WO2013087874A1 - Anticorps dirigés contre la toxine cdt de c. difficile - Google Patents

Anticorps dirigés contre la toxine cdt de c. difficile Download PDF

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Publication number
WO2013087874A1
WO2013087874A1 PCT/EP2012/075617 EP2012075617W WO2013087874A1 WO 2013087874 A1 WO2013087874 A1 WO 2013087874A1 EP 2012075617 W EP2012075617 W EP 2012075617W WO 2013087874 A1 WO2013087874 A1 WO 2013087874A1
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Prior art keywords
antigen
seq
sequences
cdr
binding polypeptide
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PCT/EP2012/075617
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German (de)
English (en)
Inventor
Friedrich Nolte
Mandy UNGER
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Universitätsklinikum Hamburg-Eppendorf
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Publication of WO2013087874A1 publication Critical patent/WO2013087874A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
    • 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
    • C07K16/1282—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Clostridium (G)
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the invention relates to antigen-binding polypeptides comprising the CDR1, CDR2 and CDR3 region of a VHH domain of a camelid heavy chain antibody.
  • the polypeptides specifically bind the CDTa subunit of the CDT toxin of Clostridi ⁇ to difficile and are therefore particularly suitable for the thera ⁇ Promotionic and prophylactic treatment of a disease caused by the CDT toxin as well as for the diagnosis of such a disease.
  • the invention relates to the use of such antigen-binding polypeptides in methods for detecting the CDTa subunit of the CDT toxin of Clostridium difficile in a biological sample. Methods for purifying and / or concentrating the CDTa subunit of the Clostridium difficile CDT toxin using the antigen-binding polypeptides are also provided.
  • C. difficile is an anaerobic Gram-positive bacterium ⁇ , which is part of the normal intestinal flora in a healthy person.
  • CDAD Clostridium difficile associated diseases
  • ToxA Toxin A
  • ToxB Toxin B
  • GT glycosyltransferase
  • CP cysteine protease
  • RBD receptor binding domain
  • the toxic activity of the ⁇ ser multidomain proteins is due to the transferase by glycosyl (GT) domain catalyzed glycosylation of huma ⁇ NEN Rho-GTPase at the position of Thr-37 (Cook Nolte, et al. (2001), J. Biotechnol., 92, 81-87).
  • GT glycosyl
  • Rho family of GTPases leading to the redistribution of the actin cyto- keletts, for the disintegration of the cell-cell contacts of the intestine ⁇ pithels and thus leads to loss of barrier function (AK tories & Barbieri (2005) Nat Rev Microbiol., 3 (5): 397-410).
  • CDT binary toxin
  • CDTb has a molecular weight of 99 kDa and binds to the cell receptor LSR (Lipolysis-stimulated lipoprotein receptor), which leads to the internalization of CDTa into the cytosol (Patapheodorou et al. (2011), Proc Natl Acad Sei USA 108 (39 ): 16422-27).
  • CDTa has a molecular weight of 48 kDa and is the enzymatically active subunit of the toxin.
  • CDTa catalyzes the ADP-ribosylation of actin in the cytosol, which ultimately leads to complete collapse of the tissue cell cytoskeleton.
  • fragments of voll miti ⁇ gen IgG antibodies were, such as Fv, Fab, F (ab ') and F (ab') 2, used for therapeutic purposes. These fragments have ever ⁇ but often compared to the complete antibody reduced specificity and / or affinity for the An ⁇ term on. Furthermore, these fragments often show only a ge ⁇ rings solubility in aqueous solutions.
  • camelid antibodies have properties which make them particularly suitable for therapeutic use in humans.
  • produ ⁇ adorn the camelids, antibodies which consist exclusively of heavy chains (Hamers-Casterman et al. (1993), Na 363: 446-448; Wesolowski et al. (2009), Med Microbiol Immunol. 198 (3): 157-74).
  • heavy chain antibodies are homodimers of two identical heavy chains that interact with the antigen through a single variable domain called VHH (variable domain of the heavy chain of heavy chain antibodies).
  • the VHH domain has three complementarity Re ⁇ regions (complementary determining regions, CDRs) and four framework regions (framework regions, FR), alternating with the CDRs in the primary sequence of the VHH domain.
  • the VHH domain of a heavy chain antibody forms a small polypeptide moiety with high antigen binding capacity. Due to an unusually long CDR3 region having a length of about 7 to 25 amino acids, the VHH domain may be different than conventional anti ⁇ body in cavities of protein antigens bind and so, for example, block the active site of an enzyme (De Genst et al. (2006), Proc Natl Acad. USA 103 (12): 4586-4591).
  • VHH domains can be produced recombinantly as soluble proteins in bacteria or mammalian cells. Such recombinant Herge ⁇ presented VHH domains are also called single domain antibodies (sin- gle-domain antibodies, sdAbs) or nano-bodies ®. Nanobody ®, a human serum protein (von Willebrand factor), is currently being tested in a Phase II clinical trial (van Loon et al. (2011), Thromb Haemost 106 (1): 165-71, Ulrichts et al. (2011)). , Blood 118 (3): 757-65).
  • VHH single domain antibodies are not only stable but also about ten times smaller than conventional antibodies and white ⁇ sen in addition to high solubility markedly better self sheep ⁇ th in tissue penetration on. For this reason, VHH single domain antibodies have been proposed for the treatment of various diseases.
  • International application WO 2006/122825 uses VHH single domain antibodies against von Willebrand factor for the treatment of arterial occlusive diseases.
  • International application WO 2010/042815 discloses VHH single domain antibodies that can be used to treat cancers.
  • the present invention provides VHH single domain antibodies prepared which are capable of binding specifically to a toxin produced by C. difficile, and to this neutra ⁇ taping.
  • the antibodies are therefore particularly suitable for use in the diagnosis and / or therapeutic treatment of diseases caused by toxins from a CDTa-expressing strain of C. difficile.
  • VHH single domain antibodies directed against ToxA and ToxB for use in neutralizing the toxins have been previously described in the prior art (Hussack et al. (2011), J Biol Chem., 286 (11): 8961-76). VHH single domain antibodies directed against the CDTa subunit of the CDT toxin are unknown in the art.
  • RNA was isolated from the peripheral blood lymphocytes of the llamas and converted into cDNA by means of reverse transcription.
  • the variable domains of the heavy chain antibodies were amplified by PCR.
  • phage display libraries were prepared. These libraries were selected using recombinant CDTa in a panning procedure. The sequences of the selected single domain antibodies were subsequently determined by sequencing and expressed recombinantly in bacterial host cells.
