells but rather by a potent neurotoxin released upon lysis of the bacterial cells.
• a single (parent) peptide is synthesised which comprises both light and heavy chains.
• an endogenous protein cleaves the peptide between a preexisting disulphide bridge to give two peptide chains linked by a disulphide bridge.
• Papain cleaves the heavy chain to give a fragment B (100 kDa) consisting of the light chain and part of the heavy chain, and a fragment C (50 kDa). consisting of the remainder of the heavy chain. It has been shown that both fragments are capable of providing protection but may be associated with residual toxicity that would be due to contamination with uncleaved toxin.
• Tetanus toxin is a member of the family of clostridial neurotoxins (CNTs) which includes the botulinum toxins (BoNTs). CNTs inhibit neurotransmitter release from presynaptic neuronal cells by proteolytic cleavage of proteins involved in the fusion of synaptic vesicles with the cell membrane. This cleavage is catalysed by the 50 kDa L-chain of CNTs which encodes a zinc dependent metaloprotease. The overall structural and functional properties of the CNTs are very similar, having a similar sub-unit structure and 30-40% amino acid identity (Minton, 1995).
• TeNT In contrast to BoNTs which remain within the peripheral nervous system, TeNT is trafficked to the central nervous system by retrograde axonal transport followed by trans-synaptic spread into inhibitory interneurons. It is this differential cellular trafficking of TeNT compared to the BoNTs which explains the distinct sites of action of the toxins and therefore the distinct clinical symptoms of the two diseases.
• TeNT and BoNT bind to sensitive cells in a variety of in vitro systems including tissue preparations, primary cell cultures and cell lines. These studies, and others, have shown that TeNT and BoNT bind to membrane receptors, and are internalised prior to the cytosolic action of the L-chain of the toxins. The steps involved in binding and trafficking of TeNT are largely uncharacterised, although it appears to involve internalisation through uptake of small synaptic vesicles during vesicle reuptake. The binding of TeNT to neuronal tissue involves the C-terminal 50 kDa domain (He domain, also known as fragment C).
• TeNT He fragment blocked uptake of TeNT as shown by inhibition of VAMP-2 cleavage and antagonism of neuromuscular transmission, indicating that the binding of He is functional.
• the He fragment alone retains the ability to undergo retrograde transport in vivo, and has been used as a carrier of foreign proteins to the CNS. Since the initial demonstration of TeNT binding to gangliosides, many studies have attempted to characterise binding of CNTs to gangliosides using in vitro and in vivo assays.
• synaptotagmin II which is localised in the lumen of small synaptic vesicles, has been identified as a protein receptor for BoNT serotype B, and serotypes A and E.
• Cross-linking experiments using primary spinal cord motoneurons and NGF-differentiated PC 12 cells have identified a 15 kDa glycoprotein as a possible neuronal receptor for TeNT (Herreros, et al. J. Neurochemsitry in press). This receptor binds only to the carboxy terminal half of the He domain of TeNT.
• Mutant molecules were produced in E.coli, purified and assayed in ganglioside binding, cell binding and in vivo retrograde transport assays. Our results identify specific residues that are required for ganglioside binding, and identify certain mutants with reduced ganglioside binding but which still retain the ability to bind primary neuronal cells and undergo retrograde axonal transport.
• the TeNT fragment C required for binding gangliosides deletion of which leads to a greater reduction in ganglioside binding than the results obtained for His 1293 alone.
• the regions are loop regions which connect two sections of ⁇ sheet.
• the present invention provides a polypeptide comprising tetanus toxin (TeNT) fragment C, or an immunogenic fragment thereof, which tetanus toxin fragment C, or immunogenic fragment thereof comprises a mutation in a loop region, which mutation results in a reduction in the binding of the tetanus toxin fragment C, or immunogenic fragment thereof, to gangliosides.
• TeNT tetanus toxin
• the present invention also provides a polypeptide comprising tetanus toxin (TeNT) fragment C, or an immunogenic fragment thereof, which tetanus toxin fragment C. or immunogenic fragment thereof comprises a mutation in a loop region, which mutation results in a reduction in the binding of the tetanus toxin fragment C, or immunogenic fragment thereof, to primary motoneurones.
• TeNT tetanus toxin
• the present invention also provides a polypeptide comprising tetanus toxin (TeNT) fragment C, or an immunogenic fragment thereof, which tetanus toxin fragment C. or immunogenic fragment thereof comprises a mutation in a loop region, which mutation results in a reduction in the ability of the tetanus toxin fragment C, or immunogenic fragment thereof, to undergo retrograde transport.
