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WO2009144240A1 - Composition et procédé de dosage et de traitement de l'anthrax - Google Patents

Composition et procédé de dosage et de traitement de l'anthrax Download PDF

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Publication number
WO2009144240A1
WO2009144240A1 PCT/EP2009/056432 EP2009056432W WO2009144240A1 WO 2009144240 A1 WO2009144240 A1 WO 2009144240A1 EP 2009056432 W EP2009056432 W EP 2009056432W WO 2009144240 A1 WO2009144240 A1 WO 2009144240A1
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Prior art keywords
antibody
bacillus anthracis
heavy chain
antibodies
camelid
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Patrick T Prendergast
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1278—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Bacillus (G)
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/40—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum bacterial
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary

Definitions

  • the present invention relates to compositions and methods for the treatment and/or prophylaxis of anthrax, which is caused by infection with the bacteria Bacillus anthracis.
  • camelid family comprises camels, alpacas, dromedaries and llamas.
  • Camelids have a unique immune system which allows them to produce immunoglobulins (antibodies) which inhibit enzymes. No other domestic animals can produce antibodies with such binding specificity.
  • heavy chain antibodies In addition to the production of a standard four-chain heterotetrameric immunoglobulin comprised of two heavy chains and two light chains, camilids produce a further subset of antibodies known as "heavy chain antibodies". These heavy chain antibodies are comprised of a dimer of two heavy chains. No light chains are present in heavy chain antibodies.
  • variable domain of the camilid heavy chain antibody differs in both structure and function to the variable heavy domain of the classical heterotetrameric four-chain antibody.
  • the heavy chain variable domain referred to as VHH
  • VHH has no interaction with an equivalent variable region on a complexed light chain due to the absence of such a domain.
  • the CH1 constant domain is absent in the camelid heavy chain antibody structure.
  • the exon encoding the CH1 domain is present in the genome, but this is spliced out.
  • the variable domain of the heavy chain (VHH) provides the complete antigen binding site of the antibody, this conferring the binding specificity of the antibody.
  • the VHH site is formed solely from the variable domain of the heavy chain as opposed to resulting from a complex of the light and heavy variable domains, as is found in the classical heterotetrameric antibody structure.
  • the heavy chain antibody has significant advantages over a heterotetrameric antibody, for example the comparative simplicity of structure, high affinity and specificity of binding, a greater potential to reach and interact with enzyme active sites and the ability to mimic substrate. All such advantages suggest that camelid heavy chain antibodies present advantages over heterotetrameric antibodies in relation to the design, production and application of clinically valuable serum and antibody products.
  • tumour- targeting vehicles due to their small size, soluble behaviour and because they are non-immunogenic and interact with epitopes that are less antigenic than conventional antibodies.
  • Anthrax is an infectious disease caused by toxigenic strains of the gram- positive bacterium Bacillus anthracis, which is mainly found in the environment in the form of highly resistant spores. If inhaled, a highly pathogenic form of generalized anthrax infection develops. In the lung alveoli, the spores are phagocytosed by alveolar macrophages and transported to regional lymph nodes where the spores germinate. The bacteria then produce anthrax toxins, which suppress and inhibit the innate and adaptive immune response. This suppression enables the capsulated bacterium to escape the lymph node defence barrier to reach the blood system causing bacteraemia and toxaemia, which rapidly kill the infected host.
  • the anthrax toxins consist of three distinct proteins, namely (i) protective antigen (PA; 87 kDa), (ii) edema factor (EF; 90 kDa) and (iii) lethal factor (LF; 87 kDa).
  • Protective antigen (PA) binds to at least two different cell- surface receptors. Binding is followed by the removal of an N-terminal fragment by a furin-like surface protease, which triggers the oligomerisation of the truncated PA into heptamers ((PA)7). This membrane-bound oligomer is capable of binding both EF andforming edema toxin and lethal toxin respectively.
  • a flexible loop of each PA subunit is inserted into the lipid bilayer and EF and LF leave their original position and translocate across the membrane of the organelle into the cytosol.
  • EF displays a calmodulin-dependent adenylate cyclase activity leading to an increase of cytosolic cAMP, which is similar to the activity observed in many other bacterial toxins.
  • LF is a metalloprotease enzyme that acts in the cytosol. It specifically cleaves most isoforms of mitogen-activated protein kinase (MAPK)- kinases (MEKs) close to the N terminus.
  • LF consists of four domains, with domain IV containing the active site which is a narrow crevice that hosts the peptide substrate.
  • LF is selective for peptides that contain the consensus sequence, with at least three positively charged residues followed by some hydrophobic residues that include the cleavage site.
  • MAPK binds its MEK partner via specific interactions mediated by the kinase domain and by the same N-terminal tail recognized by LF. It appears that LF binds MEK in a manner similar to the binding of MAPK to MEK, such that, MEKs cannot escape LF recognition and cleavage by mutation without severe consequences for the host cell physiology.