  • the present invention provides single domain VHH antibodies suitable for use in therapeutic and / or diagnostic methods.
  • the present invention provides CDR regions of VHH single domain antibodies that can be used in the construction of recombinant, antigen-binding polypeptides.
  • the invention provides an antigen-binding polypeptide comprising the complementarity-determining regions CDR1, CDR2 and CDR3 of a VHH domain of a camelid heavy chain antibody necessary for the binding of a VHH single domain antibody; Polypep ⁇ tid specifically binds to the CDTa subunit of the CDT toxin of C. difficile.
  • the polypeptides of the invention are thus characterized by CDR regions derived from heavy chain antibodies of camelids.
  • the Schwereketten- antibodies is a special group of In ⁇ munglobulinen, which consist exclusively of heavy chains.
  • a heavy chain of such antibody contains ne ⁇ ben two constant domains, a single variable domain, again comprising three CDR regions and four FR regions.
  • the interaction with the antigen occurs exclusively via this variable domain, which is also referred to as VHH domain.
  • VHH domain By pronounced loop structures in the CDR3 a VHH domain is able to NEN ⁇ erken also cryptic epitopes.
  • Heavy chain antibodies are produced in only a few species, eg camelids.
  • the group of camelids is a mammal family of the order Artiodactyla (Cloven hoofed animals).
  • the camelid family includes camels, dromedaries, llamas, alpacas and vicunas.
  • ⁇ DERS the arrival includes antigen-binding polypeptide CDR regions that are derived from a VHH domain of a llama.
  • the polypeptide according to the invention has a specific Affini ⁇ ty for CDTa subunit produced by C. difficile toxin CDT. This means that an inventive Po ⁇ lypeptid binds with a binding affinity to the CDTa-subunit, which is significantly higher than the binding affinity for the binding to another protein that is not homologous to the sequence of CDTa subunit.
  • the binding affinity of the polypeptide of the invention is at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold, 250-fold, 500-fold. 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 5000-fold, or 10000-fold higher than the binding affinity for a protein not homologous to the CDTa subunit.
  • the binding affinity is preferably in the range from min ⁇ least 10 -6 M.
  • Particularly preferred antigen-binding Polypep ⁇ tide of the present invention have a binding affinity with a dissociation constant (K d) in the range of at least 10 -6 M, and preferably in the range of at least 10 ⁇ 7 sts, 10 ⁇ 8 sts, 10 "9 sts, 10 " 10 sts, 10 "11 sts, or 10 " 12 sts.
  • K d dissociation constant
  • the CDTa-specific, antigen-binding polypeptide comprises the CDR regions CDR1, CDR2 and CDR3 of a VHH domain of a Kame ⁇ lid heavy chain antibody, these CDR regions:
  • antigen-binding polypeptides having CDRs which differ from the sequences shown in SEQ ID NO: 15-56 in individual amino acid positions. More particularly, the invention relates to antigen-binding polypeptides having CDR sequences that differ from the CDR sequences depicted in SEQ ID NOs: 15-56 in at most one amino acid per CDR.
  • the antigen-binding polypeptide of the invention comprises a complete VHH domain of a camelid heavy chain antibody.
  • VHH single domain antibody When used alone, the VHH domain is capable of binding to the corresponding antigen and is therefore also referred to herein as "VHH single domain antibody".
  • the VHH domain also includes the four framework regions, FR1-FR4, which are arranged between the CDR regions.
  • the Gerüstregi ⁇ tions can be fully preserved in the inventive VHH domain, or they can be in the form of fragments of the original framework sequences.
  • the framework sequences which delimit the VHH domain C-terminal and N-terminal can be shortened by one or more amino acids.
  • modified VHH domains are expressly encompassed by the invention.
  • VHH single domain antibody refers to an antigen-binding polypeptide that lacks the constant domains CH2 or CH3 as compared to a complete heavy chain antibody.
  • Preferred antigen-binding polypeptides comprising a VHH domain of a camelid heavy chain antibody and specific binding ⁇ fish toxin of C. difficile to the CDTa subunit of CDT are shown in SEQ ID NO: 1-14.
  • the invention also extends also to variants of SEQ ID NO: 1-14 shown polypeptides leading to the in SEQ ID NO: 14 homologous l- shown polypeptides and also the ability to bind the Weg ⁇ CDTa subunit of the CDT have toxins of C. difficile.
  • the term "variant" refers to an amino acid sequence that has been modified by the deletion, substitution or addition of amino acids to be at least 80%, 85%, 90%, 91%, 92%, 93%, 94%.
  • variants in particular in the framework regions (FR1-FR4), can deviate from the VHH regions shown in SEQ ID NO: 1-14 without adversely affecting the binding affinity of the polypeptides.
  • the variants differ from the polypeptides shown in SEQ ID NO: 1-14 only by the replacement of a few amino acids. It is particularly preferred that variants of the polypeptides shown in SEQ ID NO: 1-14 of these in less than 20, in less than 15, in less than 10, in less than 5, for example in 4, 3, 2 or in 1 amino acid position (s), different.
  • the invention thus relates to a CDTa-specific, antigen-binding polypeptide comprising a Ami ⁇ acid sequence selected from the group ⁇ be detached from:
  • the substitutions by which the variants differ from the VHH regions depicted in SEQ ID NOs: 1-14 are conservative substitutions, ie, substitutions of one or more amino acid residues by an amino acid of similar polarity that acts as a functional equivalent
  • the exchange serving as Amino Text ⁇ re-radical selected from the same group of amino acids such as to be replaced amino acid residue.
  • a hy ⁇ drophober radical may be replaced by another hydrophobic residue or a polar residue with another polar residue having the same charge.
  • Functionally homologous amino acids which are used for the conservative substitution Kgs ⁇ NEN example, include non-polar amino acids such as glycine, valine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, and tryptophan.
  • non-polar amino acids such as glycine, valine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, and tryptophan.
  • uncharged polar ami Nucleic acids include serine, threonine, glutamine, asparagine, tyrosine, and cysteine.
  • charged polar (acidic) amino acids include histidine, arginine and lysine.
  • charged polar (basic) amino acids include aspartic acid and glutamic acid.
  • variants of the sequences shown in SEQ ID NO: 1-14 also include those polypeptides in which one or more amino acids have been inserted into the amino acid sequence of one of the polypeptides shown in SEQ ID NO: 14. Such insertions may in principle to the at each position in SEQ ID NO: 1-14 shown carried polypeptides, as long as the resulting Vari ⁇ ante of the polypeptide (ie, the ability to bind and inhibit the CDTa subunit) at ⁇ retains its immunological activity. Further, in the present case also such proteins as Va ⁇ are variants of a sequence shown in SEQ ID NO: 1-14 shown polypeptides in which one or more amino acids within the sequence are missing in comparison to the Referenzpolypeptid.