• TeNT tetanus toxin
• the mutation results in a reduction in the binding of the tetanus toxin fragment C (or immunogenic fragment thereof) to gangliosides and a reduction in the binding to primary motoneurones and/or a reduction in the ability to undergo retrograde transport.
• said loop region is present in the full length wild type sequence between two ⁇ sheets. More preferably said loop region is selected from amino acid residues 1214 to 1219 and 1271 to 1282 of the amino acid sequence of TeNT fragment C.
• said mutation is at least one deletion, more preferably at least one deletion selected from ⁇ 1214 to 1219, ⁇ 1274 to 1279 and ⁇ 1271 to 1282 of the amino acid sequence of TeNT fragment C.
• the present invention further provides a polynucleotide encoding a polypeptide of the invention.
• the present invention also provides vectors comprising a polynucleotide encoding a polypeptide of the invention operably linked to a regulatory sequence.
• a regulatory sequence allows expression of the polypeptide in a host cell.
• the host cell is a bacterium, or a cell of an animal, more preferably a mammal, including primates and humans.
• polypeptides, polynucleotides and vectors of the present invention may be used in the prevention (or reduction in susceptibility to), or treatment of, Clostridium tetani infections.
• the present invention provides a pharmaceutical composition comprising a polypeptide, polynucleotide or vector of the invention together with a pharmaceutically acceptable carrier or diluent.
• the present invention also provides a method of treating or preventing C. tetani infection in a human or animal which comprises administering to the human or animal an effective amount of a polypeptide, polynucleotide or vector of the invention.
• the present invention further provides a polypeptide of the present invention for use in therapy, such as the prevention (or reduction in susceptibility to), or treatment of, C. tetani infections. Also provided is the use of a polypeptide of the invention in the manufacture of a medicament for use in the prevention (or reduction in susceptibility to), or treatment of. C. tetani infections.
• the invention provides the use of a polypeptide. polynucleotide or vector of the invention in a method for producing antibodies which recognise epitopes within a TeNT polypeptide.
• the present invention provides a method for producing antibodies which recognise epitopes within a TeNT polypeptide which method comprises administering a polypeptide, a polynucleotide or a vector of the invention to a mammal.
• Such antibodies produced by the various methods known in the art may be used in a method of treating C. tetani infection in a human or animal which comprises administering to a human or animal an effective amount of an antibody produced by the above method of the invention.
• the invention provides a method for reducing the binding affinity of a TeNT fragment C polypeptide for gangliosides which method comprises modifying one or more amino acid residues present in a surface-exposed loop region of the polypeptide. Also provided is a polypeptide produced by said method.
• fragment C is a 50 kDa polypeptide generated by papain cleavage and comprises or substantially corresponds to the 451 amino acids at the C-terminus.
• fragment C and “He” are used interchangeably herein. It is to be understood that although the description below adopts the amino acid numbering based on the sequence of tetanus toxin described herein, the present invention is equally applicable to fragment C variants found in other strains of C. tetani (which may have slightly different amino acid numbering).
• Polvpeptides of the invention comprise tetanus toxin fragment C or immunogenic g fragments thereof that have been modified to reduce their ability to bind to gangliosides, and/or their ability to bind primary motoneurones, and/or their ability to undergo retrograde transport.
• the polypeptides of the invention comprise mutations in a loop region found between 2 ⁇ sheets.
• the 3D structure of TeNT fragment C has been determined by Umland et al. (1997) the structural coordinates deposited in the Protein Data Bank under reference number laf9.
• the structural information presented in Umland et al. (1997) (see Figure la in particular) allows the skilled person to identify candidate loop regions. Preferably these loops are exposed on the surface of the molecule.
• the first loop consists of approximately amino acids 1214 to 1219 and the second loop consists of approximately amino acids 1271 to 1282.
• Mutations in the loop regions may be substitutions, insertions and/or deletions.
• the mutations are deletions, more preferably deletions which remove substantially all of a particular loop region.
• Techniques for modifying amino acids sequences are well known in the art, such as PCR-directed mutagenesis.
• mutant TeNT polypeptides of the present invention have less than 50% ganglioside binding activity compared with wild type TeNT, more preferably less than 40. 30. 20 or 10%), most preferably less than 5%.
• Ganglioside binding activity may. for example, be determined in vitro as described in the examples.
• mutant TeNT polypeptides of the present invention have less than 50% neuronal cell binding activity compared with wild type TeNT, more preferably less than 40. 30, 20 or 10%. most preferably less than 5%.