  • Non-toxigenic strains of B.anthracis are poorly pathogenic indicating that the anthrax toxins are significant mediators in all stages of the pathogenesis of the disease, from initial infection through to eventual death.
  • Anthrax is particularly dangerous as it is asymptomatic until the bacterium reaches the blood. Once in the blood system, B.anthracis undergoes cell division so rapidly that it is unlikely that antibiotic therapy could prevent death. In this context, it is essential to develop anti-toxin therapeutics to be used for preventive therapies alone or in combination with other therapeutics such as antibiotics. There are currently no completely effective therapeutic or prophylactic treatments for humans infected with anthrax. The potential severity and mortality linked to human infection with anthrax means that there exists an urgent need for an effective reliable and non-toxic treatment.
  • a method for the treatment and/or prophylaxis of infection of a subject with Bacillus anthracis comprising the step of:
  • a heavy chain camelid antibody, or an antigen binding portion thereof which has a binding specificity for at least one Bacillus anthracis protein for use in the preparation of a medicament for the prophylaxis and/or treatment of Bacillus anthracis infection in a subject.
  • a heavy chain camelid antibody or an antigen binding portion thereof, which has a binding specificity for at least one Bacillus anthracis protein for use in the prophylaxis and/or treatment of Bacillus anthracis infection in a subject.
  • a further aspect of the invention provides an isolated heavy chain camelid antibody, or an antigen binding portion thereof, which has a binding specificity for at least one Bacillus anthracis protein.
  • the present invention further provides an isolated antibody that specifically binds to at least one Bacillus anthracis protein, wherein at least the complementarity determining region sequences of said antibody are camelid.
  • an isolated nucleic acid which encodes a heavy chain camelid antibody, or an antigen binding portion thereof, which has a binding specificity for at least one Bacillus anthracis protein.
  • the invention further extends to host cells comprising said nucleic acid.
  • a vector comprising a nucleic acid which encodes a heavy chain camelid antibody, or an antigen binding portion thereof, which has a binding specificity for at least one Bacillus anthracis protein.
  • the vector optionally comprises an expression control sequence operably linked to the nucleic acid.
  • the invention further extends to host cells comprising, or transformed by, said vector.
  • an isolated cell line that produces a monoclonal heavy chain camelid antibody which has a binding specificity for at least one Bacillus anthracis protein.
  • a yet further aspect of the invention provides a method for the treatment and/or prophylaxis of infection of a subject with Bacillus anthracis, the method comprising the steps of: - administering to the subject a therapeutically effective amount of an isolated nucleic acid molecule encoding a heavy chain camelid antibody, or an antigen-binding portion thereof, which has a binding specificity for at least one Bacillus anthracis protein; and - expressing the nucleic acid molecule in the subject.
  • a yet further aspect of the present invention provides a pharmaceutical composition comprising a heavy chain camelid antibody, or an antigen- binding portion thereof, which has binding specificity for at least one Bacillus anthracis protein, and a pharmaceutically acceptable excipient or carrier.
  • a still further aspect of the present invention provides a method for producing a heavy chain camelid antibody which has a binding specificity for at least one Bacillus anthracis protein, said method comprising the steps of:
  • a further aspect of the present invention provides for the use of heavy chain camelid antibodies with a binding specificity for Bacillus anthracis proteins in the preparation of a vaccine for immunising a subject against infection with Bacillus anthracis.
  • a therapeutically effective amount of an isolated heavy chain camelid antibody, or an antigen binding portion thereof, along with a therapeutically effective amount of a suitable antibiotic in the preparation of a combined medicament for the treatment and/or prophylaxis of infection of a subject with Bacillus anthracis.
  • an assay for determining the efficacy of a candidate agent for use in the treatment and/or prophylaxis of Bacillus anthracis comprising the step of:
  • kits comprising a heavy chain camelid antibody or an antigen binding portion of the invention.
  • a kit may include diagnostic or therapeutic agents.
  • a kit can also include instructions for use in a diagnostic or therapeutic method.
  • an in-vitro assay for the determination of the amount of Bacillus anthracis proteins and/or spores in a sample, said assay comprising the steps of:
  • the heavy chain camelid antibody, or the antigen binding portion thereof inhibit the enzyme activity of the at least one Bacillus anthracis protein.
  • the Bacillus anthracis protein is a toxin protein.
  • the Bacillus anthracis protein is selected from the group consisting of protective antigen, lethal factor and edema factor.
  • the antibody inhibits the metalloprotease activity of lethal factor and/or the adenylate cyclise activity of edema factor.
  • the infection with Bacillus anthracis is caused by Bacillus anthracis spores.
  • the spores are Ame spores.
  • the spores are Sterne spores.
  • the heavy chain camelid antibody is an isolated antibody. In certain embodiments, the heavy chain camelid antibody is affinity purified.