  • deletions may involve any amino acid positions within the sequences shown in SEQ ID NO: 1-14.
  • the deletions may be those comprising two or more (eg 3, 4 or 5) contiguous amino acids from any one of the sequences of SEQ ID NOS: 1-14.
  • immunologically active fragments of the polypeptides shown in SEQ ID NO: 1-14 are understood as meaning those sequences which differ from the amino acid sequences shown in SEQ ID NO: 1-14 or from their variants by the absence of one or more amino acids at the N-terminus and / or at the C-terminus of the protein, and which are further capable of specifically binding the CDTa subunit of the CDT toxin.
  • immunologically active fragments of SEQ ID NO may play at ⁇ : l sequence shown to be fragments, in which the first two N-terminal and / or C-terminal amino acids of the SEQ ID NO: l missing polypeptide.
  • One skilled in the art will readily be able to determine the ability of fragments to bind to the CDTa subunit of the CDT toxin, eg, by competitive antibody assays.
  • polypeptides shown in SEQ ID NOs: 1-14 and their variants or fragments may be further structurally modified at one or more positions of the amino acid sequence, such as by introducing a modified amino acid.
  • these modified amino acids can be amino acids which have been modified by biotinylation, phosphorylation, glycosylation, acetylation, branching and / or cyclization.
  • the variants or fragments of the OF INVENTION ⁇ to the invention antigen-binding polypeptides maintain more than 50% of the original binding affinity for the CDTa subunit of the toxin CDT of C. difficile. Even more preferred is that the variants or fragments contain more than 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the original binding affinity of the antigen-binding polypeptides of the invention, eg the polypeptides shown in SEQ ID NO: 1-14.
  • the binding affinity for the CDTa subunit of the C. difficile CDT toxin can be routinely measured by in vitro assays.
  • the antigen-binding polypeptide is preferably of a size that results in physico-chemical properties which are particularly suitable for therapeutic and / or diagnostic applications, such as good solubility and tissue ⁇ bepenetration, thermal stability, and similar natural sheep ⁇ th.
  • the antigen is binding polypeptides of the present invention have a size of 80-250 amino acids, with a size of 90-200, 90-180, 90-150, 100-140, 100-130, 100-120 being particularly preferred.
  • binding of the antigen-binding polypeptide to CDTa results in the inhibition or reduction of enzyme activity or toxicity of the bound subunit and / or the entire toxin.
  • the enzyme activity is reduced to approximately 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less of the activity of the unbound enzyme.
  • the enzyme activity of the bound subunit and / or all of the toxin is completely neutra ⁇ llibrary by binding the polypeptide of the invention.
  • CDTa-specific antigen-binding polypeptides described above are used in conjunction with CDTb-specific antigen-binding polypeptides.
  • the binding of CDTa and CDTb-specific Polypepti ⁇ the CDT to the toxin leads to a particularly efficient inhibition or reduction of the enzyme activity or toxicity of the CDT subunit or the entire toxin.
  • the enzyme activity is reduced to approximately 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less of the activity of the unbound enzyme.
  • the invention provides in a further aspect also relates to a CDTB-specific antigen-binding polypeptide, which comprises the notwen for the binding of a VHH single-domain antibody ⁇ ended complementarity determining regions CDR1, CDR2 and CDR3 of a V HH domain of a camelid heavy chain antibody wherein the polypeptide specifically binds to the CDTb subunit of the C. difficile CDT toxin (SEQ ID NO: 85).
  • the CDTb-specific antigen-binding polypeptide comprises the CDR regions CDR1, CDR2 and CDR3 of a VHH domain of a Kame ⁇ lid heavy chain antibody, these CDR regions:
  • a CDTB specific antigen-binding polypeptide comprising a Ami ⁇ acid sequence selected from the group ⁇ be detached from:
  • variants, modifications and immunologically active fragments are also shown in SEQ ID NOS: 65-69 and 86-101 Polypeptides of the invention, wherein the terms "Va ⁇ variants", “modification” and “immunologically active fragments” belongs to the same meaning as set forth above in connection with the CDTa-specific antigen-binding polypeptides.
  • the CDTb-specific antigen-binding polypeptides can be used either together with or without CDTa-specific antigen-binding polypeptides.
  • the binding of CDTb-specific polypeptides to the CDTb subunit of the CDT toxin results in efficient inhibition or reduction of the enzyme activity or toxicity of the CDTb subunit or the entire toxin.
  • VHH single-domain antibodies are distinguished from conventional antibodies from mammals particularly characterized in that only three CDRs for the binding of the antigen are erfor ⁇ sary.
  • the invention therefore also relates to such anti ⁇ gen-binding polypeptides, the only necessary for the binding of the toxin regions CDR1, CDR2 and CDR3 in one of the SEQ ID NO: include polypeptides exemplified 1-14, 65-69 and 86-101, wherein the regions located between the CDR regions show little or even substantially no similarity to the corresponding regions of any of the polypeptides shown in SEQ ID NOs: 1-14, 65-69, and 86-101.
  • the invention provides a nucleic acid molecule which codes for an antigen-binding polypeptide as defined above.
  • the nucleic acid molecule may be DNA or RNA. Preferably, it is single-stranded or double-stranded DNA, such as - Il ⁇
  • genomic DNA or cDNA Preferably, the poly ⁇ nucleotide encoding a polypeptide having a sequence shown in SEQ ID NO: l- amino acid sequences shown 14, 65-69 and 86-101.
  • the invention further includes an expression vector comprising a nucleic acid molecule encoding an antigen-binding polypeptide of the present invention.
  • Expression vectors are usually DNA constructs that have a replication origin that allows the vector to replicate independently of the host cell.
  • expression vectors comprise regulatory control elements which are functionally linked to the nucleotide sequences to be expressed and control their transcription and / or translation.
  • Suitable control elements can Proka ⁇ ryontician promoters (constitutive or regulatable, such as lac, trp, T3, T7 or gpt promoter) eukaryotic promoters ⁇ factors (constitutive or regulatable, such as thymidine kinase promoter, SV40 promoter or CMV promoter), operators, transcriptional or translational enhancers, transcription terminators, ribosome binding sites, polyadenylation signals, selection markers, silencers and repressor sequences.
