WO2000014114A1 - Peptide fragments of cholera toxin b or enterotoxin b as vaccine adjuvants - Google Patents
Peptide fragments of cholera toxin b or enterotoxin b as vaccine adjuvants Download PDFInfo
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- WO2000014114A1 WO2000014114A1 PCT/GB1999/002970 GB9902970W WO0014114A1 WO 2000014114 A1 WO2000014114 A1 WO 2000014114A1 GB 9902970 W GB9902970 W GB 9902970W WO 0014114 A1 WO0014114 A1 WO 0014114A1
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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
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- C07K14/245—Escherichia (G)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/12—Antidiarrhoeals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- 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
- C07K14/28—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Vibrionaceae (F)
-
- 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
- the present invention relates to a substance.
- the present invention relates to a substance that is capable of displaying one or more properties that are useful in medicine.
- the substance is useful for use as an immunomodulator and/or an adjuvant and/or an inhibitor of toxin-induced diarrhoea.
- the present invention relates to the use of an immunomodulatory substance in modulating an immune response - such as that associated with an autoimmune disease.
- the present invention relates to the use of the substance as an adjuvant when given in combination with a related or an unrelated antigen.
- the present invention relates to the use of the substance for inhibiting toxin-induced diarrhoea.
- the present invention also relates to an assay for screening for agents capable of interacting with the substance of the present invention.
- Eschenchia coli heat labile enterotoxin (Etx) and its closely related homologue, cholera toxin (Ctx) from Vibrio cholerae, are examples of protein toxins which bind to glycolipid receptors on host cell surfaces.
- Each toxin consists of six noncovalently linked polypeptide chains, including a single A subunit (27 kDa) and five identical B subunits (11.6 kDa) which bind to GM-1 ganglioside receptors found on the surfaces of mammalian cells (Nashar et al 1996 Proc Natl Acad Sci 93: 226-
- the A subunit is responsible for toxicity possessing adenosine diphosphate
- ADP ADP-ribosyltransferase activity
- B subunits EtxB and CtxB
- GM-1 ubiquitous cell surface glycolipid ganglioside
- EtxB and CtxB are exceptionally potent immunogens and their respective holotoxins
- Etx and Ctx (which comprise the A and B subunits) are known to be potent adjuvants when given orally in combination with unrelated antigens (Ruedl et al 1996 Vaccine 14: 792-798; Nashar et al 1993 Vaccine 11: 235; Nashar and Hirst 1995 Vaccine 13: 803; Elson and Ealding 1984 J Immunol 133: 2892; Lycke and Holmgren 1986 Immunology 59: 301).
- EtxB and CtxB have been used as carriers for other epitopes and antigens (Nashar et al 1993 ibid) and have been used as components of vaccines against cholera and E.coli mediated diarrhoeal diseases (Jetbom et al 1992 Vaccine 10: 130).
- UK Patent Application No. 2 415 419A discloses a synthetic vaccine against cholera and against heat labile toxin of E. coli comprising a conjugate of a carrier with a synthetic polypeptide corresponding to part of the sequence of CtxB.
- WO 85/02611 discloses synthetic polypeptides corresponding to particular sequences of EtxB which are deemed useful as conjugates or as an active ingredient to raise antibodies against the B subunit and for protecting a host animal against infection by entero toxins.
- WO 89/10967 discloses an amino acid sequence which represents residue numbers
- the CtxB which can be used in combination with an epitopes of a heterologous organism, such as Flagellum and/or Salmonella, in vaccine formulations with a view to providing protection against infection by the heterologous organism or to providing protection against conditions or disorders caused by an antigen of the organism.
- a heterologous organism such as Flagellum and/or Salmonella
- WO 90/03437 relates to a hybrid protein which fuses the CtxB subunit with the active sequence of a heterologous antigen which is deemed useful for vaccination purposes, particularly to help the stabilisation of heterologous antigens in the intestinal environment.
- WO 94/06465 relates to amino acid fragments which are linked, either with or without a linker, to an appropriate carrier such that the amino acid fragment linked to the carrier generates an opsonic or protective immune response to the epitopes of the fragment.
- WO95/29701 discloses a vaccine against Vibrio cholera which comprises a conjugate of cholera toxin B subunit (CTB) or a synthetic fragment peptide which consists of a portion thereof, such as peptide CTP 3 comprising the 50-64 amino acid sequence of the B chain linked to an inert carrier.
- CTB cholera toxin B subunit
- CTP 3 synthetic fragment peptide which consists of a portion thereof, such as peptide CTP 3 comprising the 50-64 amino acid sequence of the B chain linked to an inert carrier.
- WO96/26282 discloses an expression systems for expressing gene products from recombinant Bordetella strains wherein the gene product may be a cholera toxin molecule.
- WO96/34893 discloses hybrid molecules between EtxB and CtxB which may be useful as a vaccine and to prevent and or treat enterotoxin-induced illness in an individual.
- WO 98/21344 discloses an EtxB subunit which is modified to mclude an inserted antigenic peptide. The chimeric antigen-EtxB molecule is used to elicit an antibody response against an antigenic peptide in host animals.
- WO 91/07979 discloses a chimeric protein which mcludes a portion of CtxB and an epitope region of a desired antigen which are designed for use as a vaccine to elicit an immune response in a subject to a desired antigen.
- the portion of CtxB is being used as an adjuvant but not as an immunomodulator. Accordingly, none of the above cited documents relates to the use of a CtxB/EtxB peptide or part thereof as an immunotherapeutic capable of modulating the immune response.
- EtxB subunit is capable of acting as an immunomodulator in immune disorders.
- EtxB binds to GM-1 ganglioside receptors which are found on the surfaces of mammalian cells and that this binding induces differential effects on lymphocyte populations including a specific depletion of CD8+ T cells and an associated activation of B cells.
- a substance comprising any one or more of: an amino acid sequence comprising the sequence presented as SEQ ID 5 No. 2, or a variant thereof, or a homologue thereof, or a fragment thereof, or a derivative thereof, or a mimetic thereof; which substance is capable of acting in a manner that is the same as or is similar to EtxB and/or CtxB; but wherein the substance is not capable of exhibiting GM-1 binding activity.
- a substance comprising any one or more of: an amino acid sequence comprising the sequence presented as SEQ ID No. 2, or a variant thereof, or a homologue thereof, or a fragment thereof, or a derivative thereof, or a mimetic thereof; which substance is capable of acting in a manner that is the same as or is similar to loop of EtxB and/or CtxB; but wherein the substance does not exhibit GM-1 binding activity.
- a substance comprising any one or more of: an amino acid sequence comprising the sequence presented as SEQ LD No. 2, or a variant thereof, or a homologue thereof, or a fragment thereof, or a derivative thereof, or a mimetic thereof; which substance is capable of acting in a manner that is the same as or is similar to the ⁇ 4- ⁇ 2 loop of EtxB and/or CtxB; but wherein the substance does not exhibit GM-1 binding activity.
- the substance of the present invention can be an amino acid sequence or a chemical derivative thereof.
- the substance may be a synthetic peptide or a synthetic peptide variation - such as a retroinverso D peptide.
- the substance may even be an organic compound or other chemical. The latter examples are example of mimetics of SEQ ID No. 2.
- the susbstance of the present invention is capable of acting in a manner that is the same as or is similar to EtxB and/or CtxB.
- an assay for determining whether a substance is capable of acting in a manner that is the same as or is similar to EtxB and/or CtxB would be readily determinable to those skilled in the art.
- the assay may measure and/or determine an effect on cell populations, such as lymphocyte cell populations. These effects can include but are not limited to an induction of apotosis in CD8+ T cells , the enhanced activation of CD4+ T cells and the polyclonal activation of B cells.
