DE102011121238A1 - SINGLE DOMAIN ANTIBODY AGAINST CLOSTRIDIUM DIFFICILE TOXINE - Google Patents

Abstract

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 in the region of amino acids 1-800 of a glycosyltransferase activity-bearing toxin of Clostridium difficile. The antigen-binding polypeptides are particularly useful for the therapeutic treatment of a disease caused by a glycosyltransferase activity-bearing toxin of Clostridium difficile and for the diagnosis of such a disease. Moreover, the invention relates to the use of such antigen-binding polypeptides in methods for detecting a toxin of Clostridium difficile having a glycosyltransferase activity in a biological sample. Methods for purifying and / or concentrating a Clostridium difficile toxin having glycosyltransferase activity using the antigen-binding polypeptides are also provided.

Description

The invention relates to antigen-binding polypeptides comprising the CDR1, CDR2, and CDR3 region of a VHH domain of a camelid heavy chain antibody. Specifically, the polypeptides bind in the region of amino acids 1-800 of a glycosyltransferase activity-bearing toxin of Clostridium difficile and are thus particularly useful for the therapeutic and prophylactic treatment of a disease caused by a glycosyltransferase activity-bearing toxin of Clostridium difficile for the diagnosis of such a disease. Moreover, the invention relates to the use of such antigen-binding polypeptides in methods for detecting a toxin of Clostridium difficile having a glycosyltransferase activity in a biological sample. Methods for purifying and / or concentrating a Clostridium difficile toxin having glycosyltransferase activity using the antigen-binding polypeptides are also provided.

BACKGROUND OF THE INVENTION

The use of antibiotics has been proven in the control of many bacterial infectious diseases and has become indispensable in the field of human medicine. On the other hand, the massive use of antibiotics also contributes to the spread of other potentially life-threatening diseases. For example, diseases caused by Clostridium difficile are a widespread consequence of antibiotic therapies, which often lead to significant damage to the normal intestinal flora ( Kyne (2010), New Engl. J. Med. 362 (3): 264-5 ). C. difficile is an anaerobic Gram-positive bacterium that is part of normal intestinal flora in healthy people. In the context of antibiotic therapy, there are changes in the composition of the normal flora, as individual bacteria are more influenced by the bactericidal effect of the antibiotic than others. In this way, there may be an unusually high multiplication of C. difficile associated with various diseases, collectively referred to as "CDAD" (Clostridium difficile associated diseases). A particularly serious and potentially life-threatening disease from the CDAD group is pseudomembranous colitis.

Due to the steadily increasing use of antibiotics in recent decades, the incidence of diseases caused by C. difficile has increased significantly. It is estimated that the treatment of these diseases causes healthcare costs of up to € 3 billion a year in Europe alone ( Kuijper et al. (2006), Netherlands. Emerg. Infect. Dis. 12: 827-30 ). Recently, a hypervirulent strain of C. difficile has been detected, designated BI / NAP1 / 027 ( Goorhuis et al. (2008), Clin Infect Dis. 47 (9): 1162-70 ). Some isolates of this strain show increased production of virulence factors as well as resistance to fluoroquinolone antibiotics. The control of these strains has proven to be particularly difficult.

The pathogenicity of C. difficile is caused by various enzymatically active toxins. The bacterium produces two high molecular weight exotoxins called toxin A (ToxA) and toxin B (ToxB) ( Just & Gerhard (2004), Rev Physiol Biochem Pharmacol 152: 23-47 ). These toxins each consist of a glycosyltransferase (GT) domain, a cysteine protease (CP) domain, a translocation domain, and a receptor binding domain (RBD). The general structure of ToxA and ToxB is in Jank & Aktories (2008), Trends Microbiology, 16, 222-229 , described. The toxic activity of these multidomain proteins is due to the glycosyltransferase (GT) domain catalyzed glycosylation of human Rho GTPase at position Thr-37 ( Koch-Nolte et al. (2001), J. Biotechnol. 92, 81-87 ). Glycosylation prevents Rho-GTPase from adopting its native conformation, resulting in redistribution of the actin cytokelette, disintegration of the cell-cell contacts of the intestinal epithelium, and loss of barrier function ( Aktories & Barbieri, (2005) Nat Rev Microbiol., 3 (5): 397-410 ).

In addition, 20-35% of the strains of C. difficile contain the binary toxin CDT ( Perelle et al. (1197), Immun. 65 (4): 1402-1407 ). The CDT toxin consists of two separate subunits called CDTa and CDTb. 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 ( Papatheodorou et al. (2011), Proc Natl Acad Sci 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.