  • Proka ⁇ ryontician promoters consisttitutive or regulatable, such as lac, trp, T3, T7 or gpt promoter
  • eukaryotic promoters ⁇ factors constitutive or regulatable, such as thymidine kinase promoter, SV40 promoter or CMV promoter
  • operators transcriptional or translational enhancers
  • transcription terminators transcription terminators
  • ribosome binding sites poly
  • Examples are the prokaryotic vectors pHEN2, pGEM, pBluescript and pUC, which are regularly used for heterologous expression in E. coli.
  • Examples of eukaryotic Expressi ⁇ onsvektoren include pcDNA3.1, pOG44, pSV2CAT Rc / CMV, Rc / RSV, GEM1 and the like.
  • the invention relates to a host cell comprising a nucleic acid molecule ⁇ encoding an antigen-binding poly ⁇ peptide of the present invention, or an expression vector comprising such a nucleic acid molecule.
  • a host cell comprising a nucleic acid molecule ⁇ encoding an antigen-binding poly ⁇ peptide of the present invention, or an expression vector comprising such a nucleic acid molecule.
  • It can be a prokaryotic or a Euka ⁇ ryontician host cell in particular.
  • Examples of host cells that can be used in the invention include pro-karyontician host cells, such as bacterial staem ⁇ me of Escherichia coli, Bacillus subtilis and the like.
  • Suitable eukaryotic cells include yeast cells, insect cells, mammalian cells, plant cells, and the like.
  • the arrival antigen-binding polypeptide of the present invention is expressed before ⁇ preferably in bacterial cells, such as E. coli.
  • the method of transforming or transfecting the host cell with an expression vector comprising a nucleic acid molecule encoding the antigen-binding polypeptide will depend on the type of expression vector and the host cell used. The person skilled in the art will readily be able to select suitable vectors and host cells for recombinant expression.
  • the invention also provides a method for the recombinant production of an antigen-binding polypeptide, in which a host cell as described above under Bedin ⁇ conditions culturing (a) which allow the expression of a Nukleinklaremole- CRWT that of an antigen-binding polypeptide encodes present invention; and (b) isolating the antigen-binding polypeptide from the culture.
  • the antigen-binding polypeptides of the present invention are particularly suitable for use in a method for the therapeutic treatment of a disease caused by CDT toxin of C. difficile.
  • Such therapeutically ⁇ tables treatment includes both the treatment of acute disease state and prophylactic treatment to prevent disease.
  • the polypeptides of the present invention are also useful in the diagnosis of a disease caused by CDT toxin of C. difficile.
  • the antigen-binding polypeptides of the present invention are used for the therapeutic treatment of pseudomembranous colitis.
  • the CDTa-specific antigen-binding polypeptides of the invention are preferably used in conjunction with CDTb-specific antigen-binding polypeptides for therapeutic treatment.
  • the CDTa-specific antigen-binding polypeptides are administered together with CDTb-specific antigen-binding polypeptides.
  • the CDTB-specific rule antigen-binding polypeptides are used in a further embodiment without CDTa-specific antigen-binding poly ⁇ peptide for therapeutic treatment.
  • the treatmen ⁇ lung comprises the administration of one or more of the polypeptides of the invention to a patient in whom an infection was detected with a CDTa-expressing strain of C. difficile that is suspected to be infected with the pathogen, or of a Is at risk of becoming infected with the bacterial pathogen.
  • the invention accordingly also provides pharmaceutical compositions ⁇ ratios ready comprise the antigen binding polypeptides of the vorlie ⁇ constricting invention.
  • the antigen-binding polypeptide of the present invention is then administered in a therapeutically effective amount to the patient, ie, in an amount sufficient to inhibit the CDTa subunit of the toxin CDT to ei ⁇ nem considerable extent.
  • the administration of the antigen binding polypeptide in an amount which results in a complete union in Wesent ⁇ inactivation of the toxin is performed.
  • the inactivation of the toxin can be tracked by improving one or more symptoms of the disease.
  • polypeptide that needs to be administered to achieve a therapeutic effect depends on several ren parameters. Factors that are relevant to the amount of administered polypeptide, for example, include the route of administration of the polypeptide, the size of the respective poly ⁇ peptide, the nature and severity of the disease, theiscussge ⁇ layer of the patient to be treated and the age, weight, Size and health of the patient to be treated.
  • a therapeutically effective amount of the antigen-binding polypeptide may be readily determined by one skilled in the art from the general knowledge of the art and the present disclosure.
  • the polypeptide is preferably administered in an amount to a subject sufficient to achieve a plasma concentration of from about 0.05 yg / ml to about 150 yg / ml, preferably from about 0.1 to about 50 yg / ml, more preferably from about 1 yg / ml to about 20 yg / ml and normally from about 1 yg / ml to about 10 yg / ml, eg 5 yg / ml.
  • the weekly dose may be from about 1 mg to about 500 mg polypeptide per m 2 Whyoberflä ⁇ che of the patient.
  • the weekly dose of the peptide may poly ⁇ / m 2 range from about 10-300 mg, preferably from about 100-200 mg / m 2, such as 150 mg / m 2.
  • the amount of polypeptide to be administered may be adjusted.
  • the antigen-binding polypeptide of the present invention may be formulated for various modes of administration, for example for oral administration as a tablet, capsule, powder, liquid or the like. It is however preferred that the antigen-binding polypeptide is administered parenterally by injection or intrave ⁇ nuiter intravenous infusion. The administration may, for example, by intravenous infusion, for example, within 60 minutes, within 30 Minu ⁇ th, or within 15 minutes.
  • Compositions suitable for administration by injection and / or infusion generally comprise solutions and dispersions, as well as powders, from which corresponding solutions and Dispersions can be prepared.
  • Such Caribbeanset ⁇ tongues are the immunologically active polypeptide and Minim ⁇ least contain a suitable pharmaceutically acceptable carrier.
  • Suitable pharmaceutically acceptable carriers for intravenous administration include bacteriostatic water, Ringer's solution, physiological saline, phosphate buffered saline (PBS) and Cremophor EL TM.
  • Sterile compositions for injection and / or infusion can be prepared by introducing the polypeptide in the required amount into a suitable carrier and then filter sterilizing by means of a filter.
  • Compositions for Verab ⁇ submission by injection or infusion should remain stable after their preparation under storage conditions over an extended period of time. The compositions may contain a preservative for this purpose.
  • Suitable Kon ⁇ servtechniksstoff include chlorobutanol, phenol, ascorbic acid and thimerosal.
  • the antigen-binding polypeptide of the present invention is formulated with carriers that protect the polypeptide from excretion too rapidly from the body (sustained-release formulations).