  • the heavy chain camelid antibody is a component of precipitated general gamma-globulin.
  • the heavy chain camelid antibody is provided in serum isolated from a camelid inoculated with one or more Bacillus anthracis proteins.
  • the heavy chain camelid antibody is provided as a polyclonal preparation of antibodies.
  • said isolated polyclonal mixture comprises antibodies of the classes IgGI , lgG2 and/or lgG3.
  • the heavy chain camelid antibody is a monoclonal antibody.
  • the monoclonal antibody is of the subtype lgG2.
  • the monoclonal antibody is of the subtype lgG3.
  • the monoclonal antibody is of the subtype IgGI .
  • Monoclonal antibodies can be generated using methods commonly known to the person skilled in the art through the selection of antibody-producing B cells of single specificity from the polyclonal antibody response generated following the immunisation of a camelid.
  • the method further comprises the step of administering a therapeutically effective amount of a suitable antibiotic to the subject prior to, or along with, the administration of the therapeutically effective amount of the heavy chain camelid antibody or the antigen binding portion thereof.
  • the antibiotic may be administered simultaneously with, sequentially with or separately to the heavy chain camelid antibody or the antigen binding portion thereof.
  • the antibiotic may further be administered by way of the same or a different route to the route of administration used to administer the antibody or the antigen binding portion.
  • the antibiotic may be any antibiotic suitable for use in the treatment and/or prophylaxis of anthrax.
  • the antibiotic is ciprofloxacin.
  • the composition includes an antibiotic.
  • the antibiotic may be any antibiotic suitable for use in the treatment and/or prophylaxis of anthrax.
  • the antibiotic is ciprofloxacin.
  • the antibody, or the antigen binding portion thereof is administered by the oral route.
  • the camelid is a camel, typically of the species
  • the camelid is a llama such as Lama Paccos, Lama Glama or Lama Vicugna.
  • the subject is a mammal, typically an animal or a human.
  • the camelid is immunised with an anthrax antigen.
  • the camelid is immunised with the Bacillus anthracis organism or an attenuated form thereof.
  • the camelid is immunised with the pathogenic peptides EF, LF, PA or a portion thereof.
  • the camelid is immunised with nucleic acids encoding the peptides EF, LF, PA or a portion thereof.
  • the camelid is immunised with an isolated recombinant plasmid encoded Bacillus anthracis protein.
  • the camelid is immunised with a human anthrax vaccine.
  • the camelid is immunised with the UK licensed human vaccine for anthrax (34F2) or the US licensed anthrax vaccine coded (V770-NPI-R).
  • the camelid is immunised with an anthrax virus vectored vaccine preparation constructed by cloning the protective antigen, edema factor or lethal factor gene from Bacillus anthracis into a viral vector.
  • the heavy chain camelid antibody is produced by recombinant means.
  • administration of the heavy chain camelid antibody, or the antigen binding portion thereof results in clearance of Bacillus anthracis from the subject.
  • the vaccine includes one or more adjuvants.
  • the in vitro assay includes a step of germinating the spores.
  • the unbound signal generating molecules are removed by washing.
  • the quantification of the amount of bound signal generating molecules in the assay is determined by comparing the signal generated against a pre-determined standard.
  • the assay method is a rapid assay method, which may be performed in a location other than in a laboratory, thus providing the advantage that a result can be provided immediately upon obtaining a sample and therefore the sample does not have to be stored and transported to a further location for testing.
  • a rapid camelid antibody based assay test will be well known to the person skilled in the art, who will be aware that said test can take the form of a dipstick test or a bio-barcode rapid assay test.
  • Edema factor (EF; 90 kDa) and lethal factor (LF; 87 kDa) are considered the two toxins which exhibit intracellular catalytic activity.
  • the essential requisites for potentially useful anti-LF and anti-EF heavy chain camelid antibodies which can bind to and inhibit the enzymatic activity of at least one and preferably both of these anthrax factors are: (i) at the biochemical level, the camelid antibodies should be strong inhibitors of the metalloprotease activity of LF and/or of the adenylate cyclase activity of EF; (ii) the camelid antibody or fragment thereof should act at the cellular level to be able to translocate into the cytosol, the location of EF and LF action; and (iii) at the organism level, the camelid antibodies should be delivered at sufficient concentrations to inhibit the toxicity of LF and/or EF.
  • the inventor has surprisingly shown that heavy chain antibodies or serum isolated from camelids inoculated with Bacillus anthracis (anthrax) proteins, such as protective antigen, edema factor and lethal factor, inhibit the pathogenic effects of lethal factor on cultured macrophages and protect and allow complete recovery of animals challenged with the Sterne strain spores of Bacillus anthracis.
  • Bacillus anthracis anthrax proteins
  • heavy chain camelid antibodies can be produced which act to inhibit the enzymatic and pathogenic activity of anthrax protective antigen, lethal factor and edema factor.