• Neuronal cell binding activity may, for example, be determined as described in the examples.
• mutant polypeptides of the invention retain at least 50% of the immunogenicity of the wild type sequence from which they are derived (i.e. the full WO 01/00839 .9. PCT/GBOO/02428 length fragment C sequence or immunogenic fragments thereof), more preferably at least 70, 80 or 90%.
• Immunogenicity may be determined typically by the use of in vitro techniques such as ELISA or Western blotting.
• immunogenicity may be determined in vivo by, for example, immunising animals with a polypeptide of the invention and then either testing sera by ELISA or Western blotting, or by subsequently challenging immunised individuals with active toxin.
• a polypeptide of the invention comprises TeNT fragment C, or an immunogenic fragment thereof, which TeNT fragment C, or immunogenic fragment thereof, comprise a mutation in amino acid residues 1214 to 1219 and/or 1271 to 1282.
• a polypeptide of the invention comprises TeNT fragment C, or an immunogenic fragment thereof, which TeNT fragment C. or immunogenic fragment thereof, comprises a deletion of amino acid residues 1214 to 1219 and/or at least 1274 to 1279, more preferably 1271 to 1282.
• polypeptides of the invention further comprise a modification at residue His-1293, preferably a substitution such as His ⁇ Ala or His- ⁇ Ser.
• Fragments of tetanus toxin fragment C that contain epitopes may also be used in the poK ⁇ eptides of the invention. These fragments will comprise at least 5 or 6 amino acids, preferably at least 10 amino acids, more preferably at least 15. 20. 50 or 100 amino acids. Particularly preferred fragments include from about amino acids 943 to 1023. which is a good B-cell epitope, and from about amino acids 946 to 966 which is a good T-cell epitope. All amino acid numbering is with reference to the full-length toxin gene.
• amino acid sequence of tetanus toxin fragment C can be further modified to provide polypeptides of the invention. For example, this may be carried out to enhance the immunogenicity of the polypeptides of the invention. Amino acid substitutions may be made, for example from 1, 2 or 3 to 10. 20 or 30 substitutions provided that the modified polypeptide retains epitopes.
• Polypeptides of the invention may further comprise heterologous amino acid sequences, typically at the N-terminus or C-terminus, preferably the N-terminus.
• Heterologous sequences may include sequences that affect intra or extracellular protein targeting (such as leader sequences).
• Heterologous sequences may also include sequences that increase the immunogenicity of the polypeptide of the invention and/or which facilitate identification, extraction and/or purification of the polypeptides.
• Another heterologous sequence that is particularly preferred is a polyamino acid sequence such as polyhistidine which is preferably N-terminal.
• a polyhistidine sequence of at least 10 amino acids, preferably at least 17 amino acids but fewer than 50 amino acids is especially preferred.
• heterologous amino acid sequences includes immunogenic sequences from other pathogenic organisms such as bacteria or viruses. Examples include pathogenic E. coli, Neiserria sp., B. pertussis, C. difficile, Salmonella sp., Campylobacter sp., P. falciparum, hepatitis B virus, hepatitis C virus and human papilloma virus. Conveniently, these immunogenic sequences may be inserted into a loop region which it is desired to disrupt (such as amino acids 1214 to 1219).
• Polypeptides of the invention are typically made by recombinant means, for example as described below. However they may also be made by synthetic means using techniques well known to skilled persons such as solid phase synthesis. Polypeptides of the invention may also be produced as fusion proteins, for example to aid in extraction and purification. Examples of fusion protein partners include glutathione-S-transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and ⁇ -galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences, such as a thrombin cleavage site. Preferably the fusion protein will not hinder the function of the protein of interest sequence.
• Polypeptides of the invention may be in a substantially isolated form. It will be understood that the protein may be mixed with carriers or diluents which will not interfere with the intended purpose of the protein and still be regarded as substantially isolated.
• a polypeptide of the invention may also be in a substantially purified form, in which case it will generally comprise the protein in a preparation in which more than 90%o, e.g. 95%. 98% or 99%o of the protein in the preparation is a polypeptide of the invention.
• Polynucleotides of the invention comprise nucleic acid sequences encoding the polypeptides of the invention.
• Polynucleotides of the invention may comprise DNA or RNA. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule.
• the polynucleotides described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of the invention.
• Preferred polynucleotides of the invention also include polynucleotides encoding any of the polypeptides of the invention described above. It will be understood by a skilled person that numerous different polynucleotides can encode the same polypeptide as a result of the degeneracy of the genetic code.