It has been shown that administration of monoclonal antibodies to ToxA and ToxB can prevent recurrence of infection with C. difficile. For example, the recurrence rate was reduced by more than 70% through a single infusion of antibodies ( Lowy et al. (2010) New Engl J Med. 362 (3): 197-205 ). One Advantage in the use of antibodies over other therapeutic approaches, such as. B. Use of vaccines, is that an active immune system is not a mandatory requirement for successful therapy. Therefore, the administration of antibodies also in people with weakened immune system therapy success.

Disadvantages in the use of polyclonal and / or monoclonal antibodies in the field of human medicine arise from the often insufficient stability after administration. In addition, the costs incurred in the production of monoclonal antibodies are very high. In particular, the production of humanized antibodies, which have a lower immunogenicity and are therefore particularly suitable for use in humans, is labor-intensive and cost-intensive. In addition, complete antibodies have limited tissue permeability due to their size. A significant improvement of these properties which are disadvantageous for the therapy is therefore necessary for an efficient, yet financially acceptable therapy.

Various strategies have been developed in the art to solve the problems associated with the administration of antibodies. Thus, fragments of complete IgG antibodies, such as. B. Fv, Fab, F (ab ') and F (ab') ₂ , used for therapeutic purposes. However, these fragments often have reduced specificity and / or affinity for the antigen compared to the complete antibody. Furthermore, these fragments often show only a low solubility in aqueous solutions.

It is known that fragments of certain camelid antibodies have properties which make them particularly suitable for therapeutic use in humans. In addition to conventional antibodies, which each have two heavy and two light chains, the camelids also produce antibodies that consist exclusively of heavy chains ( Hamers-Casterman et al. (1993), Nature 363: 446-448 ; Wesolowski et al. (2009), Med Microbiol Immunol. 198 (3): 157-74 ). These so-called 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 determining regions (CDRs) and four framework regions (FR) that alternate 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 exceptionally long CDR3 region about 7 to 25 amino acids in length, unlike conventional antibodies, the VHH domain can bind in cavities of protein antigens. B. block the active site of an enzyme ( De Genst et al. (2006), Proc Natl Acad Sci USA 103 (12): 4586-4591 ). VHH domains can be produced recombinantly as soluble proteins in bacteria or mammalian cells. Such recombinantly produced VHH domains are also called single domain antibodies (single-domain antibodies, sdAbs) or nano-bodies ^®. Nanobody ^®, which targets a human serum protein (von Willebrand factor), is currently being tested in a Phase II clinical trial ( by Loon et al. (2011), Thromb Haemost 106 (1): 165-71, Ulrichs et al. (2011), Blood 118 (3): 757-65 ).

VHH single domain antibodies are not only more stable but also about ten times smaller than conventional antibodies and, in addition to high solubility, have significantly better tissue penetration properties. For this reason, VHH single domain antibodies have been proposed for the treatment of various diseases. In the international application WO 2006/122825 For example, VHH single domain antibodies against von Willebrand factor are used to treat arterial occlusive diseases. The 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 capable of specifically binding to and neutralizing a toxin produced by C. difficile. The antibodies are therefore particularly suitable for use in the diagnosis and / or therapeutic treatment of diseases caused by the toxa ToxB and ToxB of C. difficile.

VHH single domain antibodies directed against ToxA and ToxB for use in neutralizing the toxins have already been described in the prior art ( Hussack et al. (2011) J Biol Chem., 286 (11): 8961-76 ). However, these toxins are directed against the receptor binding domain of the toxins, thus preventing their binding to the target cells. However, it has now surprisingly been found within the scope of the present invention that VHH single domain antibodies directed against the glycosyltransferase domain or the cysteine protease domain of toxin A or toxin B of C. difficile have particularly advantageous properties in the neutralization of toxin-mediated tissue damage.

DESCRIPTION OF THE INVENTION

In the context of the present invention, firstly, by immunizing llamas with peptides from the toxin A cysteine protease domain and toxin B glycosyltransferase domain, heavy chain antibodies were generated. Subsequently, RNA was isolated from the peripheral blood lymphocytes of the llamas and converted into cDNA by means of reverse transcription. Starting from the isolated RNA, the variable domains of the heavy chain antibodies were amplified by PCR. By cloning the VHH fragments into a phagemid vector, phage display libraries were prepared. These libraries were selected using recombinant ToxA and ToxB, respectively, in a panning procedure. The sequences of the selected single domain antibodies were subsequently determined by sequencing and expressed recombinantly in bacterial host cells. In this way, the present invention provides single domain VHH antibodies suitable for use in therapeutic and / or diagnostic procedures. In addition, the present invention provides CDR regions from VHH single domain antibodies against ToxA and ToxB, respectively, which can be used in the construction of recombinant, antigen-binding polypeptides.