  • These enzyme inhibitory camelid antibodies have particular utility in the inhibition of Bacillus anthracis infection in subjects.
  • these camelid antibodies do not produce the side effects which have been observed to result from the use of chemical inhibitors currently in development, for example, the treatment of snake bite using horse antibodies and serum directed to the specific venom peptide.
  • Bacillus anthracis protein or “Bacillus anthracis proteins” as used herein is intended to refer to protein(s) derived from Bacillus anthracis in addition to recombinantly produced versions of these proteins.
  • antigen binding portion is used herein to refer to any portion of the antibody which retains the ability of the antibody to bind and inhibit the at least one Bacillus anthracis protein.
  • terapéuticaally effective amount means an amount capable of inducing one or more of the following effects: (i) inhibition of the functional activity of at least one of the Bacillus anthracis proteins; (ii) at least partial inhibition or killing of Bacillus anthracis; (iii) enhancement of an anti-bacterial immune response; and (iv) prophylaxis or relief, to some extent, of one or more of the symptoms associated with infection by Bacillus anthracis.
  • treatment is used herein to refer to any regime that can benefit a human or non-human animal.
  • the treatment may be in respect of an existing condition or may be prophylactic (preventative treatment).
  • Treatment may include curative, alleviation or prophylactic effects. More specifically, reference herein to "therapeutic” and “prophylactic” treatment is to be considered in its broadest context. The term “therapeutic” does not necessarily imply that a subject is treated until total recovery.
  • prophylactic does not necessarily mean that the subject will not eventually contract a disease condition.
  • therapeutic and prophylactic treatment includes amelioration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition.
  • prophylactic may be considered as including a reduction in the severity or the onset of a particular condition.
  • “Therapeutic” also includes a reduction in the severity of an existing condition.
  • isolated antibody as used herein means an antibody which (1 ) is free of at least some of the proteins with which it would normally be found, (2) is essentially free of other proteins from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, or (4) does not occur in nature.
  • serum refers to serum comprising heavy chain camelid antibodies having a binding specificity for at least one of the Bacillus anthracis proteins.
  • an “antibody” is an immunoglobulin, whether natural or partly or wholly synthetically produced.
  • the term also covers any polypeptide, protein or peptide having a binding domain that is, or is homologous to, an antibody binding domain. These can be derived from natural sources, or they may be partly or wholly synthetically produced.
  • heterotetrameric four-chain antibodies are the immunoglobulin isotypes and their isotypic subclasses and fragments which comprise an antigen binding domain such as Fab, scFv, Fv, dAb, Fd, and diabodies.
  • heavy chain camelid antibodies differ in the structures of heavy chain camelid antibodies, and in particular the specific absence of a light chain and a CH1 constant domain, mean that many of the fragments which result in classical four-chain antibodies cannot be extended to heavy chain camelid antibodies.
  • fragments and antigen binding portions of heavy chain camelid antibodies can be produced, such as chimeric antibodies and diabodies, as well as polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide.
  • An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or may be different. For instance, a naturally-occurring immunoglobulin has two identical binding sites, a single-chain antibody or Fab fragment has one binding site, while a "bispecific” or “bifunctional” antibody (diabody) has two different binding sites.
  • Chimeric and bispecific antibodies can be generated from heavy chain camelid antibodies.
  • a chimeric antibody may be made that comprises complementarity determining regions (CDRs) and framework regions from different antibodies.
  • CDRs complementarity determining regions
  • the CDRs of the chimeric antibody comprise all of the CDRs of the variable region of the heavy chain of a camelid anti- ⁇ ac/7/us anthracis protein antibody, while the framework regions are derived from one or more different antibodies.
  • the CDRs of the chimeric antibody comprise all of the CDRs of the variable regions of the heavy chain of a camelid anti- Bacillus anthracis protin antibody.
  • the framework regions may be from another species and may, in certain embodiments, be humanized. Alternatively, the framework regions may be from another human antibody.
  • a bispecific antibody can be generated that binds specifically to at least one Bacillus anthracis protein through one binding domain and to a second molecule through a second binding domain.
  • the bispecific antibody can be produced through recombinant molecular biological techniques, or may be physically conjugated together.
  • a single chain antibody containing more than one VHH may be generated that binds specifically to the Bacillus anthracis protein.
  • Such bispecific antibodies can be generated using techniques that are well known, for example see Fanger et al., Immunol Methods 4: 72-81 (1994) and Wright and Harris, supra, and in connection with (iii) see, e.g., Traunecker et al., Int. J. Cancer (Suppl.) 7: 51 -52 (1992).
  • antibody should be construed as covering any binding member or substance having a binding domain with the required specificity.
  • the antibody of the invention may be a monoclonal antibody, or a fragment, derivative, functional equivalent or homologue thereof.
  • the term includes any polypeptide comprising an immunoglobulin binding domain, whether natural, wholly or partially synthetic.