• Polynucleotides of the invention can be incorporated into a recombinant replicable vector.
• the vector may be used to replicate the nucleic acid in a compatible host cell.
• the invention provides a method of making polynucleotides of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector.
• the vector may be recovered from the host cell.
• Suitable host cells include bacteria such as E. coli, yeast, mammalian cell lines and other eukaryotic cell lines, for example insect Sf9 cells.
• a polynucleotide of the invention in a vector is operably linked to a regulator ⁇ ' sequence that is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector.
• the term "operably linked'" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
• a regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
• Such vectors ma)' be transformed or transfected into a suitable host cell as described above to provide for expression of a polypeptide of the invention.
• This process may comprise culturing a host cell transformed with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the polypeptides. and optionally recovering the expressed polypeptides.
• the vectors may be, for example, plasmid or virus vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter.
• the vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector.
• Vectors may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell.
• the vector may also be adapted to be used in vivo, for example in a method of gene therapy.
• Promoters/enhancers and other expression regulation signals may be selected to be compatible with the host cell for which the expression vector is designed.
• prokaryotic promoters may be used, in particular those suitable for use in E. coli strains (such as E. coli HB101).
• an htrA or nirB promoter may be used.
• mammalian promoters may be used.
• Tissue-specific promoters may also be used.
• Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR), the promoter rous sarcoma virus (RSV) LTR promoter, the SV40 promoter, the human cytomegalovirus (CMV) IE promoter, herpes simplex virus promoters or adenovirus promoters. All these promoters are readily available in the art.
• MMLV LTR Moloney murine leukaemia virus long terminal repeat
• RSV promoter rous sarcoma virus
• CMV human cytomegalovirus
• IE herpes simplex virus promoters or adenovirus promoters. All these promoters are readily available in the art.
• Vectors and polynucleotides of the invention may be introduced into host cells for the purpose of replicating the vectors/polynucleotides and/or expressing the polypeptides of the invention encoded by the polynucleotides of the invention.
• Suitable host cells include prokaryotes such as eubacteria, for example E. coli and B. subtilis and eukaryotes such as yeast, insect or mammalian cells.
• Vectors/polynucleotides of the invention may be introduced into suitable host cells using a variety of techniques known in the art, such as transfection, transformation and electroporation. Where vectors/polynucleotides of the invention are to be administered to animals, several techniques are known in the art, for example infection with recombinant viral vectors such as retroviruses, herpes simplex viruses and adenoviruses. direct injection of nucleic acids and biolistic transformation.
• Host cells comprising polynucleotides of the invention may be used to express polypeptides of the invention.
• Host cells may be cultured under suitable conditions which allow expression of the proteins of the invention.
• Expression of the polypeptides of the invention may be constitutive such that they are continually produced, or inducible, requiring a stimulus to initiate expression. In the case of inducible expression, protein production can be initiated when required by. for example, addition of an inducer , A
• -14- substance to the culture medium, for example dexamethasone or IPTG.
• Suitable methods for producing tetanus toxin fragment C polypeptides are described in WO-A-9015871 and
• Polypeptides of the invention can be extracted from host cells by a variety of techniques known in the art, including enzymatic, chemical and/or osmotic lysis and physical disruption.
• polypeptides may optionally be performed using well known techniques such as affinity chromatography. including immuno affinity chromatography, ion-exchange chromatography and the like.
• affinity chromatography including immuno affinity chromatography, ion-exchange chromatography and the like.
• a particularly preferred technique is to express the polypeptide of the invention as a fusion protein with polyhistidine tag (for example 6xHis) and purify cell extracts using Ni-NTA agarose (Qiagen).
• polyhistidine tag for example 6xHis
• Qiagen Ni-NTA agarose
• Polypeptides of the invention may also be produced recombinantly in an in vitro cell-free system, such as the TnTTM (Promega) rabbit reticulocyte system.
• the polypeptides of the invention may be administered by direct injection.
• the polypeptides are combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition, which may be for human or veterinary use.
• Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline.
• the composition may be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration.
• each polypeptide is administered at a dose of from 0.01 to 30 ⁇ g/kg body weight, preferably from 0.1 to 10 ⁇ g/kg. more preferably from 0.1 to 1 ⁇ g/kg body weight.
• antibodies prepared using the polypeptides of the invention can be administered in a similar manner to the polypeptides of the invention.
• the polynucleotides of the invention may be administered directly as a naked nucleic acid construct, preferably further comprising flanking sequences homologous to the host cell genome.