  • Such formulations may contain biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acids, collagen, polyorthoesters and polylactic acids.
  • Methods for the preparation of sustained release formulations are well known in the art. Retardformulie ⁇ conclusions can be present as semipermeable films of solid hydrophobic polymer, eg in the form of microcapsules. The preparation of corresponding administration forms and suitable auxiliaries are described, for example, in “Remington: The Science and Practice of Pharmacy", Lippincott Williams &Wilkins; 21. On ⁇ position (2005) described.
  • the polypeptides according to the invention are also suitable for the detection of the CDTa or CDTb subunit of the CDT toxin in biological samples, in particular in samples obtained from a Individuals who have been found to be infected with a strain of C. difficile that produces CDT toxin, such as stool samples, tissue specimens, etc. However, other samples, such as water samples for exposure to toxin, may also be obtained to be examined.
  • the invention relates to a method for detecting the CDTa subunit of the CDT toxin of C. difficile in a biological sample, wherein
  • the invention relates to a method for detecting the CDTb subunit of the CDT toxin of C. difficile in a biological sample, in which
  • the method first comprises a step wherein the antigen-binding polypeptide of the invention is brought into contact with the biological sample ⁇ rule.
  • This step is performed un ⁇ ter conditions of the formation of binding complexes from the antigen-binding polypeptide and be detected CDTa or CDTb subunit of the CDT toxin, are suitable.
  • Conditions permitting the formation of binding complexes of antigen-binding polypeptides (particularly antibodies) and toxin subunit are known in the art. Such conditions may include a temperature in the range of 4-37 ° C, a pH in the range of 6-8, and an incubation time in the range of several minutes to several hours.
  • suitable binding conditions for a particular pair of antigen-binding polypeptide and toxin subunit depend on various factors, such as the concentration of the antigen-binding polypeptide, the anticipated concentration of the toxin subunit in the sample, and the like.
  • concentration of the antigen-binding polypeptide the concentration of the antigen-binding polypeptide
  • concentration of the toxin subunit in the sample the concentration of the antigen-binding polypeptide
  • One skilled in the art will be able to determine suitable conditions for binding between polypeptide and CDTa or CDTb subunit using routine experimentation to optimize the formation of binding complexes.
  • the antigen-binding poly ⁇ peptide of the invention may, for example, prior to contacting with the sample suspected of containing it contains the CDT toxin, be provided with a detectable label.
  • the marking may be, for example, a fluorescent, chemiluminescent or radioactive label. Labeling with enzymes such as is also possible with horseradish peroxidase or alkaline-phosphatase ⁇ .
  • Metho ⁇ de which allows detection of the complexes, is the recombinant expression of the antigen-binding polypeptides as fusion proteins, the add on one or more peptide epitopes ver ⁇ , which can be visualized by means of labeled antibodies.
  • the invention relates to a process for the purification and / or concentration of the CDTa subunit of the CDT toxin of C. difficile, in which
  • the employed in the above process CDTa-specific polypeptide is a such, which ei ⁇ ne or more of the SEQ ID NO: 15-56 listed CDR regions or modifications thereof, as described above, comprising.
  • the CDTa-specific polypeptides comprise the sequences or fragments listed in SEQ ID NO: 1-14 or immunologically active fragments or variants thereof, as described above.
  • the invention relates to a process for the purification and / or concentration of the CDTb subunit of the CDT toxin of C. difficile, in which
  • CDTB-specific antigen-binding polypeptide is brought into contact with the biological sample under Be ⁇ conditions which allow the formation of complexes of the antigen-binding polypeptide and the CDTb- subunit;
  • the CDTb-specific polypeptide used is one containing one or more of the CDR regions listed in SEQ ID NOs: 70-84 or 102-149, or modifications thereof, as described above in connection with the CDTa. specific polypeptides.
  • the CDTB specific polypeptides include those described in SEQ ID NO: 65-69 or SEQ ID NO: 86-101 ⁇ be led sequences or fragments or immunologically active fragments or variants thereof, as described above in connection with the specific CDTa Polypeptides were described in more detail.
  • the purification and / or concentration of the toxin subunit is preferably carried out with antigen-binding polypeptides are immobilized on a solid support, such as on a material which is suitable for use in chroma ⁇ tographischen separation process.
  • Suitable sheulenma ⁇ terialien include Sepharose (for example, Q-Sepharose, DEAE-Sepharose, SP-Sepharose) and the like.
  • the CDTa- or CDTb- binding polypeptides can be on beads immobi ⁇ lêt beyond, eg on agarose or glass beads.
  • the beads are magnetizable, whereby the separation of the binding complexes, which adhere to the beads after incubation in toxin-containing samples, is facilitated.
  • Methods of coupling polypeptides to a solid support are well known in the art.
  • a geeigne ⁇ tes for the coupling process can provide, for example, immobilization of polypeptides were coupled to biotin or derivatives of biotin, to a carrier material which has previously been coated with streptavidin or derivatives thereof. Such compounds are commercially available from various suppliers.
  • the protein can be bound directly to the carrier by chemical reaction.
  • the sample in which the toxin is contained is prepared by the appropriately prepared Column, so that the toxin is specifically adsorbed by the immobilized antigen-binding polypeptides.
  • a suitable wash buffer By using a suitable wash buffer, all non-specifically bound molecules are washed off the column while the toxin is retained in the column.
  • the toxin can be eluted from the column with a suitable elution buffer (eg, a high salt buffer) to thereby obtain the purified and / or concentrated toxin.
  • a suitable elution buffer eg, a high salt buffer
  • the toxin bound to the matrix or column can be detected by an immunological detection method.
  • fusion polypeptides having an affinity tag in addition to an antigen-binding polypeptide of the invention.
  • fusion polypeptides can be by means of a material which has a particularly high affinity for the affinity tag used, purified effi cient ⁇ .
  • the affinity tag may have 6-12 histidine residues that specifically interact with a chelating nickel ion matrix.
  • the affinity tag can be stand ⁇ also from a glutathione-S-transferase, which can be purified using a glutathione matrix.
  • affinity tag and affinity tag pairs include maltose binding protein (MBP) and maltose; Avidin and biotin; Streptag and streptavin or neutravidin.
  • Affinity tags may be prepared, for example, by ligation of a DNA encoding an antigen-binding polypeptide of the invention with a sequence encoding the affinity tag.
  • the affinity tag may be coupled by chemical coupling. tion reactions are conjugated to the antigen-binding polypeptide.