The invention thus provides, in a first aspect, 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, which polypeptide is specific in the art of amino acids 1-800 of a toxin with glycosyltransferase activity of C. difficile. Toxins of this pathogen having glycosyltransferase activity are toxins A and B (ToxA and ToxB) as shown in SEQ ID NOs: 69 and 70, respectively. The binary toxin CDT of C. difficile has no glycosyltransferase activity. In both ToxA and ToxB, the N-terminally located 800 amino acids of the unprocessed toxin comprise the GT and CP domains.

Thus, the invention specifically discloses toxA binding polypeptides which comprise the CDR1, CDR2 and CDR3 regions of a VHH domain of a camlide heavy chain antibody required for binding to ToxA. Binding to ToxA occurs in the region of amino acids 1-800 of the native, unprocessed sequence of ToxA, d. H. the epitope for binding of the ToxA-binding polypeptide is within amino acids 1-800 of ToxA. In a preferred embodiment, the epitope is in the cysteine protease domain region of ToxA. This extends in unprocessed ToxA from amino acid 547 to amino acid 779.

Furthermore, the invention also discloses polypeptides that specifically bind to ToxB. These polypeptides include the CDR1, CDR2 and CDR3 regions of a VHH domain of a camelid heavy chain antibody, which regions are required for binding to ToxB. Binding to ToxB occurs in the region of amino acids 1-800 of the native, unprocessed sequence of ToxB, d. H. the epitope for binding of the ToxB binding polypeptide is within the amino acids 1-800 of ToxB. In a preferred embodiment, the epitope is in the region of the glycosyltransferase domain of ToxB. This extends in the unprocessed ToxB from amino acid 1 to amino acid 546.

The polypeptides of the invention are thus characterized by CDR regions derived from camelid heavy chain antibodies. The heavy chain antibodies are a special group of immunoglobulins consisting exclusively of heavy chains. A heavy chain of such an antibody contains two constant domains and a single variable domain, which in turn consists of 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. Due to pronounced loop structures in the CDR3, a VHH domain is able to recognize cryptic epitopes. Heavy chain antibodies are produced in only a few species, e.g. In 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. In a particularly preferred embodiment of the invention, the antigen-binding polypeptide comprises CDR regions derived from a VHH domain of a lamase.

The polypeptide of the invention has a specific affinity for the cysteine protease (CP) domain or the glycosyltransferase (GT) domain of ToxA of C. difficile. In addition, that can polypeptide of the invention also have a specific affinity for the cysteine protease (CP) domain or the glycosyltransferase (GT) domain of ToxB of C. difficile.

This means that a polypeptide of the invention having a binding affinity binds to the corresponding domain of ToxA or ToxB, which is significantly higher than the binding affinity for binding to another protein, which is not homologous to the sequence of these toxins.

Preferably, 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. tray, 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 ToxA or ToxB. The binding affinity is preferably in the range of at least 10 ^-6 M. Particularly preferred antigen-binding polypeptides of the present invention have a binding affinity with dissociation constants (K _d ) in the range of at least 10 ^-6 M, and preferably in the range of at least 10 ^-7 M , 10 ^-8 M, 10 ^-9 M, 10 ^-10 M, 10 ^-11 M, or 10 ^-12 M. Methods for measuring the binding affinities of antibodies are well known in the art and include e.g. As the determination of K _d values by flow cytometry.

• (a) die in SEQ ID NO: 18-20 dargestellten CDR-Sequenzen;
• (b) die in SEQ ID NO: 21-23 dargestellten CDR-Sequenzen;
• (c) die in SEQ ID NO: 24-26 dargestellten CDR-Sequenzen;
• (d) die in SEQ ID NO: 27-29 dargestellten CDR-Sequenzen;
• (e) die in SEQ ID NO: 30-32 dargestellten CDR-Sequenzen;
• (f) die in SEQ ID NO: 33-35 dargestellten CDR-Sequenzen;
• (g) die in SEQ ID NO: 36-38 dargestellten CDR-Sequenzen;
• (h) die in SEQ ID NO: 39-41 dargestellten CDR-Sequenzen; aufweisen.