• the amount of nucleic acid administered is typically in the range of from 1 ⁇ g to 10 mg, preferably from 100 ⁇ g to 1 mg.
• Uptake of naked nucleic acid constructs by mammalian cells is enhanced by several known transfection techniques for example those including the use of transfection agents.
• transfection agents include cationic agents (for example calcium phosphate and DEAE-dextran) and lipofectants (for example lipofectamTM and transfectamTM).
• cationic agents for example calcium phosphate and DEAE-dextran
• lipofectants for example lipofectamTM and transfectamTM.
• nucleic acid constructs are mixed with the transfection agent to produce a composition.
• a nucleotide of the invention is introduced into an attenuated strain of Salmonella sp. such that a polypeptide of the invention is expressed by the transformed bacterial strain and the live bacteria administered, typically orally, to a patient.
• the polynucleotide or vector of the invention is combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition.
• Suitable carriers and diluents include isotonic saline solutions, for example phosphate- buffered saline.
• the composition may be formulated for parenteral. intramuscular, intravenous, subcutaneous, intraocular or transdermal administration.
• Vaccines may be prepared from one or more polypeptides of the invention.
• the preparation of vaccines which contain an immunogenic polypeptide(s) as active ingredient(s), is known to one skilled in the art.
• such vaccines are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
• the preparation may also be emulsified, or the protein encapsulated in liposomes.
• the active immunogenic ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
• the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine.
• auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine.
• adjuvants which may be effective include but are not limited to: aluminum hydroxide, N-acetyl-muramyl-L-tlrreonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 1 1637.
• nor-MDP N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(T-2'-dipalmitoyl-sn- glycero-3-hydroxyphosphoryloxy)-ethylamine
• CGP 19835 A referred to as MTP-PE
• RIBI N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(T-2'-dipalmitoyl-sn- glycero-3-hydroxyphosphoryloxy)-ethylamine
• MTP-PE N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(T-2'-dipalmitoyl-sn- glycero-3-hydroxyphosphoryloxy)-ethylamine
• MTP-PE N-acetylmuramyl-L-alanyl-
• adjuvants and other agents include aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions, oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin, lipid X, Corynebacterium parvum (Propionobacterium acnes), Bordetella pertussis, polyribonucleotides. sodium alginate, lanolin, lysolecithin, vitamin A, saponin, liposomes. levamisole, DEAE-dextran, blocked copolymers or other synthetic adjuvants.
• Such adjuvants are available commercially from various sources, for example, Merck Adjuvant 65 (Merck and Company, Inc.. Rahway, N.J.) or Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Michigan).
• adjuvants such as Amphigen (oil-in-water).
• Alhydrogel aluminum hydroxide
• Amphigen oil-in-water
• Alhydrogel aluminum hydroxide
• the proportion of immunogen and adjuvant can be varied over a broad range so long as both are present in effective amounts.
• aluminum hydroxide can be present in an amount of about 0.5% of the vaccine mixture (Al O 3 basis).
• the vaccines are formulated to contain a final concentration of immunogen in the range of from 0.2 to 200 ⁇ g/ml, preferably 5 to 50 ⁇ g/ml, most preferably 15 ⁇ g/ml.
• the vaccine may be incorporated into a sterile container which is then sealed and stored at a low temperature, for example 4°C. or it may be freeze-dried. Lyophilisation permits long-term storage in a stabilised form.
• the effectiveness of an adjuvant may be determined by measuring the amount of antibodies directed against an immunogenic polypeptide containing a TeNT antigenic sequence resulting from administration of this polypeptide in vaccines which are also comprised of the various adjuvants.
• the vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly.
• Additional formulations which are suitable for other modes of administration include suppositories and. in some cases, oral formulations.
• suppositories traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%. preferably 1% to 2%.
• Oral formulations include such normally employed excipients as. for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate. sodium saccharine, cellulose, magnesium carbonate, and the like.
• compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10% to 95% of active ingredient, preferably 25% to 70%.
• the vaccine composition is lyophilised.
• the lyophilised material may be reconstituted prior to administration, e.g. as a suspension. Reconstitution is preferably effected in buffer
• Capsules, tablets and pills for oral administration to a patient may be provided with an enteric coating comprising, for example, Eudragit "S”, Eudragit "L”, cellulose acetate, cellulose acetate phthalate or hydroxypropylmethyl cellulose.
• the polypeptides of the invention may be formulated into the vaccine as neutral or salt forms.
• Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric and maleic. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine. 2-ethylamino ethanol. histidine and procaine.