  • the antigen-binding polypeptide can be chemically coupled with biotin. Labeling with biotin can also be achieved by attaching to the antigen-binding polypeptide a peptide recognized by a biotin protein ligase (EC 6.3.4.15), such as BirA from E. coli (see, for example, Smith et al.
  • Suitable peptides include, for example, the 13 amino acid minimum biotinylation sequence of the biotin carboxy carrier protein (BCCP) (Beckett et al. (1999), Protein Sci., 8, 921-929), the 15 amino acid variant thereof, called AviTag TM Macromol., 39 (1-3): 66-76), and the 76 amino acid BioEase TM sequence (Tirat et al., (2006), Int. J. Biol. Macromol., 39 (1-3): 66-76).
  • BCCP biotin carboxy carrier protein
  • biotinylated by the biotin protein ligase BirA at a central lysine residue are biotinylated by the biotin protein ligase BirA at a central lysine residue.
  • streptagems such as Streptag II (SA-WSHPQFEK) and One-STrEP-tag (SA-WSHPQFEK (GGGS) 2GGSAWSHPQFEK) (IBA BioTAGnology, IBA GmbH, Germany).
  • antigen-binding polypeptides of the present invention can also be coupled with enzymes (such as alkaline phosphatase) or with recognition sequences for enzymes (such as the above BirA biotinylation sequences). Also, coupling of various antigen-binding polypeptides, such as VHHs, directed against different epitopes of the CDTa or CDTb subunit is possible, thereby enhancing the efficiency of binding to the target antigen.
  • enzymes such as alkaline phosphatase
  • recognition sequences for enzymes such as the above BirA biotinylation sequences
  • two or more of the VHH single domain antibodies described above can be coupled to biotin via a BirA recognition sequence and subsequently linked via the high affinity binding of biotin to avidin or streptavidin, or to modified streptavidin such as neutravidin or Strep-Tactin , Avidin and streptavidin are tetrameric molecules that contain four molecules of Biotin can bind.
  • Such multimerized, single-domain VHH tetravalent antibodies have an increased affinity for the CDTa and CDTb subunits, respectively.
  • streptavidin-coated beads eg magnetic or sepharose beads, can be used.
  • Suitable beads include Strep-Tactin Magnetic Nanobeads (IBA BioTAGnology, IBA GmbH, Germany).
  • coupling of CDTa or CDTb directed VHH domains to VHH domains directed against albumin or against a transcytotic receptor is also useful.
  • the VHH single domain antibodies of the present invention may also be coupled to immunoglobulins, eg to the Fc region of an immunoglobulin.
  • step (b) detecting complexes of the polypeptide and the CDTa subunit; wherein the detection of the complexes in step (b) indicates ⁇ comprises that the patient is suffering from a verstoff by C. difficile ⁇ th disease.
  • a method for diagnosing a disease which is caused by CDT toxin of Clostridium difficile, reliefge ⁇ provides, in which: (a) contacting an above described defined CDTB-specific, antigen-binding polypeptide with the biological sample from a patient under conditions that ermögli the formation of complexes of the antigen-binding polypeptide and the CdtB subunit of CDT toxin ⁇ chen; and
  • step (b) detecting complexes of the polypeptide and the CDTb subunit; wherein the detection of the complexes in step (b) indicates ⁇ comprises that the patient is suffering from a verstoff by C. difficile ⁇ th disease.
  • the disease may be diarrhea, especially antibiotic-associated diarrhea (ADD), colitis, especially pseudomembranous colitis, enterocolitis, or a toxic megacolon.
  • ADD antibiotic-associated diarrhea
  • colitis especially pseudomembranous colitis, enterocolitis, or a toxic megacolon.
  • the antigen-binding polypeptides of the present invention are used for the therapeutic treatment of pseudomembranous colitis.
  • the disease caused by C. difficile is pseudomembranous colitis.
  • the disease caused by C. difficile is pseudomembranous colitis.
  • the invention further relates to the use of an antigen-binding polypeptide as described above and defined in the claims for the therapeutic treatment or diagnosis of a disease caused by CDT toxin of Clostridium difficile.
  • kits for the detection of C. difficile which kit includes a comprises the antigen-binding polypeptide described above.
  • the kit may also further reagents and means umfas ⁇ sen that are required for the detection, for example reagents for the visualization of a marker.
  • FIG. 1 shows the schematic representation of the cloning strategy for the VHH repertoire in the phage display vector pHEN2.
  • L signal peptide (leader); VHH: variable region of the heavy chain antibody; h: Hinge region; CH2: constant Re gion ⁇ 2 of the heavy chain antibody; CH3: constant region 3 of the heavy chain antibody; amber stop: stop codon UAG; H6x: His-tag; g3p: phage protein g3p.
  • FIG. 2 shows the amino acid sequences of the VHHs selected by CDTa.
  • the three CDR regions are highlighted (CDR1: just underlined, CDR2: underlined twice, and CDR3: underlined in bold). All underlines also apply to the amino acid positions in the other sequences of the same block. In bold letters are present in the VHHs vorkom ⁇ ing cysteine residues.
  • Figure 3 shows the results of an ELISA analysis of the Regenti ⁇ ty of the selected VHHs against different antigens.
  • Figure 4 shows the results of an ELISA analysis of the Regenti ⁇ ty of the selected VHHs against CDTa at various concentrations VHH.
  • coVHH Control VHH.
  • FIG. 5 shows the inhibition of CDTa-mediated P-ADP-ribosylation of actin by means of specific VHHs in the cell assay.
  • coVHH Control VHH.
  • FIG. 6 shows the protection of MDCKII cells mediated by the VHHs according to the invention before the CDTa-induced protection Collapse of transepithelial cohesion.
  • coVHH Control VHH.
  • Figure 7 shows the specificity of the selected VHHs L-3a (# 3230, SEQ ID NO: 65), L-15.
  • Id (# 3231, SEQ ID NO: 67) and L-15.
  • IIb (# 3240, SEQ ID NO : 68) in an ELISA analysis in biva ⁇ lentem format against CDTb (the receptor-binding B subunit of the CDT toxin), ART2 and milk powder (MP).
  • Figure 8 shows the specificity of the selected VHHs L-15.IC (# 3345, SEQ ID NO: 66) and L-15.III (# 3352, SEQ ID NO: 69) in an ELISA analysis in bivalent format against ToxA- Holotoxin, ToxB holotoxin, and CDTb (the receptor-binding B subunit of the CDT toxin).
  • FIG. 9 shows the amino acid sequences of the VHHs selected by means of CDTb.