- the assay could be based on determining and/or measuring particular cell surface marker(s) indicative of activation of certain intracellular events (e.g. meauring an increase in CD25 expression).
- the susbstance of the present invention is not capable of exhibiting GM-1 binding activity.
- the assay may utilise GM-1 bound to a solid support and wherein the substance is then passed across the bound GM-1. Elution of the substance is indicative that it does not bind to GM-1.
- the assay is that described in WO 97/02045.
- the binding activity of the substance is not necessarily dependent on a primary binding event as is found with full length Ctx and EtxB subunits. With full length Ctx and EtxB, the primary binding activity is GM-1 binding activity.
- the substance may exhibit a single binding event. However, for some cases, the substance may possess the capability of having more than one binding activity.
- the substance is substantially isolated and/or substantially pure.
- isolated and purified refer to molecules, either nucleic or amino acid sequences, that are removed from their natural environment and/or isolated or separated from at least one other component with which they are naturally associated.
- a protein may be mixed with carriers or diluents which will not interfere with the intended purpose of the substance and still be regarded as substantially isolated.
- the present invention is based on the suprising finding that there are mutants which are capable of binding to the GM-1 receptor but which lack an immunomodulatory effect. These mutants facilitate the elucidatation of the mechanism by which the B subunits of Ctx and/or Etx act, particularly vis-a-vis an immunomodulatory effect.
- Other aspects of the present invention are as follows.
- a substance according to the present invention for use in medicine for use in medicine.
- a substance according to the present invention for use as an immunomodulator for use as an immunomodulator.
- a substance according to the present invention for use as an adjuvant for use as an adjuvant.
- a substance according to the present invention for use as an inhibitor of toxin-induced diarrhoea for use as an inhibitor of toxin-induced diarrhoea.
- a substance according to the present invention wherein the substance additionally comprises an antigen or an antigenic determinant.
- a pharmaceutical composition comprising the substance according to the present invention, optionally admixed with one or more pharmaceutically acceptable carrier(s), diluent(s) or excipient(s).
- a substance according to the present invention for use in the manufacture of a medicament that is capable of treating and/or preventing and/or modulating a disease and/or condition associated with an immune disorder and/or a toxin mediated disorder.
- An assay method for determing one or agents that are capable of interacting with and/or affecting the substance according to the present invention comprising contacting the substance with an agent to be tested, and then determining whether or not the agent affects the substance.
- a method of treatment comprising administering to a subject in need of treatment of and/or prevention of and/or modulation of a disease and/or condition associated with an immune disorder and/or a toxin mediated disorder a substance according to the present invention.
- the term "immunomodulator” means a substance that is capable of modulating the immune response by inducing, for example, a differential effect on cells, such as lymphocyte cells - preferably leading to induction of apoptosis in CD8+ T cells and/or enhanced activation of CD4+ cells and or the polyclonal activation fo B cells.
- the term "differential effect on leukocyte cells” may include but is not limited to a specific depletion of CD8+ cells (through for example apotosis), the enhanced activation of CD4+ T cells and/or an associated activation of B cells.
- the immunomodulator is capable of downregulating the pathological response of Thl and/or Th2-associated immune responses.
- the immunomodulator is capable of upregulating the production of antibodies at mucosal surfaces.
- an “adjuvant” is a substance which non-specifically enhances the immune response to an antigen.
- the adjuvant is capable of acting as a mucosal adjuvant.
- the adjuvant is capable of prolonging antigen presentation and providing a sustained immuno logic memory in a mammalian subject.
- an "antigen” means an entity which, when introduced into an immunocompetent host, stimulates the production of a specific antibody or antibodies that can combine with the entity.
- the antigen may be a pure substance, a mixture of substance or soluble or paniculate material (including cells or cell fragments).
- the term includes any suitable antigenic determinant, auto-antigen, self-antigen, cross reacting antigen, alloantigen, xenoantigen, tolerogen, allergen, hapten, and immunogen, or parts thereof, as well as any combination thereof, and these terms are used interchangeably throughout the text.
- An "allergen” includes any antigen that stimulates an allergic reaction, inducing a Type I hypersensitivity reaction.
- Examples of common allergen sources include but are not limited to ragweed, rye, couch, wild oat, timothy, Bermuda, Kentucky blue, mugwortalder, birch, hazel, beech, Cupressae, oak, olive, Aspergillus spp., Cladosporium spp., Alternaria spp., Basidospores, Ascomyceteswheat, rye, oatcat, dog, horse, rabbit, guinea pig, hamsterbudgerigar, parrot, pigeon, duck, chicken, Dermatophagoides pteronyssinus, D.farinae, Euroglyphus maynei, cockroach, fly, locust, midge, seafood, legumes, peanuts, nuts, cereals, dairy products, eggs, fruits, tomatoes, mushrooms, alcoholic beverages, coffee, chocolate, penicillins, sulphonamides and other antibiotics, sulphasalazine, carbamazepine,
- antigenic determinant refers to a site on an antigen which is recognised by an antibody or T-cell receptor. Preferably it is a short peptide derived from or as part of a protein antigen. However the term is also intended to include glycopeptides and carbohydrate epitopes. The term also includes modified sequences of amino acids or carbohydrates which stimulate responses which recognise the whole organism.
- the antigenic determinant is an antigenic determinant of the infectious agent (such as a bacterium or virus) which causes the infectious disease.
- the antigenic determinant may be an antigenic determinant of gp340 or gp350 or of a latent protein (for example EBNAs 1,2 3 A, 3B, 3C and -LP, LMP-1, -2A and 2B or an EBER).
- a latent protein for example EBNAs 1,2 3 A, 3B, 3C and -LP, LMP-1, -2A and 2B or an EBER.
- the infectious agent is an influenza virus
- the antigenic determinant may be an antigenic determinant of a viral coat protein (for example haemagglutinin and neuraminidase) or of an internal protein (for example, nucleoprotein).
- the antigenic determinant may be an antigenic determinant of a bacterial toxin or adhesion factor.
- the antigenic determinant is an antigenic determinant from an autoantigen.
- the agent can be an amino acid sequence or a chemical derivative thereof.
- the substance may even be an organic compound or other chemical.
- the agent may be a nucleotide sequences - which may be sense or anti-sense sequences.
- the agent may be an antibody.
- the agent is a cell receptor that is engageable by the substance.
- inhibitor of toxin-induced diarrhoea includes any substance which is capable of affecting the activity of Etx/Ctx holotoxins such that the pathological consequences of Etx/Ctx, such as diarrhoea, may be avoided.
- the present invention demonstrates the highly surprising finding that:
- the substance of the present invention is capable of acting as an immunomodulator and/or an adjuvant and/or an inhibitor of toxin-induced diarrhoea which is capable of affecting enterotoxin mediated diarrhoeal diseases.
- the substance of the present invention is capable of acting in a manner that is the same as or is similar to EtxB and or CtxB.
- the activity of the substance of the present invention may be mediated by the "so-called" B4- ⁇ 2 loop of EtxB and CtxB, which is a flexible loop included within amino acid residues 45-65.
- EtxB molecules with point mutations at three separate sites within the B4- ⁇ 2 loop retain GM-1 binding activity, but lack other activities, such as toxicity and the capacity to upregulate CD25 and trigger apoptosis of CD8- positive T-cells.
- Ctx holotoxins comprising B subunits with mutations also show a defect in an ability to trigger electrogenic chloride secretion, the primary secretory event responsible for mediating diarrhorea.
- the binding activity of the substance is not necessarily dependent on a primary binding event as is found with full length Ctx and EtxB. With full length Ctx and EtxB, the primary binding activity is GM-1 binding activity.