  • An antigen binding portion or a fragment of an antibody or of a polypeptide for use in the present invention generally means a stretch of amino acid residues of at least 5 to 7 contiguous amino acids, often at least about 7 to 9 contiguous amino acids, typically at least about 9 to 13 contiguous amino acids, more preferably at least about 20 to 30 or more contiguous amino acids and most preferably at least about 30 to 40 or more consecutive amino acids.
  • the present invention extends to derivatives of heavy chain camelid antibodies or antigen binding portions thereof.
  • a "derivative" of such an antibody or polypeptide, or of antigen binding portions thereof means an antibody or polypeptide modified by varying the amino acid sequence of the protein, e.g. by manipulation of the nucleic acid encoding the protein or by altering the protein itself.
  • Such derivatives of the natural amino acid sequence may involve insertion, addition, deletion and/or substitution of one or more amino acids, preferably while providing a peptide having interferon alpha binding activity.
  • such derivatives involve the insertion, addition, deletion and/or substitution of 25 or fewer amino acids, more preferably of 15 or fewer, even more preferably of 10 or fewer, more preferably still of 4 or fewer and most preferably of 1 or 2 amino acids only.
  • antibody includes antibodies which have been "humanised”.
  • a humanised antibody may be a modified antibody having the hypervariable region or the variable region of a camelid monoclonal antibody and the constant region of a human antibody.
  • the binding member may comprise a human constant region.
  • variable region other than the hypervariable region may also be derived from the variable region of a human antibody and/or may also be derived from a monoclonal antibody. In such case, the entire variable region may be derived from a camelid antibody and the antibody is said to be chimerised.
  • Methods for making chimerised antibodies are known in the art. Such methods include, for example, those described in U.S. patents by Boss (Celltech) and by Cabilly (Genentech). See U.S. Patent Nos. 4,816,397 and 4,816,567, respectively.
  • polyclonal antibodies refers to a heterogeneous mixture of antibodies that bind to different epitopes of the same antigen.
  • monoclonal antibodies refers to a collection of antibodies encoded by the same nucleic acid molecule. In certain embodiments, monoclonal antibodies are produced by a single hybridoma or other cell line, or by a transgenic mammal. Monoclonal antibodies typically recognize the same epitope.
  • monoclonal is not limited to any particular method for making an antibody.
  • humanized antibody refers to an antibody in which all or part of an antibody framework region is derived from a human, but all or part of one or more CDRs is derived from another species, for example a mouse.
  • the antibodies and the antigen binding portions thereof of, and for use in, the present invention may be generated wholly or partly by chemical synthesis.
  • the antibodies can be readily prepared according to well- established, standard liquid or, preferably, solid-phase peptide synthesis methods, general descriptions of which are broadly available (see, for example, in J. M. Stewart and J. D. Young, (1984), in M. Bodanzsky and A. Bodanzsky, (1984); and Applied Biosystems 430A Users Manual, ABI Inc., Foster City, California), or they may be prepared in solution, by the liquid phase method or by any combination of solid-phase, liquid phase and solution chemistry.
  • Another convenient way of producing antibodies or antibody fragments suitable for use in the present invention is to express nucleic acids encoding them by use of nucleic acids in an expression system.
  • the present invention further provides the use of an isolated nucleic acid encoding antibodies or antigen binding portions thereof which bind to the proteins of Bacillus anthracis and inhibit their activity.
  • Nucleic acids for use in accordance with the present invention may comprise DNA or RNA and may be wholly or partially synthetic.
  • the nucleic acid for use in the invention codes for antibodies or antigen binding portions of the invention as defined above. The skilled person will be able to determine substitutions, deletions and/or additions to such nucleic acids which will still provide the antibodies or antigen binding portions of the present invention.
  • Nucleic acid sequences encoding antibodies or antigen binding portions for use in the present invention can be readily prepared by the skilled person using the information and references contained herein and techniques known in the art (for example, see Sambrook et al.(1989), and Ausubel et al, (1992)), given the nucleic acid sequences and clones available. These techniques include (i) the use of the polymerase chain reaction (PCR) to amplify samples of such nucleic acid, e.g. from genomic sources, (ii) chemical synthesis or (iii) preparing cDNA sequences.
  • PCR polymerase chain reaction
  • DNA encoding antibody fragments may be generated and used in any suitable way known to those of skill in the art, including by taking encoding DNA, identifying suitable restriction enzyme recognition sites either side of the portion to be expressed and cutting out said portion from the DNA. The portion may then be operably linked to a suitable promoter in a standard commercially available expression system. Another recombinant approach is to amplify the relevant portion of the DNA with suitable PCR primers. Modifications to the sequences can be made, e.g. using site directed mutagenesis, to lead to the expression of modified peptide or to take account of codon preferences in the host cells used to express the nucleic acid.