• the vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective.
• the quantity to be administered which is generally in the range of 5 ⁇ g to 250 ⁇ g of antigen per dose, depends on the subject to be treated, capacity of the subject's immune system to synthesize antibodies, and the degree of protection desired. A preferable range is from about 20 ⁇ g to about 40 ⁇ g per dose.
• a suitable dose size is about 0.5 ml. Accordingly, a dose for intramuscular injection, for example, would comprise 0.5 ml containing 20 ⁇ g of immunogen in admixture with 0.5%o aluminum hydroxide.
• Precise amounts of active ingredient required to be administered may depend on the judgement of the practitioner and may be peculiar to each subject.
• the vaccine may be given in a single dose schedule, or preferably in a multiple dose schedule.
• a multiple dose schedule is one in which a primary course of vaccination may be with 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reinforce the immune response, for example, at 1 to 4 months for a second dose, and if needed, a subsequent dose(s) after several months.
• the dosage regimen will also, at least in part, be determined by the need of the individual and be dependent upon the judgement of the practitioner.
• the vaccine containing the immunogenic TeNT antigen(s) may be administered in conjunction with other immunoregulatory agents, for example, immunoglobulins.
• the immunogenic polypeptides prepared as described above can be used to produce antibodies, both polyclonal and monoclonal. If polyclonal antibodies are desired, a selected mammal (e.g.. mouse, rabbit, goat, horse, etc.) is immunised with an immunogenic polypeptide bearing a TeNT epitope(s). Serum from the immunised animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to a TeNT epitope contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art.
• Monoclonal antibodies directed against TeNT epitopes in the polypeptides of the invention can also be readily produced by one skilled in the art.
• the general methodology for making monoclonal antibodies by hybridomas is well known.
• Immortal antibody- producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein- Barr virus.
• Panels of monoclonal antibodies produced against TeNT epitopes can be screened for various properties; i.e., for isotype and epitope affinity.
• An alternative technique involves screening phage display libraries where, for example the phage express scFv fragments on the surface of their coat with a large variety of complementarity determining regions (CDRs). This technique is well known in the art.
• Antibodies both monoclonal and polyclonal, which are directed against TeNT epitopes are particularly useful in diagnosis, and those which are neutralising are useful in passive immunotherapy.
• Monoclonal antibodies in particular, may be used to raise anti-idiotype antibodies.
• Anti-idiotype antibodies are immunoglobulins which carry an "internal image" of the antigen of the infectious agent against which protection is desired.
• anti-idiotype antibodies may also be useful for treatment of C. tetani, as well as for an elucidation of the immunogenic regions of C. tetani antigens.
• the term "antibody”, unless specified to the contrary, includes fragments of whole antibodies which retain their binding activity for a target antigen. Such fragments include Fv, F(ab') and F(ab') 2 fragments, as well as single chain antibodies (scFv). Furthermore, the antibodies and fragments thereof may be humanised antibodies, for example as described in EP-A-239400.
• Figure 1 is a 3D representation of the structure of tetanus toxin fragment C.
• Figure 2 is a graph showing % binding to gangliosides compared with wild type for various TeNT fragment C mutants.
• E. coli strain BL21 ( ⁇ DE3, ompT, hsdS H (r B -m B -), gal, dcm) was used as the host for the plasmids described below.
• Plasmid pKSl contains a codon-optimised gene for the expression of the He fragment of TeNT under the control of the T7 promoter.
• the Ndel - BamHl fragment was purified by agarose gel electrophoresis using a Qiaex II gel purification kit (Qiagen, West Wales, UK), and sublconed into pET28a (Novagen, Cambridge UK) which had previously been digested with Ndel and BamHl (Roche Molecular Biochemicals, East Wales, UK). DNA manipulations were performed by standard procedures.
• Mutagenesis was performed using the QuikChangeTM site-directed mutagenesis kit (Stratagene). Pairs of complementary oligonucleotides were used to construct mutant He molecules (see Table 1). To create mutants containing single amino acid mutations (Ml 5, M24. M667, M564 and M57), the following cycles were used with pKSl as template DNA: Step 1 : 95°C 30 sec, step 2: 95 °C 30 sec, step 3: 55 °C 1 min. step 4: 68 °C 12 min. Steps 2 to 4 were repeated for 12 cycles. TABLE 1 Mutants of TeNT Re constructed
• Step 1 95°C 30 sec.
• step 2 95 °C 30 sec.