  • the three CDR regions are highlighted (CDR1: just underlined, CDR2: underlined twice, and CDR3: underlined in bold). All underlines also apply to the amino acid positions in the other sequences of the same block. In bold letters are present in the VHHs vorkom ⁇ ing cysteine residues.
  • Lama # 6 received a CDTa-derived polypeptide
  • Lama # 5037 each received a polypeptide derived from ToxA, ToxB and CDTa.
  • ToxA cysteine protease domain
  • ToxB glycosyltransferase domain amino acids 1-546, 60 kDa
  • CDTa For immunization with the complete CDTa CDTa was Unterein ⁇ uniform as shown in SEQ ID NO: 57, the CDT holotoxin ver ⁇ spent.
  • Lama no. 180 received each a ToxA and ToxB polypeptide consisting of the GT and the CPD-domain of the toxin jewei ⁇ time, and a CDTB polypeptide (the hexameric receptor binding B subunit of CDT).
  • the toxin fragments were added to 500 ⁇ l of the Specol adjuvant (Cedi Diagnostics, Lelystad, The Netherlands) in 400 ⁇ l PBS and ultrasonicated three times at 4 ° C.
  • the Injektio ⁇ nen were made subcutaneously in the neck of the Lamas. Immunization of the llamas was enhanced by three more consecutive injections of the antigens. Finally, ten days after the last injection, the llamas were bled 20-100 ml of blood for phage display library preparation (see Example 2).
  • the detection was carried out with 3, 3 ', 5, 5' -Tetramethylbenzidine (MB) and was stopped by the addition of 0.5 M sulfuric acid.
  • the substrate turnover was quantified at 450 nm on the ELISA reader.
  • the preimmune serum was compared with the immune serum of the llamas after the last injection.
  • the preimmune sera showed no reactivity against the antigens tested.
  • the llama No. 6 showed a high reactivity against CDTa.
  • the llama no. 5037 showed high reactivity to all antigens are Toxinan ⁇ (ToxA, and ToxB CDTa).
  • the immune serum of lamase # 180 showed reactivity against ToxB and CDTb.
  • Example 2 Selection and sequence analysis of CDTa and CDTb-specific VHHs from phage display libraries of the immunized llamas
  • RNA was isolated from the peripheral blood lymphocytes of the llamas after the last injection and converted into cDNA by reverse transcription.
  • RH6 random hexamers
  • RNA was amplified by PCR with the specific and partially degenerate oligonucleotides SHFmu / SHRmu and LHFmu / LHRmu:
  • the amplified VHH fragments were cloned after digestion with the restriction endonucleases Sfil and Notl in the phagemid vector pHEN2 (see Figure 1).
  • the ligation products were transformed into E. coli so that a phage library was ent ⁇ expressing VHH fragment.
  • Single domain specific antibodies were selected from the phage display libraries by panning, whereby the single domain antibodies bound via their VHHs to immobilized CDTa or CDTb antigen previously immobilized in the wells of an ELISA plate.
  • CDTa or CDTb was biotinylated with the EZ-Link sulfo-NHS biotinylation kit Thermo Scientific (Fisher Scientific, Schrö, Germany). The biotin is to tie in the La ⁇ ge, to streptavidin-agarose. 20 ⁇ streptavidin agarose were incubated with 2 ⁇ g of the biotinylated antigens and then incubated with 10 ⁇ of the phage library.
  • the phages were eluted competitively with 2 yg of un- conjugated antigen, and the supernatant was used for ei ⁇ ne reinfection of E. coli with the phage.
  • the Bib ⁇ liotheken were subjected to 2 to 3 such selection cycles. After each round, 12 clones were sequenced to verify the success of the round of selection. The sequences of the final selected clones are listed in FIGS. 2 and 9.
  • the VHHs identified as described above were produced recombinantly in E. coli.
  • Single domain soluble antibodies were generated by transformation of the vector construct of Example 2 into E. coli HB2151 (genotype: K12 D (lac-pro), ara, nalr, thi / F '[proAB, laclq, lacZDM15]).
  • This strain is capable of recognizing the stop codon between the VHH coding sequence and the M13 phage coding sequence for the g3 capsid protein.
  • coli were mixed in 100 ml 2xYT (BD Difco, Heidelberg, 16 g / 1 trypsin, 5 g / 1 NaCl, 10 g / 1 yeast extract) with 100 yg / ml carbenicillin up to an optical Density of 0.5 cultivated.
  • Pro ⁇ teinexpression was induced by isopropyl-ß-D-thiogalactopyranoside (IPTG) and terminated after three hours by centrifugation. By osmotic shock, the outer cell wall was broken.
  • IPTG isopropyl-ß-D-thiogalactopyranoside
  • IPTG isopropyl-ß-D-thiogalactopyranoside
  • the VHHs were purified via their histidine tag (SEQ ID NO: 64) by metal ion affinity chromatography in a yield of between 0.5 mg / l and 1 mg / l.
  • the VHHs were then dialysed against PBS and stored after determination
  • the binding of the VHHs recombinantly produced in Example 3 at CDTa or CdtB was ge ⁇ tested by ELISA in the bivalent format. Single-domain antibodies were detected by peroxidase or fluorochrome-conjugated antibodies against their C-terminal myc-tag (SEQ ID NO: 63).
  • the antigens used were 500 ng of CDTa (the catalytic domain of CDT), salmo nella plasmid virulence protein B (SpvB), ToxA holotoxin and milk powder (MPBS) ( Figure 3); or 200 ng CDTb, ART2 and milk powder (MP) ( Figure 7); or 500 ng ToxB (holotoxin), 500 ng ToxA (holotoxin) or 100 ng CDTB ( Figure 8) overnight at 4 ° C in the wells of a microtiter / ELISA plate immobi ⁇ larra.
  • VHH anti-Myc antibody complexes (compare Example 3) were incubated in the wells for 1 hour.
  • the VHHs 100 ng each
  • a mouse anti-c-Myc antibody 700 ng
  • a sheep anti-mouse peroxidase conjugate (1: 5000) was used.
  • the detection was carried out with 3, 3 ', 5, 5' -Tetramethylbenzidine (TMB) and was stopped by the addition of 0.5 M sulfuric acid.
  • the substrate turnover was quantified at 450 nm on the ELISA reader.
  • the controls used were SpvB, ToxA and ToxB holotoxin, ART2 and milk powder (MPBS).
  • the VHHs L + 8 (# 2621, SEQ ID NO: 1), L-14 (# 2622, SEQ ID NO: 2), L-15a (# 2169, SEQ ID NO: 3), L + 18a (# 2173 , SEQ ID NO: 5) and S-20 (# 2352, SEQ ID NO: 11) showed strong specific reactions with CDTa (see Fi gur ⁇ 3).