In a particularly preferred embodiment of the invention, the antigen-binding polypeptide comprises the CDR regions CDR1, CDR2 and CDR3 of a VHH domain of a camelid heavy chain antibody, said CDR regions being:

• (a) the CDR sequences shown in SEQ ID NOs: 18-20;
• (b) the CDR sequences shown in SEQ ID NOs: 21-23;
• (c) the CDR sequences shown in SEQ ID NOs: 24-26;
• (d) the CDR sequences shown in SEQ ID NOs: 27-29;
• (e) the CDR sequences shown in SEQ ID NOs: 30-32;
• (f) the CDR sequences shown in SEQ ID NOs: 33-35;
• (g) the CDR sequences shown in SEQ ID NOS: 36-38;
• (h) the CDR sequences shown in SEQ ID NOS: 39-41; exhibit.

• (a) die in SEQ ID NO: 42-44 dargestellten CDR-Sequenzen;
• (b) die in SEQ ID NO: 45-47 dargestellten CDR-Sequenzen;
• (c) die in SEQ ID NO: 48-50 dargestellten CDR-Sequenzen;
• (d) die in SEQ ID NO: 51-53 dargestellten CDR-Sequenzen;
• (e) die in SEQ ID NO: 54-56 dargestellten CDR-Sequenzen;
• (f) die in SEQ ID NO: 57-59 dargestellten CDR-Sequenzen;
• (g) die in SEQ ID NO: 60-62 dargestellten CDR-Sequenzen;
• (h) die in SEQ ID NO: 63-65 dargestellten CDR-Sequenzen;
• (i) die in SEQ ID NO: 66-68 dargestellten CDR-Sequenzen; aufweisen.

In yet another particularly preferred embodiment of the invention, the antigen-binding polypeptide comprises the CDR regions CDR1, CDR2 and CDR3 of a VHH domain of a camelid heavy chain antibody, which CDR regions:

• (a) the CDR sequences shown in SEQ ID NO: 42-44;
• (b) the CDR sequences shown in SEQ ID NOs: 45-47;
• (c) the CDR sequences shown in SEQ ID NOs: 48-50;
• (d) the CDR sequences shown in SEQ ID NOs: 51-53;
• (e) the CDR sequences shown in SEQ ID NOs: 54-56;
• (f) the CDR sequences shown in SEQ ID NOs: 57-59;
• (g) the CDR sequences shown in SEQ ID NOs: 60-62;
• (h) the CDR sequences shown in SEQ ID NOs: 63-65;
• (i) the CDR sequences shown in SEQ ID NOs: 66-68; exhibit.

It will be apparent to those skilled in the art that within the presently disclosed CDRs, some modification of the amino acid sequence is possible without affecting the binding ability of the VHH single domain antibody. For example, it may be possible to alter a CDR of the single domain antibody by exchanging 1, 2 or 3 amino acids. In general, any of the amino acid residues within the CDRs depicted in SEQ ID NOs: 18-68 may be replaced with another residue as long as the resulting CDR is still capable (along with the other two CDRs) of the specific toxin (ie ToxA or ToxB). Further, 1, 2 or 3 amino acids within any of the sequences shown in SEQ ID NO: 18-68 may also have been deleted or added by addition, as long as the binding ability to the corresponding toxin (i.e., ToxA or ToxB) is not completely eliminated.

Also encompassed by the invention are thus antigen-binding polypeptides having CDRs which differ from the sequences shown in SEQ ID NO: 18-68 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: 18-68 in at most one amino acid per CDR.

In a further preferred embodiment, the antigen-binding polypeptide of the invention comprises a complete VHH domain of a camelid heavy chain antibody. Such a VHH domain, when used alone, is capable of binding to the corresponding antigen and is therefore also referred to herein as "VHH single domain antibody". The VHH domain, besides the three CDR regions responsible for antigen binding, also includes the four framework FR1-FR4 regions located between the CDR regions. The framework regions may be fully conserved in the VHH domain of the invention, or they may be in the form of fragments of the original framework sequences. So z. B. the framework sequences which limit the VHH domain C-terminal and N-terminal, one or more amino acids are shortened. Such modified VHH domains are expressly encompassed by the invention. As used herein, "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 specifically binding to C. difficile ToxA are shown in SEQ ID NOs: 1-8. Preferred antigen-binding polypeptides comprising a VHH domain of a camelid heavy chain antibody which specifically bind to C. difficile ToxB are shown in SEQ ID NOs: 9-17. The invention also extends to variants of the polypeptides shown in SEQ ID NO: 1-17 that are homologous to the polypeptides shown in SEQ ID NOs: 1-17 and also have the ability to bind the corresponding toxin of C. have difficile. As used herein, the term "variant" refers to an amino acid sequence that has been modified by deletion, substitution or addition of amino acids to be at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% %, 96%, 97%, 98%, 99% sequence identity to any of the sequences shown in SEQ ID NO: 1-17 when these sequences are optimally aligned, e.g. B. with the programs GAP or BESTFIT using the default gap method. Computer programs for determining amino acid sequence identity are well known in the art.