  • the nucleic acid may be comprised as constructs in the form of a plasmid, vector, transcription or expression cassette which comprises at least one nucleic acid as described above.
  • the construct may be comprised within a recombinant host cell which comprises one or more constructs as above.
  • Expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the nucleic acid. Following production by expression, the antibody or antigen binding portions may be isolated and/or purified using any suitable technique, and then used as appropriate.
  • Suitable host cells include bacteria, mammalian cells, yeast, and baculovirus systems.
  • Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells and NSO mouse myeloma cells.
  • a common bacterial host is E. coli.
  • antibodies or antibody fragments for use in the invention may be produced in transgenic organisms, for example primates, camelids, avians, fish, insects or plants using methods known in the art.
  • a nucleic acid encoding the antibodies or the antigen binding portions thereof may be introduced to the cell or embryo by methods including, but not limited to, direct injection, electroporation, nuclear transfer techniques or by use of vectors, e.g. viral vectors such as lentiviral vectors.
  • Suitable vectors can be chosen or constructed containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • Vectors may be plasmids, viral e.g. bacteriophage, or phagemid, as appropriate.
  • plasmids viral e.g. bacteriophage, or phagemid, as appropriate.
  • Many known techniques and protocols for manipulation of nucleic acids for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Ausubel et al. eds., (1992).
  • the nucleic acid may be introduced into a host cell by any suitable means.
  • the introduction may employ any available technique.
  • suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus.
  • suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage.
  • Marker genes such as antibiotic resistance or sensitivity genes may be used in identifying clones containing the nucleic acid of interest, as is well known in the art.
  • the introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells under conditions for expression of the gene.
  • the nucleic acid may be integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences which promote recombination with the genome in accordance with standard techniques.
  • the nucleic acid may be on an extra-chromosomal vector within the cell, or otherwise identifiably heterologous or foreign to the cell.
  • Antibodies and antigen binding portions thereof of, and for use in, the present invention may be administered to a subject in need of treatment via any suitable route.
  • the camelid antibodies, or the antigen binding portions thereof, of the invention may be administered orally without the conditions of the stomach or gastro-intestinal tract adversely affecting the ability of the antibody or the antigen binding portion to effect its use as defined herein.
  • Some suitable routes of administration include (but are not limited to) oral, rectal, nasal, topical (including buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) administration or administration via oral or nasal inhalation.
  • the composition is deliverable as an injectable composition, is administered orally or is administered to the lungs as an aerosol via oral or nasal inhalation.
  • the active ingredient will be in a suitable pharmaceutical formulation and may be delivered using a mechanical form including, but not restricted to an inhaler or nebuliser device.
  • a SPAG small particulate aerosol generator
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as sodium chloride injection, Ringer's injection and Lactated Ringer's injection.
  • Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
  • compositions for oral administration may be in tablet, capsule, powder or liquid form.
  • a tablet may comprise a solid carrier such as gelatin.
  • Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • composition may also be administered via microspheres, liposomes, other microparticulate delivery systems or sustained release formulations placed in certain tissues including blood.
  • sustained release carriers include semipermeable polymer matrices in the form of shared articles, e.g. suppositories or microcapsules.
  • Implantable or microcapsular sustained release matrices include polylactides (US Patent No.
  • EP-A-0058481 copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al, Biopolymers 22(1 ): 547-556, 1985), poly (2-hydroxyethyl-methacrylate) or ethylene vinyl acetate (Langer et al, J. Biomed. Mater. Res. 15: 167-277, 1981 , and Langer, Chem. Tech. 12:98-105, 1982).
  • Antibodies and antigen binding portions thereof of, and for use in, the present invention may be administered alone but will preferably be administered as a pharmaceutical composition, which will generally comprise a suitable pharmaceutical excipient, diluent or carrier selected dependent on the intended route of administration.
  • the present invention extends to a pharmaceutical composition for the treatment of Bacillus anthracis in a subject, wherein the composition comprises at least one isolated camelid heavy chain antibody or an antigen binding portion thereof.
  • compositions according to the present invention and for use in accordance with the present invention may comprise, in addition to the active ingredient (i.e. the isolated heavy chain camelid antibody or antigen binding portion thereof), a pharmaceutically acceptable excipient, carrier, buffer stabiliser or other material well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • a pharmaceutically acceptable excipient i.e. the isolated heavy chain camelid antibody or antigen binding portion thereof
  • carrier i.e. the isolated heavy chain camelid antibody or antigen binding portion thereof
  • buffer stabiliser such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier or other material will depend on the route of administration, which may be, for example, oral, intravenous, and intranasal or via oral or nasal inhalation.
  • the formulation may be a liquid, for example, a physiologic salt solution containing non-phosphate buffer at pH 6.8-7.6, or a lyophilised or freeze dried powder.