- Ctx refers to the cholera toxin and the term “CtxB” refers to the B subunit of the cholera toxin. In other texts, these may sometimes be identified as CT or Ct or CTB or CtB respectively.
- Etx herein means the E. coli heat labile enterotoxin and the term “EtxB” is the B subunit of Etx. In other texts, these may sometimes be identified as LT or Lt and LTB or LtB respectively.
- the present invention relates to an substance comprising the sequence EVPGSQH (SEQ LD No 2) which is capable of acting in a manner that is the same or is similiar to EtxB and/or CtxB or a variant thereof, or a homologue thereof, or a fragment thereof or a derivative thereof or a mimetic thereof but which is not capable of exhibiting GM-1 binding activity.
- EVPGSQH SEQ LD No 2
- each B subunit of Etx or Ctx consists of a small N-terminal helix ( l), two three-stranded anti-parallel sheets (sheet I, composed of strands B2, B3, B4 and sheet II, composed of strands Bl, B5 and R6), and a long ⁇ -helix ( ⁇ 2).
- the two B sheets form a B barrel.
- the loops joining these elements of secondary structure in the B subunit can be divided into two classes, referring to the two ends of the sheets. On one end of the subunit, the "narrow" (or "A") end, the loops are generally short, involving the connections B1-B2, B3-B4, and ⁇ 2-B5 as well as the C-terminus.
- the subunit widens at the other end, with much longer loops connecting secondary structure elements ⁇ l-Bl, B2-B3, B4- ⁇ 2.
- the longest loop connects B4 and ⁇ 2 (hereinafter the "B4- ⁇ 2 loop"), includes the residues Glu 51 to Asp 59, and extends below the plane of the ⁇ sheets.
- This loop is quite flexible, but according to Sixma et al (Nature (1992) 355; 561-564) becomes distinctly less mobile after lactose binding.
- the present invention demonstrates that the B4- ⁇ 2 loop of EtxB/CtxB is responsible for the secondary binding activity and so the use of this loop in isolation from the rest of the EtxB/CtxB molecule (for example as a peptide), may permit the secondary binding activity to occur in the absence of the first.
- Selective mutation of the B4- ⁇ 2 loop, or a peptide derived from this loop may be exploited with a view to increasing the affinity of the secondary binding activity. By increasing the affinity of the secondary binding activity, the interaction with GM-1 may be further obviated.
- B4- ⁇ 2 loop of EtxB/CtxB is the entity which is responsible for the secondary binding activity of the B subunits of toxins such as the cholera toxin and heat labile E.coli toxin.
- the secondary binding activity may occur in the absence of the first and is herein after referred to as an activity or binding activity.
- the substance of the present invention comprises an isolated ⁇ 4- ⁇ 2 loop of EtxB/CtxB.
- the substance comprises a mimetic of the isolated B4- ⁇ 2 loop of EtxB/CtxB.
- the substance comprises a mimetic of the isolated B4- ⁇ 2 loop of EtxB/CtxB with a high affinity binding activity.
- the substance comprise a peptide of from about 5 to about 40 amino acids.
- the peptide has less than 25 amino acids.
- the peptide is a fusion protein, preferably the peptide has greater than 25 amino acids.
- the substance comprises the sequence VEVPGSQHIDSQ (SEQ ID No 3).
- the substance comprises the sequence GATFQVEVPGSQHIDSQKKAI (SEQ ID No 4).
- the substance comprises the sequence GETFQVEVPGSQHIDSQKKAI (SEQ ID No 5) derivable from residues 45-65 of porcine E.coli.
- the substance comprises residues 45-65 derivable from EtxB of the human variant of E.coli derivable from EtxB of the porcine variant of E.coli.
- the present invention provides a substance comprising the amino acid sequences of the present invention which is capable of acting as an immunomodulator and/or an adjuvant and/or an an inhibitor of toxin-induced diarrhoea which is capable of affecting enterotoxin mediated diarrhoeal diseases.
- the substance may also be used in assays for the identification of one or more agents capable of interacting with and/or affecting the substance activity.
- amino acid sequence refers to peptide, polypeptide sequences, protein sequences or portions thereof.
- affect includes modulation, such as treatment, prevention, suppression, alleviation, restoration, elevation, modification of the substance activity.
- modification includes but is not limited to disabling, silencing, mutating, removing, enhancing, increasing, agonising, antagonising, decreasing or blocking the substance activity.
- Preferred amino acid sequences of the invention are SEQ ID No 2 or SEQ ID No 3 or SEQ ID No 4 or SEQ LD No 5 or sequences obtainable from the substance of the present invention but also include homologous sequences obtained from any source, for example related viral/bacterial proteins, cellular homologues and synthetic peptides, as well as variants or derivatives thereof.
- the present invention covers variants, homologues or derivatives of the amino acid sequences presented herein, as well as variants, homologues or derivatives of the nucleotide sequence coding for those amino acid sequences.
- a homologous sequence is taken to include an amino acid sequence which may be at least 75, 85 or 90% identical, preferably at least 95 or 98% identical at the amino acid level over at least the 7 amino acids of SEQ ID No 2, for example as shown in the sequence listing herein.
- homology should typically be considered with respect to those regions of the sequence (such as amino acids at positions 51, 56 and 57) known to be essential for an activity which is the same or is similiar to EtxB and/or CtxB rather than non-essential neighbouring sequences.
- homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity. Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.
- % homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
- the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension. Calculation of maximum % homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties.
- a suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package
- a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
- An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs.
- GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). It is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
- % homology preferably % sequence identity.
- the software typically does this as part of the sequence comparison and generates a numerical result.
- variant or “derivative” in relation to the amino acid sequences of the present invention presented as SEQ LD No 2, SEQ ID No 3, SEQ LD No 4 and SEQ ID No 5 includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acids from or to the sequence providing the resultant entity retains an activity, preferably having at least the same and/or similiar activity as CtxB and/or EtxB.
- SEQ ID No 2 or SEQ ID No 3 or SEQ ID No 4 or SEQ LD No 5 may be modified for use in the present invention. Typically, modifications are made that maintain the activity of the sequence. Amino acid substitutions may be made, for example from 1,
- the substance of the present invention may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance.
- Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained.
- negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
- the present invention provides nucleotide sequences encoding the substance of the present invention capable of acting as a template or as targets in assays (such as a yeast two hybrid assay) for the identification of one or more agents and/or derivatives thereof capable of affecting the substance.
- nucleotide sequence refers to nucleotide sequences, oligonucleotide sequences, polynucleotide sequences and variants, homologues, fragments and derivatives thereof (such as portions thereof).
- the nucleotide sequence may be DNA or RNA of genomic or synthetic or recombinant origin which may be double-stranded or single-stranded whether representing the sense or antisense strand or combinations thereof.
- the term nucleotide sequence is prepared by use of recombinant DNA techniques (e.g. recombinant DNA).
- nucleotide sequence means DNA.
- the substance encoding nucleotide sequence may be the same as the naturally occuring form for this aspect.
- the nucleotide sequence encoding the substance is a non-native nucleotide sequence - or is a variant, homologue, fragment or derivative thereof.
- the present invention does not cover the native nucleotide coding sequence according to the present invention in its natural environment when it is under the control of its native promoter which is also in its natural environment.
- this preferred embodiment we have called this preferred embodiment the "non-native nucleotide sequence".
- naturally occurring refers to an substance with an amino acid sequence found in nature.
- biologically active refers to an substance having regulatory or biochemical functions of the naturally occurring substance.
- variant in relation to the nucleotide sequence SEQ ID No 1 of the present invention include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence providing the resultant nucleotide sequence codes for an substance having an activity, preferably having at least the same activity as the SEQ SEQ LD No 2, SEQ LD No 3, SEQ LD No 4 and SEQ LD No 5 presented in the sequence listings.