• step 3 55 °C 1 min
• step 4 68 °C 18 min. Steps 2 to 4 were repeated
• DNA sequencing The DNA sequence of the entire insert of pKSl encoding HC and of all mutants in the regions altered was determined by automated DNA sequencing.
• cell pellets were resuspended in 10 ml of buffer A (300 mM NaCl, 50 mM NaH 2 P0 4 , pH 8.0) containing lysozyme (1 mg/ml) (Roche Molecular Biochemicals) and 10 mM imidazole (Sigma, Dorset, UK).
• buffer A 300 mM NaCl, 50 mM NaH 2 P0 4 , pH 8.0
• lysozyme (1 mg/ml)
• 10 mM imidazole 10 mM imidazole
• His-tagged proteins were eluted by application of elution buffer (buffer A containing 250 mM imidazole). The protein concentration of the eluted fractions was monitored by measurement at OD 280 . Peak fractions were pooled and dialysed in buffer A, and protein concentration was measured using a Micro BCA protein assay (Pierce, Chester, UK). Between 20 and 32 mg of purified protein was obtained per litre of bacterial culture.
• Biotinylation of HC protein was carried out using the EZ-Link Sulfo-NHS-LC- biotinylation kit (Pierce).
• ELISA plates (Life Technologies. Paisley, UK) were coated with 10 ⁇ g/ml bovine gangliosides GTl b (Sigma.
• GTlb binding activity of mutant proteins (Table 2) is expressed as a percentage of binding of wild-type He protein, and was calculated from the concentration of protein required to give an optical density at 490nm of 1.5, which represents half maximal binding of wild-type He protein.
• Negative controls for non-specific binding included incubation of cells with all reagents except either He protein or primary antibody.
• a variation of the direct binding assay was performed as follows. Coating of plates with gangliosides and blocking was as in the direct assay, and all subsequent incubations were at 37°C in 50 ⁇ l volumes. All washes were for 3 min in PBS/T and repeated three times. Proteins and secondary antibodies were diluted in PBS/TB. A constant amount of biotinylated His-tagged He (prepared as described above) at a concentration of 340 ⁇ g/ml in a volume of 25 ⁇ l was mixed with 25 ⁇ l of test protein at decreasing concentrations (highest concentration of 500 ⁇ g/ml).
• N18 RE-105 cells (ECACC number 881 12301) were cultured in DMEM (Life Technologies) supplemented with 4 mM glutamine (Sigma), 10% FBS and lOOu penicillin, 100 ⁇ g/ml streptomycin (Life Technologies).
• DMEM Dulbecco's modified Eagle's medium
• FBS FBS
• lOOu penicillin 100 ⁇ g/ml streptomycin
• 3 x IO 4 cells in 0.5 ml were plated in each well of an 8 well LabTek chambered slide (Life Technologies) pretreated overnight with 5 ⁇ g/ml poly-L-lysine. Cells were fixed in 4%o paraformaldehyde in PBS for 10 minutes at room temperature. All subsequent incubations were carried out at 4°C.
• Rat spinal cord motoneurons were purified from El 4 rat embryos as previously described. Briefly, ventral spinal cords were dissected, dissociated with 0.025% trypsin (GIBCO- BRL. USA) for 10 minutes at 37 °C and triturated. Cells were centrifuged through a BSA cushion and then tlirough a metrizamide density gradient.
• the interphase and pellet cells were collected tlirough a BSA cushion and resuspended in 50 ⁇ l of phosphate-buffered saline (PBS) with 0.5% BSA (PBS-BSA) plus 50 ⁇ l of mouse anti-rat p75 hybridoma supernatant, which recognises the p75 low affinity nerve growth factor (NGF) receptor specifically expressed by rat motoneurons at E14.
• PBS phosphate-buffered saline
• PBS-BSA BSA
• NGF nerve growth factor
• Cells were incubated with the p75 hybridoma supernatant at 12°C for 15 minutes, washed with PBS-BSA and centrifuged through a BSA cushion.
• the pellet was resuspended in 80 ⁇ l of PBS-BSA and incubated with 20 ⁇ l of goat anti-mouse IgG magnetic microbeads (Miltenyi Biotec, Bergisch -26-
• Cultures were maintained in a humidified 7.5% C0 2 incubator at 37°C in Neurobasal medium (Life Technologies Gibco- BRL, USA) containing B27 supplement (Gibco-BRL, USA), 2% horse serum, 0.5 mM L- glutamine, 25 ⁇ M 2-mercaptoefhanol (Fluka, UK), 10 ng/ml rat ciliary neurotrophic factor and 100 pg/ml rat glial cell line-derived neurotrophic factor (both from R&D systems, USA). L-glutamate (25 ⁇ M) was added to the medium for the first four days in culture.