  • the isolated blood serum, or specifically isolated antibody or antibodies of the invention, or the antigen binding portions which may be derived there from, are preferably administered to an individual in a therapeutically effective amount, this being sufficient to show benefit to the subject.
  • the optimal dose can be determined by physicians based on a number of parameters including, for example, age, sex, weight, severity of the condition being treated, the active ingredient being administered and the route of administration.
  • An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of camelid serum or an isolated antibody or antigen binding portion of the invention is 0.025 to 50 mg/kg, more preferably 0.1 to 50 mg/kg, more preferably 0.1 -25, 0.1 to 10 or 0.1 to 3 mg/kg.
  • a formulation contains 5 mg/mL of antibody in a buffer of 20 mM sodium acetate, pH 5.5, 140 mM NaCI, and 0.2 mg/mL polysorbate 80. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated.
  • chimeric antibodies that comprise at least a portion of a human sequence and another species' sequence are provided.
  • such a chimeric antibody may result in an immune response in a host which is reduced in comparison with the immune response observed with an antibody without that host's antibody sequences.
  • an animal of interest may be used as a model for a particular human disease. In order to study the effect of an antibody on that disease in the animal host, one could use an antibody from a different species, but, in certain instances such antibodies from another species may elicit an immune response to the antibodies themselves in the host animal, thus impeding evaluation of these antibodies.
  • replacing part of the amino acid sequence of a camelid heavy chain antibody with antibody amino acid sequences from the host animal may decrease the magnitude of the host animal's anti-antibody response.
  • an active agent or "a pharmacologically active agent” includes a single active agent as well a two or more different active agents in combination
  • references to “an antibody” includes mixtures of two or more antibodies as well as a single antibody, and the like.
  • the antigen used for the camelid inoculation programme was an isolated recombinant plasmid encoded toxin protein from Bacillus anthracis that contributes to pathogenesis.
  • the protein was selected from the group consisting of protective antigen, edema factor and lethal factor.
  • Another antigen that may be used is the available human anthrax vaccine, or an anthrax virus vectored vaccine preparation constructed by cloning the protective antigen gene or the lethal factor gene from Bacillus anthracis into a viral vector.
  • the immune response of the inoculated camels was monitored by Elisa analysis of anti- Bacillus anthracis IgG's. These antibody titers rose rapidly after the 25th day reaching a maximum on day 65. These high titers were maintained by boosting at 30 day intervals with 1 mg of the Bacillus anthracis antigens. Blood was withdrawn on day 65 from the jugular vein prior to each inoculation injection for examination of its ability to inhibit edema factor and lethal factor enzymes.
  • Serum was obtained by centrifugation of blood at 4000 rpm for 10 minutes. This was stored at -20 C until further use.
  • Isolation of immunoglobulin concentrate from the camelid blood Isolation of immunoglobulin was achieved by utilizing the following procedure.
  • Anticoagulant was added to whole blood after it was extracted from the jugular vein of the animal. The blood was centhfuged to separate the plasma. Any anticoagulant may be used for this purpose, including sodium citrate and heparin. Calcium was then added to the plasma to promote clotting, the conversion of fibrinogen to fibrin. However, other methods known to the skilled person are also acceptable. This mixture was then centrifuged to remove the fibrin portion. Once the fibrin was removed from plasma resulting in serum, the serum was used as a principal source of IgG for use directly as a therapeutic. Alternatively, this portion of the clotting mechanism may be inactivated through the use of various anticoagulants.
  • the defibrinated plasma was next treated with an amount of salt compound or polymer sufficient to precipitate the albumin or globulin fraction of the plasma.
  • salt compound or polymer examples include all polyphosphates, including sodium hexametaphosphate and potassium polyphosphate.
  • the globulin may also be precipitated through the addition of polyethylene glycol or ammonium sulphate.
  • the pH of the plasma solution was lowered to stabilize the albumin precipitate.
  • the pH should not be lowered below 3.5, as this will cause the proteins in the plasma to become damaged.
  • Any type of acid can be used for this purpose, so long as it is compatible with the plasma solution. Examples of suitable acids are HCI, acetic acid, H 2 SO 4 , citric acid, and H 2 PO 4 .
  • the acid was added in an amount sufficient to lower the pH of the plasma to the designated range. Generally, this amount will range from a ratio of about 1 :4 to 1 :2 acid to plasma.
  • the plasma was then centrifuged to separate the globulin fraction from the albumin fraction.
  • IgG immunoglobulin G
  • the conventional IgG antibodies (IgGI ) were found in the second eluted fraction (pH 2.7) of the protein G column.
  • This IgG consists, under reducing condition, of heavy chains with an apparent molecular weight of 55 kDa and light chains with an apparent molecular weight of 30 kDa that bind to protein A and protein G under non-reducing condition.
  • the apparent 160 kDa molecular weight matches the hetero-tetrameric IgG composed of two H and two L chains.
  • the 100 kDa apparent molecular weight corresponds to heavy chain antibodies (HC-Abs).