- sequence homology preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% homology to the sequences shown in the sequence listing herein. More preferably there is at least 95%, more preferably at least 98%, homology. Nucleotide homology comparisons may be conducted as described above. A preferred sequence comparison program is the GCG Wisconsin Bestfit program described above. The default scoring matrix has a match value of 10 for each identical nucleotide and -9 for each mismatch. The default gap creation penalty is -50 and the default gap extension penalty is -3 for each nucleotide.
- the present invention also encompasses nucleotide sequences that are capable of hybridising selectively to the sequences presented herein, or any variant, fragment or derivative thereof, or to the complement of any of the above.
- Nucleotide sequences are preferably at least 15 nucleotides in length, more preferably at least 20, 30, 40 or 50 nucleotides in length.
- deletion is defined as a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent.
- an "insertion” or “addition” is that change in a nucleotide or amino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues, respectively, as compared to the naturally occurring substance.
- substitution results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively.
- hybridization shall include “the process by which a strand of nucleic acid joins with a complementary strand through base pairing” as well as the process of amplification as carried out in polymerase chain reaction (PCR) technologies.
- Nucleotide sequences of the invention capable of selectively hybridising to the nucleotide sequences presented herein, or to their complement, will be generally at least 75%, preferably at least 85 or 90% and more preferably at least 95% or 98% homologous to the corresponding nucleotide sequences presented herein over a region of at least 20, preferably at least 25 or 30, for instance at least 40, 60 or 100 or more contiguous nucleotides.
- Preferred nucleotide sequences of the invention will comprise regions homologous to nucleotides comprising SEQ ID No 1 preferably at least 80 or 90% and more preferably at least 95% homologous to SEQ LD No 1.
- selective hybridizable means that the nucleotide sequence used as a probe is used under conditions where a target nucleotide sequence of the invention is found to hybridize to the probe at a level significantly above background.
- the background hybridization may occur because of other nucleotide sequences present, for example, in the cDNA or genomic DNA library being screened.
- background implies a level of signal generated by interaction between the probe and a non-specific DNA member of the library which is less than 10 fold, preferably less than 100 fold as intense as the specific interaction observed with the target DNA. The intensity of interaction
- 32 may be measured, for example, by radiolabelling the probe, e.g. with P.
- Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152, Academic Press, San Diego
- Maximum stringency typically occurs at about Tm-5°C (5°C below the Tm of the probe); high stringency at about 5°C to 10°C below Tm; intermediate stringency at about 10°C to 20°C below Tm; and low stringency at about 20°C to 25°C below Tm.
- a maximum stringency hybridization can be used to identify or detect identical nucleotide sequences while an intermediate (or low) stringency hybridization can be used to identify or detect similar or related nucleotide sequences.
- both strands of the duplex either individually or in combination, are encompassed by the present invention.
- the nucleotide sequence is single-stranded, it is to be understood that the complementary sequence of that nucleotide sequence is also included within the scope of the present invention.
- the nucleotide sequences of the present invention can be incorporated into a recombinant replicable vector.
- the vector may be used to replicate and express the nucleotide sequence in and/or from a compatible host cell. Expression may be controlled using control sequences which include promoters/enhancers and other expression regulation signals. Prokaryotic promoters and promoters functional in eukaryotic cells may be used. Tissue specific or stimuli specific promoters may be used. Chimeric promoters may also be used comprising sequence elements from two or more different promoters described above.
- the substance produced by a host recombinant cell may be secreted or may be contained intracellularly depending on the sequence and/or the vector used.
- the substance coding sequences can be designed with signal sequences which direct secretion of the substance coding sequences through a particular prokaryotic or eukaryotic cell membrane.
- the substance of the invention may also be produced as fusion proteins, for example to aid in extraction and purification.
- 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.
- the fusion protein will not hinder the activity of the substance comprising the amino acid sequence of the present invention.
- the fusion protein comprises an antigen or an antigenic determinant fused to the substance of the present invention.
- the fusion protein is a non-naturally occuring fusion protein comprising a substance which may act as an adjuvant in the sense of providing a generalised stimulation of the immune system.
- the antigen or antigenic determinant may be attached to either the amino or carboxy terminus of the substance.
- the substance of the invention may be ligated to a heterologous sequence to encode a fusion protein.
- a heterologous sequence For example, for screening of peptide libraries for agents capable of affecting the substance activity, it may be useful to encode a chimeric substance expressing a heterologous epitope that is recognized by a commercially available antibody.
- an assay for identifying an agent, such as a target receptor, or for an agent capable of regulating CD25 transcriptional activity may be conducted using a bound fusion protein.
- the substance of the present invention may be an antibody.
- This antibody may be capable of acting as a mimetic of the present invention.
- Antibodies may be produced by standard techniques, such as by immunisation with the substance of the invention or by using a phage display library.
- the term "antibody”, unless specified to the contrary, includes but is not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library.
- Such fragments include fragments of whole antibodies which retain their binding activity for a target substance, Fv, F(ab') and F(ab') 2 fragments, as well as single chain antibodies (scFv), fusion proteins and other synthetic proteins which comprise the antigen-binding site of the antibody.
- the antibodies and fragments thereof may be humanised antibodies, for example as described in substance-A-239400. Neutralizing antibodies, i.e., those which inhibit biological activity of the substance polypeptides, are especially preferred for diagnostics and therapeutics.
- the invention also provides monoclonal or polyclonal antibodies to substances of the invention such as polypeptides or fragments thereof.
- the present invention further provides a process for the production of monoclonal or polyclonal antibodies to substances, such as polypeptides of the invention.
- a selected mammal e.g., mouse, rabbit, goat, horse, etc.
- an immunogenic polypeptide bearing a epitope(s) obtainable from an identifed agent and/or substance of the present invention.
- various adjuvants may be used to increase immunological response.
- adjuvants include, but are not limited to, Freund's, mineral gels such as aluminium hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
- BCG Bacilli Calmette-Gueri ⁇
- Corynebactenum parvum are potentially useful human adjuvants which may be employed if purified the substance polypeptide is administered to immunologically compromised individuals for the purpose of stimulating systemic defence.
- Serum from the immunised animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to an epitope obtainable from an identifed agent and/or substance of the present invention 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. In order that such antibodies may be made, the invention also provides polypeptides of the invention or fragments thereof haptenised to another polypeptide for use as immunogens in -- ⁇ imals or humans.
- Monoclonal antibodies directed against epitopes obtainable from an identifed agent and/or substance of the present 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 orbit epitopes can be screened for various properties; i.e., for isotype and epitope affinity.
- Monoclonal antibodies to the substance and or identified agent of the present invention may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique originally described by Koehler and Milstein (1975 Nature 256:495-497), the human B-cell hybridoma technique (Kosbor et al (1983) Immunol Today 4:72; Cote et al (1983) Proc Natl Acad Sci 80:2026-2030) and the EBV-hybridoma technique (Cole et al (1985) Monoclonal Antibodies and Cancer Therapy, Alan R Liss Inc, pp 77-96).
- Antibodies both monoclonal and polyclonal, which are directed against epitopes obtainable from an identifed agent and/or substance of the present invention 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 substance and/or agent against which protection is desired. Techniques for raising anti-idiotype antibodies are known in the art. These anti- idiotype antibodies may also be useful in therapy.
- Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al (1989, Proc Natl Acad Sci 86: 3833-3837), and Winter G and Milstein C (1991; Nature 349:293-299).
- Antibody fragments which contain specific binding sites for the substance may also be generated.