• mice 10 week old B6D2F1 mice were obtained from Harlan-Olac and were anaesthetised by intramuscular injection with Vetalar (l OOmg/ml) and Rompun (20 mg/ml) in a 2:1 ratio.
• mice Whilst under general anaesthesia, groups of three mice were injected in the tongue with 22 ⁇ l of PBS or PBS containing 70 - 85 ⁇ g protein using a 25 ⁇ l Hamilton syringe with a 33 gauge needle. All animals recovered quickly and were left for 24 hrs to allow the injected protein to be transported to the brainstem. The mice were killed by transcardiac perfusion with PBS, followed by 4% paraformaldehyde in PBS whilst under deep anaesthesia. Brains were dissected and post-fixed for at least 1 hour in 4% formaldehyde before being transferred to 15% sucrose/PBS at 4°C for at least 16 lus. In experiment 1 (Fig. 6, panels a-c), brains were mounted in Tissue-Tek before sectioning into 20 ⁇ m slices using a cryostat. In experiment 2 (Fig. 6, panels d-h), brains were frozen in liquid nitrogen before sectioning.
• Sections were collected on slides which were precoated with poly-L-lysine (Sigma). All subsequent washes were in PBS for 5 minutes and repeated three times. After ringing with a wax pen. the sections were washed. After blocking for 30 mins with PBS/5%) normal swine sera (Dako) sections were incubated with PBS/1% BSA/0.1% Triton X -100 containing rabbit anti-Hc polyclonal antibody diluted 1 :500 for 2 hours. After washing, sections were incubated with biotinylated swine anti-rabbit antibody (Dako) diluted 1 :500 in PBS for 2 hours.
• Dako biotinylated swine anti-rabbit antibody
• mutant M13 ⁇ 131 1-1315
• mutant M13 ⁇ 131 1-1315
• mutant M13 ⁇ 131 1-1315
• mutant M13 ⁇ 131 1-1315
• mutant M13 ⁇ 131 1-1315
• mutant M13 ⁇ 131 1-1315
• mutant M13 ⁇ 131 1-1315
• mutant M13 ⁇ 131 1-1315
• mutant M13 ⁇ 131 1-1315
• mutant M13 ⁇ 131 1-1315
• mutant M13 ⁇ 131 1-1315
• Table 2 To assess the role of individual amino acids within these 10 residues, several mutants containing single amino acid substitutions were constructed and GTlb ganglioside binding measured. These mutant proteins, T1308A. D1309A and E1310A all retained between 80 - 100% of the GTlb binding activity of wild-type He (Table 2).
• a number of other single amino acid substitutions within the ⁇ trefoil domain were also constructed and in all cases the proteins were found to have wild-type levels of GTlb binding activity (data not shown).
• Mutant M28 retained only 5.19% of the GTlb binding activity of wild type He fragment, while mutant M37 displayed an even lower level of binding, having only 1.06% of the activity of wild-type He fragment (Fig. 2 and Table 2).
• deletion of six residues resulted in 5% of wild type ganglioside binding, and deletion of the twelve residues resulted in only 1 % of wild-type binding.
• a second surface exposed loop was identified in the region of D1214 - N1219, connecting two ⁇ sheets within the ⁇ trefoil domain.
• Mutant M58 ( ⁇ D1214 - N1219) exhibited substantially reduced ganglioside GTlb binding, at a level of 0.6 % of wild type He fragment (Fig. 2 and Table 2).
• Example 3 Binding to primary motoneurones is retained by Histidine 1293 mutants and reduced in loop mutants.
• Mutant M37 ( ⁇ H1271-D1282) containing a 12 amino acid deletion in the second loop studied also showed very much reduced binding to mon ⁇ ' spinal cord cells, as did mutant M28 ( ⁇ Q1274-P1279) which contains a 6 amino acid deletion within the same loop region.
• mutant M58. M37 and M28 the reduced binding of gangliosides GTlb (Fig. 2) is consistent with reduced binding to neuronal cells suggesting that residues within loops 1214-1219 and 1274-1279 are important for both ganglioside and cell binding.
• the cell binding activities of the Histidine 1293 mutants were also studied using primary motoneurones. Both the HI 293 A and H1293S mutant proteins appeared to bind as strongly to primary cells as wild-type He protein as.
• mice were injected in the tongue with wild-type or mutant proteins O 01/00839

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