  • the anti ToxA VHH L-lOa showed a specific reaction with the fish ⁇ ToxA holotoxin.
  • L-3a (# 3230, SEQ ID NO: 65), L-15. Id (# 3231, SEQ ID NO: 67), L-15.IIb (# 3240, SEQ ID NO: 68), L-3a. 15.IC (# 3345, SEQ ID NO: 66) and L-15.III (# 3352, SEQ ID NO: 69) showed strong specific reactions with CDTb (see Figures 7 and 8).
  • VHHs concentration-dependent binding of VHHs to CDTa was tested analogously with monovalent VHHs.
  • ⁇ be signed ELISA performed with the VHHs have not been pre-incubated with anti-Myc antibodies.
  • the VHHs were incubated in 1: 3,3 dilution steps for one hour in the wells of the ELISA plate. Subsequently, the wells were 2 times washed.
  • a mouse anti-c-Myc antibody was used (700 ng / well).
  • As teritär An ⁇ tilik a sheep anti-mouse IgG peroxidase (PO) conjugate (1: 5000) was used.
  • VHHs ( Figure 4, 8-1000 ng) were tested in 1: 3.3 dilution steps. Detection was as described above.
  • CDTa VHHs mediated CDTa transfer of ADP-ribose from nicotinamide adenine dinucleotide (NAD) can inhibit amino acids of a target protein ⁇ .
  • ADP-ribosylation of actin by CDTa was visualized by using radioactively labeled nicotinamide adenyl- [adenylate-P] -dmukleotid- (P-NAD) as a substrate.
  • CDT transferred thereby to the ADP ribose group and the radioactive label to actin so that these detected at ⁇ closing by SDS-PAGE autoradiography who could ⁇ .
  • CDTa 100 ng of recombinantly produced CDTa (having differing ⁇ chen amounts of VHHs S-20a (# 2352, SEQ ID NO: ll), S-20c (# 2529, SEQ ID NO: 13), L + 8 (# 2621, SEQ ID NO: 1), L + 18e (# 2171, SEQ ID NO: 9), L-14 (# 2622, SEQ ID NO: 2), L-15a (# 2169, SEQ ID NO: 3), and two controls -VHHs (coVHH)) for 20 minutes at 37 ° C and then incubated with a HEK cell lysate as a substrate m in the presence of P-NAD for 10 minutes at room temperature. The reaction was stopped by adding an SDS loading buffer.
  • reaction production ⁇ te were fractionated by SDS-PAGE and autoradiographed by exposure to x-ray film (Amersham Hyperfilm Screen kit, GE Healthcare) (exposure time 1-4 hours).
  • the CDTa-specific VHHs S-20a, S-20c, L + 8, L + 18e, L-14 and L-15a were able to block the CDTa-mediated ADP-ribosylation of actin while the control VHHs not affect the CDTa activity showed (see fi gure ⁇ 5).
  • the inhibition of CDTa by the VHHs of the invention was further analyzed at the cellular level.
  • the conditional by the collapse of the cytoskeleton rounding of adherent growing cells after toxin treatment was an indicator of the cytotoxic activity of the toxin subunit USAGE ⁇ det.
  • 3 ⁇ g of the VHHs were preincubated with 0.5 ⁇ g CDTa and 1 ⁇ g CDTb for 30 minutes at 37 ° C. and then added to ad ⁇ tenently growing MDCKII (Madin Darby canine kidney) cells for 1 hour. Subsequently, the cells were fixed with 4% paraformaldehyde for 10 minutes at 4 ° C. The nucleus was stained with DAPI.
  • the actin cytoskeleton was stained with phalloidin, and the morphology was analy ⁇ Siert on immunofluorescence microscopy. The normal structure of the actin cytoskeleton was clearly visible in the negative control. After adding CDTa and CDTB the actin cytoskeleton appeared strongly modifi ⁇ ed, and the number of cells had decreased significantly. The reduction in cell number is due to a rounding off and subsequent detachment of the MDCKII cells by a loss of integrity of the cytoskeleton. This effect was reduced interpreting ⁇ Lich by preincubation of the toxin with the V HH L + 8. This means that the CDH-directed VHHs can effectively inhibit the action of the toxin subunit.
  • VHH L + 8 The effect of VHH L + 8 on MDCKII cells was also examined by means of a TEER (transepithelial electrical resistance) test.
  • MDCKII cells were allowed to reach one stable cell assembly in the wells of a microtiter plate Transwell system of Corning Costar (Amsterdam, Netherlands).
  • CDTa (10 nM) and CDTb (20 nM) were preincubated for 20 minutes at 37 ° C. with 6 ⁇ g of VHH L + 8 (# 2621, SEQ ID NO: 1) or with a control VHH (coVHH) and then added given to the MDCKII cells. Further controls were medium and CDTa.
  • the TEER values were determined by measuring the transepithelial resistance with a volt-ohm meter (Millicell-ERS, Millipore) over a period of six stun ⁇ at intervals of 60 minutes. Without toxin, the TEER value remained constant over six hours (FIG. 6). After addition of CDTa, after two hours rounding off the cells as a result of the collapse of the actin cytoskeleton, the TEER value dropped significantly. This effect could be minimized by adding VHH L + 8, but not by adding a control VHH. This clearly shows that the cytotoxic activity of CDTa can be reduced by the VHHs according to the invention.

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Abstract

L'invention concerne des polypeptides de liaison antigénique comprenant la région CDR1, CDR2 et CDR3 d'un domaine VHH d'un anticorps à chaînes lourdes de camélidés. Les polypeptides se lient spécifiquement à la sous-unité CDTa de la toxine cdt de Clostridium difficile et ils conviennent donc notamment pour le traitement thérapeutique et prophylactique d'une pathologie provoquée par la toxine cdt ainsi que pour le diagnostic d'une telle pathologie. L'invention concerne en outre d'utilisation de ces polypeptides de liaison antigénique dans des procédés de détermination de la sous-unité CDTa de la toxine cdt de Clostridium difficile dans un échantillon biologique. Elle concerne également des procédés de purification et/ou de concentration de la sous-unité CDTa de la toxine cdt de Clostridium difficile, lesdits procédés utilisant les polypeptides de liaison antigénique.
PCT/EP2012/075617 2011-12-14 2012-12-14 Anticorps dirigés contre la toxine cdt de c. difficile WO2013087874A1 (fr)

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