- fragments include, but are not limited to, the F(ab') 2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the
- Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity
- the immunomodulation of the immune response may be measured by transcriptional profiling, for example, by assaying for activation of transcription of CD25 cell surface marker by measuring the signal from a linked reporter gene.
- a wide variety of reporters may be used in the assay methods of the present invention with preferred reporters providing conveniently detectable signals (eg. by spectroscopy).
- a reporter gene may encode an enzyme which catalyses a reaction which alters light absorption properties.
- reporter molecules include but are not limited to ⁇ -galactosidase, invertase, green fluorescent protein, luciferase, chloramphenicol, acetyltransferase, ⁇ - glucuronidase, exo-glucanase and glucoamylase.
- radiolabeled or fluorescent tag-labeled nucleotides can be incorporated into nascent transcripts which are then identified when bound to oligonucleotide probes.
- the production of the reporter molecule is measured by the enzymatic activity of the reporter gene product, such as ⁇ -galactosidase.
- ASSAYS FOR INHIBITORS OF TOXIN-INDUCED DIARRHOEA is measured by the enzymatic activity of the reporter gene product, such as ⁇ -galactosidase.
- the substance of the present invention or a derivative or homologue thereof and/or a cell line that expresses the substance of the present invention or a derivative or homologue thereof may be used to screen for agents (such as antibodies, peptides, organic or inorganic molecules) capable of affecting the activity of the substance.
- agents such as antibodies, peptides, organic or inorganic molecules
- any agent capable of inhibiting the activity of the substance may be screened for inhibitors of toxin-induced diarrhoea thereby identifying agents capable of affecting the cholera and/or enterotoxin mediated diarrhoeal diseases.
- the screens of the present invention may identify antagonists of the substance of the present invention, such as antibodies, peptides or small organic molecules which are capable of acting as inhibitors of toxin-induced diarrhoea
- Phage display may be employed in the identification of agents, such as a cell surface receptor that is engageable by the substance of the present invention.
- agents such as a cell surface receptor that is engageable by the substance of the present invention.
- the positive identification of such a receptor may faciliate the use of combinatorial libraries to identify mimetics capable of acting in the same or a similiar manner as the substance of the present invention.
- Phage display is a protocol of molecular screening which utilises recombinant bacteriophage.
- the technology involves transforming bacteriophage with a gene that encodes an appropriate ligand (in this case a candidate agent) capable of reacting with a target substance (or a derivative or homologue thereof) or the nucleotide sequence (or a derivative or homologue thereof) encoding same.
- the transformed bacteriophage (which preferably is tethered to a solid support) expresses the appropriate ligand (such as the candidate agent) and displays it on their phage coat.
- the entity or entities (such as cells) bearing the target substance molecules which recognises the candidate agent are isolated and amplified.
- the successful candidate agents are then characterised.
- Phage display has advantages over standard affinity ligand screening technologies.
- the phage surface displays the candidate agent in a three dimensional configuration, more closely resembling its naturally occuring conformation. This allows for more specific and higher affinity binding for screening purposes.
- the screens of the present invention may identify mimetics of the substance of the present invention, such as antibodies, or other chemical compounds which have an immunomodulatory and/or adjuvant effect.
- Such mimetics can be administered alone or in combination with other therapeutics for the treatment of diseases of the present invention.
- the substance of the present invention to be used for identifying immunomodulators, adjuvants, mimetics and/or inhibitors of toxin- induced diarrhoea in any of a variety of drug screening techniques.
- the substance employed in such a test may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The abolition of substance activity or the formation of binding complexes between the substance and the agent being tested may be measured.
- HTS high throughput screening
- the present invention also provides a pharmaceutical composition
- a pharmaceutical composition comprising administering a therapeutically effective amount of the substance of the present invention and a pharmaceutically acceptable carrier, diluent or excipients (including combinations thereof).
- the pharmaceutical compositions may be for human or animal usage in human and veterinary medicine and will typically comprise any one or more of a pharmaceutically acceptable diluent, carrier, or excipient.
- Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).
- the choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.
- the pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
- Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition.
- preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
- Antioxidants and suspending agents may be also used.
- the pharmaceutical composition of the present invention may be formulated to be delivered using a a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestable solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route.
- the formulation may be designed to be delivered by both routes.
- the agent is to be delivered mucosally through the gastrointestinal mucosa, it should be able to remain stable during transit though the gastrointestinal tract; for example, it should be resistant to proteolytic degradation, stable at acid pH and resistant to the detergent effects of bile.
- compositions can be administered by inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intravenously, intramuscularly or subcutaneously.
- compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood.
- compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.
- the substance is an adjuvant which is incorporated into a vaccine composition used to treat or prevent autoimmune disease, human T cell leukaemia, transplant rejection or graft-versus-host disease (GVHD), allergic or infectious disease.
- a vaccine composition used to treat or prevent autoimmune disease, human T cell leukaemia, transplant rejection or graft-versus-host disease (GVHD), allergic or infectious disease.
- the vaccine composition may additionally comprise an antigen(s) or antigenic determinant(s). Suitable such antigens and/or antigenic determinants are disclosed in WO 99/34817.
- the vaccine composition comprises an antigen and/or antigenic determinant.
- the antigen is a self-antigen or a homologue thereof.
- the one or more substances of the present invention is used in the preparation of a therapeutic or prophylactic vaccine.
- a “prophylactic vaccine” is a vaccine which is administered to naive individuals to prevent disease development.
- a “therapeutic vaccine” is a vaccine which is administered to individuals with an existing infection to reduce or minimise the infection or to abrogate the immunopathological consequences of the disease.
- vaccines which contain one or more substances as an 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 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 and pH buffering agents.
- the vaccine composition may also comprise a combination of adjuvants which enhance the effectiveness of the vaccine.
- additional adjuvants which , in combination, may be effective include but are not limited to: 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, Corynebactenum 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., Rah
- adjuvants such as Amphigen (oil-in-water), Alhydrogel (aluminum hydroxide), or a mixture of Amphigen and Alhydrogel are used. Only aluminum hydroxide is approved for human use.
- a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response of the particular patient.
- the dosages below are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited.
- compositions of the present invention may be administered by direct injection.
- the composition may be formulated for parenteral, mucosal, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration.
- each protein may be administered at a dose of from 0.01 to 30 mg/kg body weight, preferably from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight.
- the term "administered” includes delivery by viral or non-viral techniques.
- Viral delivery mechanisms include but are not limited to adenoviral vectors, adeno-associated viral (AAV) vectos, herpes viral vectors, retroviral vectors, lentiviral vectors, and baculoviral vectors.
- Non-viral delivery mechanisms include lipid mediated transfection, liposomes, immunoliposomes, lipofectin, cationic facial amphiphiles (CFAs) and combinations thereof.
- the routes for such delivery mechanisms include but are not limited to mucosal, nasal, oral, parenteral, gastrointestinal, topical, or sublingual routes.
- administered includes but is not limited to delivery by a mucosal route, for example, as a nasal spray or aerosol for inhalation or as an ingestable solution; a parenteral route where delivery is by an injectable form, such as, for example, an intravenous, intramuscular or subcutaneous route.
- co-administered means that the site and time of administration of each of the substance of the present invention and an additional entity such as an antigen and/or antigenic determinants are such that the necessary modulation of the immune system is achieved.
- an additional entity such as an antigen and/or antigenic determinants
- the substance and the antigen may be administered at the same moment in time and at the same site, there may be advantages in administering the substance at a different time and to a different site from the antigen.
- the substance and antigen may even be delivered in the same delivery vehicle - and the substance and the antigen may be coupled and/or uncoupled and/or genetically coupled and/or uncoupled.