  • the first eluted fraction at pH 3.5 through the same protein G affinity column corresponds to the heavy chain subclass lgG3 with apparent heavy chain molecular weight of 43 kDa.
  • Heavy chain antibodies with molecular weight of 46 kDa (lgG2) were purified from protein G non adsorbed fraction through elution at pH 2.7 on protein A column. This lgG2 purified fraction, that failed to interact with protein G, was also composed of homodimeric H-chains.
  • Example 2 Experimental anti-anthrax effects in a rabbit model administered with Camilid affinity purified IgG antibodies to Anthrax toxin proteins
  • camel serum from inoculated animals contained 7mgs/ml of IgG and it had a half-life of 8hrs in the rabbit circulation so that necessitated administrating affinity purified IgG at 100 mg/kg every six hours.
  • the initial rabbit efficacy test consisted of 12 DB rabbits: six were dosed intravenously (i.v.) with affinity purified camel IgG to the anthrax toxin proteins at 100 mg/kg for 7 days and six control rabbits were dosed i.v. with physiological saline at the same times. Two hours after the first dose, all rabbits were challenged subcutaneously (s.c.) with 104 Ames spores.
  • Cipro ciprofloxacin
  • the first experimental groups received Cipro monotherapy at 5 mg/kg s.c. b.i.d (twice daily), for 2 days starting at 56 hours, and the second experimental group received combination therapy with Cipro by following the same protocol as the first group on one side of the body and affinity purified IgG at 100 mg/kg IV four times a day for one day on the other side.
  • the three rabbits that received nothing or saline died by 94 hours and, when their peritoneal cavities were swabbed and cultured, each was positive for Bacillus anthracis ames.

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Abstract

La présente invention concerne un procédé de traitement et/ou de prophylaxie d'une infection d'un sujet par Bacillus anthracis, le procédé comprenant l'étape consistant à administrer au sujet une quantité thérapeutiquement efficace d'un anticorps de camélidé à chaîne lourde, ou une partie de celui-ci de liaison à un antigène, qui a une spécificité de liaison pour au moins une protéine de Bacillus anthracis. De préférence, la protéine de Bacillus anthracis est une protéine toxine choisie dans le groupe comprenant un antigène protecteur, un facteur létal et un facteur d'œdème. Dans certains modes de réalisation, le procédé comprend en outre l'étape consistant à administrer une quantité thérapeutiquement efficace d'un antibiotique au sujet. L'invention concerne également un anticorps isolé de camélidé à chaîne lourde, ou une partie de celui-ci de liaison à un antigène, qui a une spécificité de liaison pour au moins une protéine de Bacillus anthracis.
PCT/EP2009/056432 2008-05-27 2009-05-27 Composition et procédé de dosage et de traitement de l'anthrax Ceased WO2009144240A1 (fr)

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
WO2011117886A1 (fr) * 2010-03-25 2011-09-29 Rakesh Bhatnagar Anticorps monoclonal bispécifique pouvant former une réaction croisée avec le facteur létal (lf) et le facteur œdématogène (ef), et neutraliser la toxine œdématogène (et) ainsi que la toxine létale (lt) du bacille du charbon

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EP0739981A1 (fr) * 1995-04-25 1996-10-30 Vrije Universiteit Brussel Fragments variables d'immunoglobulines-utilisation thérapeutique ou vétérinaire
US20040018193A1 (en) * 2002-03-29 2004-01-29 Ken Alibek Rapid-acting broad spectrum protection against biological threat agents
US20060258842A1 (en) * 2004-03-03 2006-11-16 Herman Groen Human anthrax toxin neutralizing monoclonal antibodies and methods of use thereof

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EP0739981A1 (fr) * 1995-04-25 1996-10-30 Vrije Universiteit Brussel Fragments variables d'immunoglobulines-utilisation thérapeutique ou vétérinaire
US20040018193A1 (en) * 2002-03-29 2004-01-29 Ken Alibek Rapid-acting broad spectrum protection against biological threat agents
US20060258842A1 (en) * 2004-03-03 2006-11-16 Herman Groen Human anthrax toxin neutralizing monoclonal antibodies and methods of use thereof

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V. KARGINOV ET AL.: "Treatment of anthrax infection with combination of ciprofloxacin and antibodies to protective antigen of Bacillus anthracis.", FEMS IMMUNOLOGY AND MEDICAL MICROBIOLOGY, vol. 40, no. 1, 15 January 2004 (2004-01-15), The Netherlands, pages 71 - 74, XP002539789 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011117886A1 (fr) * 2010-03-25 2011-09-29 Rakesh Bhatnagar Anticorps monoclonal bispécifique pouvant former une réaction croisée avec le facteur létal (lf) et le facteur œdématogène (ef), et neutraliser la toxine œdématogène (et) ainsi que la toxine létale (lt) du bacille du charbon

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