- the antigenic determinant and peptide or homologue or mimetic thereof may be administered separately or co-administered to the host subject as a single dose or in multiple doses.
- the vaccine composition of the invention may be administered by a number of different routes such as injection (which includes parenteral, subcutaneous and intramuscular injection) intranasal, mucosal, oral, intra- vaginal, urethral or ocular administration.
- the vaccines comprising the substance of the present invention 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%o, may be 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 lyophilised material may be reconstituted prior to administration, e.g. as a suspension. Reconstitution is preferably effected in buffer
- the substance of the present invention is used to treat or prevent autoimmune disease, human T cell leukaemia, transplant rejection, allogeneic or xenogeneic transplant, graft-versus-host disease (GVHD), allergic or infectious diseases.
- infectious diseases are diseases in which, during infection, the infectious agent binds to, colonises or gains access across the mucosa are particularly preferred, as are diseases in which immunopathological mechanisms are commonly involved.
- infectious diseases of the present invention include but are not limited to HSV-1, HSV-2, EBV, VZV, CMV, HHV-6, HHV-7 and HHN-8, hepatitis A, B, C, D and E, Neisseria meningitides, Haemophilus influenzae type B and Streptococcus pneumoniae, Legionella pneumophila and Mycobacterium tuberculosis, Neisseria gonnorheae, HIV-1, HIV-2 and Chlamydia trachomatism, E.coli, rotavirus, Salmonella enteritidis, Salmonella typhi, Hehcobacter pylori, Bacillus cereus, Campylobacter jejuni and Vibrio cholerae, Staphylococcus aureus, Streptococcus pyogenes and Streptococcus mutans, malaria, Trypanasoma spp., Taxoplasma gondii, Leishmania donovani and
- allergic disorders of the present invention include but are not limited to diseases include asthma, allergic cough, allergic rhinitis and conjunctivitis, atopic eczema and dermatitis, urticaria, hives, insect bite allergy, dietary and certain drug allergies.
- autoimmune diseases include but are not limited to diseases such as rheumatoid arthritis, multiple sclerosis and diabetes.
- the present invention further provides diagnostic assays and kits comprising the substance of the present invention. Such kits may be used to prevent and/or treat and/or modulate the diseases of the present invention.
- the kit may also comprise an antigen and or antigenic determinant and/or a separate adjuvant for coadministration with said therapeutic or prophylactic composition.
- kits comprising mimetics of the present invention in the form of antibodies of the invention bound to a solid support and/or packaged into kits in a suitable container along with suitable reagents, controls, instructions and the like.
- the present invention relates to a substance comprising any one or more of: an amino acid sequence comprising the sequence presented as SEQ ID No. 2, or a variant thereof, or a homologue thereof, or a fragment thereof, or a derivative thereof, or a mimetic thereof; which substance is capable of acting in a manner that is the same as or is similar to EtxB and/or CtxB; but wherein the substance does not exhibit GM-1 binding activity.
- the present invention also relates to an assay method for determing one or agents that are capable of interacting with and/or affecting the substance of the present invention wherein the assay comprises contacting the substance with an agent to be tested, and then determining whether or not the agent affects the substance.
- the assay comprises contacting the substance with an agent to be tested, and then determining whether or not the agent affects the substance.
- GATFQVEVPGSQHLDSQKKAI or the sequence GETFQVEVPGSQHLDSQKKAI.
- a prophylactic or therapeutic composition which comprises a peptide according to any preceding paragraph or a homologue or mimetic thereof.
- a prophylactic or therapeutic composition according to paragraph 4 which also comprises an antigen or an antigenic determinant.
- a prophylactic or therapeutic composition according to paragraph 4 or 5, wherein the therapeutic or prophylactic agent is used to prolong antigen presentation and give sustained immunological memory in a mammalian subject.
- the therapeutic or prophylactic agent is used to downregulate the pathological components of Thl and Th2-associated immune responses.
- a prophylactic or therapeutic composition which comprises an agent which binds specifically to the B4- ⁇ 2 loop of EtxB or CtxB.
- a prophylactic or therapeutic composition according to paragraph 11 wherein the agent is an antibody.
- a vaccine composition for use against a disease comprising a peptide according to any of paragraphs 1 to 3 or a homologue or a mimetic thereof.
- a vaccine composition according to paragraph 14 which also comprises an antigenic determinant.
- a vaccine composition according to paragraph 14 or 15 which is used to treat or prevent mfections diseases, autoimmune disease, human T cell leukaemia, transplant rejection or graft-versus-host disease (GNHD) diseases.
- kits comprising a therapeutic or prophylactic composition according to any of paragraphs 4 to 13.
- Figure 1 which shows a stereo ribbon drawing showing a B subunit of Etx/Ctx (from
- FIG. 2 which shows the identification of loop residues in CtxB involved in CD8+ T- cells apoptosis
- Figure 3 which shows mutant B subunits defective in CD8+ T-cell apoptosis retain ability to bind to cell surface receptors
- Figure 4 which shows total immunoglobulin levels against EtxB and EtxB (H57S) in sera from mice immunised intranasally with lOug of each B-subunit;
- FIG. 6 which shows E51-158 B subunit peptide exhibits an ability to induce immunomodulation of CD8+ T-cells.
- mice were sacrificed and mesenteric lymph node tissue was subsequently removed into Hanks balanced salt solution (HBSS without Calcium and Magnesium and supplemented with 20mM Hepes). Lymphocytes were then dispersed into the solution and away from fibrous tissue by gently pressing the tissue through a wire mesh.
- Hanks balanced salt solution HBSS without Calcium and Magnesium and supplemented with 20mM Hepes. Lymphocytes were then dispersed into the solution and away from fibrous tissue by gently pressing the tissue through a wire mesh.
- the lymph node cells were resuspended in modified Eagle's medium (Gibco) ( -MEM) containing 20 mM Hepes, 4mM L- Glutamine, 100 IU/ml penicillin, lOOug/ml streptomycin, and 5xl0 -5 M2- mercaptoethanol, at a concentration 2 x 10° " cells/ml and then mixed without (PBS control) or with 3.45uM (40 ug/ml) of wild-type CtxB or wild-type EtxB, or various mutant B-subunits, namely EtxB(G33D), CtxB(E51A), CtxB(V52A), CtxB(P53A), CtxB(G54A), CtxB(S55A), CtxB(Q56A), CtxB(H57A), or CtxB(I58A) and incubated at 37°C for 96h
- the cells were washed and resuspended in 0.4ml HBSS/20mM Hepes/0.1% sodium azide/10%> rat serum.
- Phycoerythrin (PE) conjugated anti-CD8 (PharMingen) and FITC-conjugated anti-CD4 (PharMingen) were added at a dilution of 1/400 and the cells incubated on ice for a period of 30 min. Following antibody incubations, the cell suspensions were washed once in ISOTON (Becton-Dickinson) and resuspended in 0.4ml ISOTON. FACS analysis was carried out, with 10,000 events collected for each sample and then plotted using WinMDI software.
- the cells with FITC-bound anti-CD4 are depicted in the top-left hand quadrant of each figure; the cells with PE-bound anti-CD8 are depicted in the bottom right-hand quadrant, with the percentage number of events in each quadrant shown.
- Mutant B-subunits defective in CD8+ T-cell apoptosis retain ability to bind to cell surface receptors
- mice were sacrificed and mesenteric lymph node tissue was subsequently removed into Hanks balanced salt solution (HBSS without Calcium and Magnesium and supplemented with 20mM Hepes). Lymphocytes were then dispersed into the solution and away from fibrous tissue by gently pressing the tissue through a wire mesh. Following 3 washes in HBSS, the lymph cells were resuspended in 300 ml of pre-cooled and de-gassed MACS buffer (PBS, 5mM EDTA, 0.5% BSA, pH7.2). 50ml each of anti CD4 and anti-B220 MACS antibodies were added to the cells and the CD8 T-cell population purified by negative selection in a magnetic MACS column. The CD8+ T-cells containing population were washed and resuspended in modified Eagle's medium (Gibco) (cc-MEM) containing 20 mM Hepes, 4mM L-
- cc-MEM modified Eagle's medium
- Glutamine 100 IU/ml penicillin, lOOug/ml streptomycin, and 5xl0 -5 M2- mercaptoethanol, at a concentration 2 x 10° " cells/ml and then mixed without (PBS control) or with 3.45uM (40 ug/ml) of wild-type CtxB or wild-type EtxB, or various mutant B-subunits, namely EtxB(G33D), CtxB(E51A), CtxB(V52A), CtxB(P53A), CtxB(G54A), CtxB(S55A), CtxB(Q56A), CtxB(H57A), EtxB(H57S), or CtxB(I58A) and incubated on ice for 20 min.
- Anti-EtxB monoclonal antibody 118-8 was added at a dilution of 1/500 to cells incubated EtxB, EtxB(G33D) or EtxB(H57S) and anti-CtxB monoclonal antibody LT-39 was added at a dilution of 1/800 to cells incubated with CtxB and CtxB mutants.
- FITC-labelled anti-mouse IgG antibody After 30 min, the cells were washed and resuspended in HBSS/20mM Hepes/0.1%) sodium azide/10%) rat serum followed by addition of a FITC-labelled anti-mouse IgG antibody. Following incubation with the secondary antibody for 30 min, the cell suspensions were washed once in ISOTON (Becton-Dickinson) and resuspended in 0.4ml ISOTON. Levels of FITC fluorescence as a representative of the extent of binding of EtxB, CtxB and the various mutants to CD8+ T-cells was assessed by FACS analysis.
- two groups of mice were intranasally immunised with lOug EtxB or CtxB as negative controls. All mice were sacrificed 14 days after the third immunisation and the blood removed by cardiac puncture.
- the sera were then analysed for levels of anti-Ova IgG antibodies by an ELISA using microtitre plates coated with 5ug/ml Ova. End point titres were determined (equivalent to a dilution giving an absorbance of 0.1 above background).
- mesenteric lymph node cells were isolated as described in Example 1, and incubated with various concentrations (0.1 uM - 20uM) of the EVPGSQHI peptide or a randomly selected control peptide, LRNETTTTKGDYC. After 96h incubation at 37°C the cells were washed and resuspended in 0.4ml HBSS/20mM Hepes/0.1%) sodium azide/10% rat serum and then assessed for the relative proportion of CD4+ and CD 8+ cells by FACS analysis, in an identical fashion to that reported in Example 1.
Abstract
Description
Claims
Priority Applications (15)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
IL14169599A IL141695A0 (en) | 1998-09-07 | 1999-09-07 | Peptide fragments of cholera toxin b or enterotoxin b as vaccine adjuvants |
JP2000568871A JP2002524469A (en) | 1998-09-07 | 1999-09-07 | Peptide fragment of cholera toxin B or enterotoxin B as vaccine adjuvant |
EP99944696A EP1109828A1 (en) | 1998-09-07 | 1999-09-07 | Peptide fragments of cholera toxin b or enterotoxin b as vaccine adjuvants |
AU57516/99A AU772015B2 (en) | 1998-09-07 | 1999-09-07 | Peptide fragments of cholera toxin B or enterotoxin B as vaccine adjuvants |
GB0107375A GB2357507A (en) | 1998-09-07 | 1999-09-07 | Peptide fragments of cholera toxin B or enterotoxin B as vaccine adjuvants |
CA002338384A CA2338384A1 (en) | 1998-09-07 | 1999-09-07 | Peptide fragments of cholera toxin b or enterotoxin b as vaccine adjuvants |
PL99346549A PL346549A1 (en) | 1998-09-07 | 1999-09-07 | Peptide fragments of cholera toxin b or enterotoxin b as vaccine adjuvants |
MXPA01002387A MXPA01002387A (en) | 1998-09-07 | 1999-09-07 | Peptide fragments of cholera toxin b or enterotoxin b as vaccine adjuvants. |
EA200100314A EA200100314A1 (en) | 1998-09-07 | 1999-09-07 | PEPTIDE FRAGLES OF CHOLER EXOTOXIN IN OR ENTEROXINE IN AS AN ADJUVANT VACCINE |
NZ509631A NZ509631A (en) | 1998-09-07 | 1999-09-07 | Peptide fragments of cholera toxin B or enterotoxin B as vaccine adjuvants |
HU0104090A HUP0104090A2 (en) | 1998-09-07 | 1999-09-07 | Peptide fragments of cholera toxin b or enterotoxin b as vaccine adjuvants |
BR9913501-9A BR9913501A (en) | 1998-09-07 | 1999-09-07 | Cholera toxin b peptide fragments or enterotoxin b as vaccine adjuvants |
KR1020017002217A KR20010044842A (en) | 1998-09-07 | 1999-09-07 | Peptide fragments of cholera toxin B or enterotoxin B as vaccine adjuvants |
IS5829A IS5829A (en) | 1998-09-07 | 2001-01-26 | Peptide fragments of cholera toxin B or Iratatoxin B as vaccine adjuvants |
NO20011075A NO20011075L (en) | 1998-09-07 | 2001-03-02 | Peptide fragments of cholera toxin or enterotoxin as vaccine adjuvants |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB9819484.8 | 1998-09-07 | ||
GBGB9819484.8A GB9819484D0 (en) | 1998-09-07 | 1998-09-07 | Therapeutic agents |
Publications (1)
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WO2000014114A1 true WO2000014114A1 (en) | 2000-03-16 |
Family
ID=10838466
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB1999/002970 WO2000014114A1 (en) | 1998-09-07 | 1999-09-07 | Peptide fragments of cholera toxin b or enterotoxin b as vaccine adjuvants |
Country Status (19)
Country | Link |
---|---|
EP (1) | EP1109828A1 (en) |
JP (1) | JP2002524469A (en) |
KR (1) | KR20010044842A (en) |
CN (1) | CN1317013A (en) |
AU (1) | AU772015B2 (en) |
BR (1) | BR9913501A (en) |
CA (1) | CA2338384A1 (en) |
CZ (1) | CZ2001839A3 (en) |
EA (1) | EA200100314A1 (en) |
GB (2) | GB9819484D0 (en) |
HU (1) | HUP0104090A2 (en) |
IL (1) | IL141695A0 (en) |
IS (1) | IS5829A (en) |
MX (1) | MXPA01002387A (en) |
NO (1) | NO20011075L (en) |
NZ (1) | NZ509631A (en) |
PL (1) | PL346549A1 (en) |
WO (1) | WO2000014114A1 (en) |
ZA (1) | ZA200100758B (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2003000899A1 (en) * | 2001-06-22 | 2003-01-03 | University Of Bristol | MUTANT FORMS OF EtxB AND CtxB AND THEIR USE AS CARRIERS |
US7588763B2 (en) | 1995-07-05 | 2009-09-15 | Trident Pharmaceuticals, Inc. | Methods for the prevention against or treatment of diabetes with ETXB and/or insulin |
US8329196B2 (en) | 2002-11-26 | 2012-12-11 | Alk-Abello A/S | Allergen dosage form |
US20130345114A1 (en) * | 1998-01-09 | 2013-12-26 | Trident Pharmaceuticals, Inc. | Agent for Treating Allergic or Hypersensitivity Condition |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104402974B (en) * | 2014-12-10 | 2017-11-14 | 重庆医科大学 | A kind of polypeptide with mucosal adjuvant activity and its purposes in mucosal adjuvant is prepared |
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DE3430894A1 (en) * | 1983-08-23 | 1985-03-14 | Yeda Research And Development Co., Ltd., Rehovot | VACCINE AGAINST CHOLERA AND AGAINST HEAT LABILES E. COLI-TOXIN |
WO1985002611A1 (en) * | 1983-12-12 | 1985-06-20 | Scripps Clinic And Research Foundation | Synthetic polypeptide corresponding to a portion of the heat-labile enterotoxin of escherichia coli, compositions and methods therewith |
WO1990003437A1 (en) * | 1988-09-27 | 1990-04-05 | L'universite De L'etat A Liege | Proteins for fusing the sub-unit b of the choleraic toxin and heterologous antigen, and nucleic acids encoding them |
WO1995020657A1 (en) * | 1994-01-27 | 1995-08-03 | Gx Biosystems A/S | Receptor specific bacterial adhesins and their use |
WO1995029701A1 (en) * | 1994-05-03 | 1995-11-09 | Yeda Research And Development Co., Ltd. | Vaccines for oral immunization against infecting agents |
WO1996006627A1 (en) * | 1994-08-26 | 1996-03-07 | The Administrators Of The Tulane Educational Fund | Mutant enterotoxin effective as a non-toxic oral adjuvant |
-
1998
- 1998-09-07 GB GBGB9819484.8A patent/GB9819484D0/en not_active Ceased
-
1999
- 1999-09-07 AU AU57516/99A patent/AU772015B2/en not_active Ceased
- 1999-09-07 CZ CZ2001839A patent/CZ2001839A3/en unknown
- 1999-09-07 CA CA002338384A patent/CA2338384A1/en not_active Abandoned
- 1999-09-07 IL IL14169599A patent/IL141695A0/en unknown
- 1999-09-07 HU HU0104090A patent/HUP0104090A2/en unknown
- 1999-09-07 NZ NZ509631A patent/NZ509631A/en unknown
- 1999-09-07 WO PCT/GB1999/002970 patent/WO2000014114A1/en not_active Application Discontinuation
- 1999-09-07 GB GB0107375A patent/GB2357507A/en not_active Withdrawn
- 1999-09-07 JP JP2000568871A patent/JP2002524469A/en not_active Withdrawn
- 1999-09-07 EP EP99944696A patent/EP1109828A1/en not_active Withdrawn
- 1999-09-07 CN CN99810666A patent/CN1317013A/en active Pending
- 1999-09-07 BR BR9913501-9A patent/BR9913501A/en not_active IP Right Cessation
- 1999-09-07 EA EA200100314A patent/EA200100314A1/en unknown
- 1999-09-07 KR KR1020017002217A patent/KR20010044842A/en not_active Application Discontinuation
- 1999-09-07 PL PL99346549A patent/PL346549A1/en not_active Application Discontinuation
- 1999-09-07 MX MXPA01002387A patent/MXPA01002387A/en unknown
-
2001
- 2001-01-26 ZA ZA200100758A patent/ZA200100758B/en unknown
- 2001-01-26 IS IS5829A patent/IS5829A/en unknown
- 2001-03-02 NO NO20011075A patent/NO20011075L/en unknown
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EP0095426A1 (en) * | 1982-05-26 | 1983-11-30 | CNRS, Centre National de la Recherche Scientifique | Synthetic polypeptides containing at least one sequence of the choleric B1 sub-unit toxin, and medicines containing them |
DE3430894A1 (en) * | 1983-08-23 | 1985-03-14 | Yeda Research And Development Co., Ltd., Rehovot | VACCINE AGAINST CHOLERA AND AGAINST HEAT LABILES E. COLI-TOXIN |
WO1985002611A1 (en) * | 1983-12-12 | 1985-06-20 | Scripps Clinic And Research Foundation | Synthetic polypeptide corresponding to a portion of the heat-labile enterotoxin of escherichia coli, compositions and methods therewith |
WO1990003437A1 (en) * | 1988-09-27 | 1990-04-05 | L'universite De L'etat A Liege | Proteins for fusing the sub-unit b of the choleraic toxin and heterologous antigen, and nucleic acids encoding them |
WO1995020657A1 (en) * | 1994-01-27 | 1995-08-03 | Gx Biosystems A/S | Receptor specific bacterial adhesins and their use |
WO1995029701A1 (en) * | 1994-05-03 | 1995-11-09 | Yeda Research And Development Co., Ltd. | Vaccines for oral immunization against infecting agents |
WO1996006627A1 (en) * | 1994-08-26 | 1996-03-07 | The Administrators Of The Tulane Educational Fund | Mutant enterotoxin effective as a non-toxic oral adjuvant |
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7588763B2 (en) | 1995-07-05 | 2009-09-15 | Trident Pharmaceuticals, Inc. | Methods for the prevention against or treatment of diabetes with ETXB and/or insulin |
US20130345114A1 (en) * | 1998-01-09 | 2013-12-26 | Trident Pharmaceuticals, Inc. | Agent for Treating Allergic or Hypersensitivity Condition |
US8992940B2 (en) * | 1998-01-09 | 2015-03-31 | Trident Pharmaceuticals, Inc. | Agent for treating allergic or hypersensitivity condition |
WO2003000899A1 (en) * | 2001-06-22 | 2003-01-03 | University Of Bristol | MUTANT FORMS OF EtxB AND CtxB AND THEIR USE AS CARRIERS |
US7422752B2 (en) | 2001-06-22 | 2008-09-09 | Hunter Immunology Ltd. | Mutant forms of EtxB and CtxB and their use as carriers |
US8425917B2 (en) | 2001-06-22 | 2013-04-23 | Hunter Immunology Ltd | Mutant forms of EtxB and CtxB and their use as carriers |
US8329196B2 (en) | 2002-11-26 | 2012-12-11 | Alk-Abello A/S | Allergen dosage form |
US9408805B2 (en) | 2002-11-26 | 2016-08-09 | Alk-Abello A/S | Allergen dosage form |
US9415015B2 (en) | 2002-11-26 | 2016-08-16 | Alk-Abello A/S | Allergen dosage form |
US10080719B2 (en) | 2002-11-26 | 2018-09-25 | Alk-Abelló A/S | Allergen dosage form |
US10471008B2 (en) | 2002-11-26 | 2019-11-12 | Alk-Abelló A/S | Allergen dosage form |
Also Published As
Publication number | Publication date |
---|---|
IL141695A0 (en) | 2002-03-10 |
NZ509631A (en) | 2003-11-28 |
GB2357507A (en) | 2001-06-27 |
EA200100314A1 (en) | 2001-10-22 |
CA2338384A1 (en) | 2000-03-16 |
AU772015B2 (en) | 2004-04-08 |
MXPA01002387A (en) | 2002-05-08 |
GB0107375D0 (en) | 2001-05-16 |
CZ2001839A3 (en) | 2001-08-15 |
GB9819484D0 (en) | 1998-10-28 |
JP2002524469A (en) | 2002-08-06 |
EP1109828A1 (en) | 2001-06-27 |
IS5829A (en) | 2001-03-07 |
NO20011075L (en) | 2001-05-07 |
ZA200100758B (en) | 2001-07-16 |
KR20010044842A (en) | 2001-06-05 |
NO20011075D0 (en) | 2001-03-02 |
HUP0104090A2 (en) | 2002-01-28 |
PL346549A1 (en) | 2002-02-11 |
CN1317013A (en) | 2001-10-10 |
AU5751699A (en) | 2000-03-27 |
BR9913501A (en) | 2001-06-05 |
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