Thus, variants, in particular in the framework regions (FR1-FR4), can deviate from the VHH regions shown in SEQ ID NO: 1-17 without adversely affecting the binding affinity of the polypeptides. Preferably, the variants differ from the polypeptides shown in SEQ ID NOs: 1-17 only by the replacement of a few amino acids. It is particularly preferred that variants of the polypeptides shown in SEQ ID NO: 1-17 of these in less than 20, in less than 15, in less than 10, in less than 5, z. In 4, 3, 2 or 1 amino acid position (s).

• (a) den Aminosäuresequenzen von SEQ ID NO: 1-8; und
• (b) einer Aminosäuresequenz, die mindestens 80% oder mehr Sequenzidentität zu einer der Aminosäuresequenzen von SEQ ID NO: 1-8 aufweist.

In a further aspect, the invention thus relates to an antigen-binding polypeptide comprising an amino acid sequence selected from the group consisting of:

• (a) the amino acid sequences of SEQ ID NO: 1-8; and
• (b) an amino acid sequence having at least 80% or more sequence identity to any of the amino acid sequences of SEQ ID NOs: 1-8.

• (a) den Aminosäuresequenzen von SEQ ID NO: 9-17; und
• (b) einer Aminosäuresequenz, die mindestens 80% oder mehr Sequenzidentität zu einer der Aminosäuresequenzen von SEQ ID NO: 9-17 aufweist.

The invention further relates to an antigen-binding polypeptide comprising an amino acid sequence selected from the group consisting of:

• (a) the amino acid sequences of SEQ ID NO: 9-17; and
• (b) an amino acid sequence having at least 80% or more sequence identity to any of the amino acid sequences of SEQ ID NOS: 9-17.

Preferably, the substitutions by which the variants differ from the VHH regions depicted in SEQ ID NOs: 1-17 are conservative substitutions, i. H. Substitutions of one or more amino acid residues by an amino acid of similar polarity that acts as a functional equivalent. Preferably, the replacement amino acid residue is selected from the same group of amino acids as the amino acid residue to be replaced. For example, one hydrophobic residue may be replaced by another hydrophobic residue or a polar residue with another polar residue having the same charge. Functionally homologous amino acids which can be used for conservative substitution include e.g. B. non-polar amino acids such. Glycine, valine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, and tryptophan. Examples of uncharged polar amino acids include serine, threonine, glutamine, asparagine, tyrosine, and cysteine. Examples of charged polar (acidic) amino acids include histidine, arginine and lysine. Examples of charged polar (basic) amino acids include aspartic acid and glutamic acid.

Further variants of the sequences shown in SEQ ID NO: 1-17 also include those polypeptides in which one or more amino acids in the amino acid sequence of one of the sequences shown in SEQ ID NO: 1-17 Polypeptides were inserted. Such insertions may in principle be made at any position of the polypeptides shown in SEQ ID NOs: 1-17 as long as the resulting variant of the polypeptide retains its immunological activity (ie, the ability to bind and inhibit ToxA or ToxB). Furthermore, in the present case, such proteins also count as variants of one of the polypeptides shown in SEQ ID NO: 1-17, in which one or more amino acids are missing within the sequence in comparison to the reference polypeptide. Such deletions may involve any amino acid positions within the sequences shown in SEQ ID NO: 1-17. In particular, the deletions may be those comprising two or more (eg 3, 4 or 5) contiguous amino acids from any of the sequences of SEQ ID NOS: 1-17.

Also encompassed by the invention are immunologically active fragments of the polypeptides shown in SEQ ID NO: 1-17 (or variants thereof). In the present case, immunologically active fragments are understood as meaning those sequences which differ from the amino acid sequences shown in SEQ ID NO: 1-17 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 ToxA and ToxB, respectively. Thus, for example, immunologically active fragments of the sequence shown in SEQ ID NO: 1 may be fragments lacking the first two N-terminal and / or C-terminal amino acids of the polypeptide shown in SEQ ID NO: 1. One skilled in the art will readily be able to determine the ability of fragments to bind to ToxA or ToxB, e.g. By competitive antibody assays.

The polypeptides shown in SEQ ID NO: 1-17 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. According to the invention, these modified amino acids may be amino acids which have been modified by biotinylation, phosphorylation, glycosylation, acetylation, branching and / or cyclization.

It is preferred that the variants or fragments of the antigen-binding polypeptides of the invention retain greater than 50% of the original binding affinity for ToxA or ToxB 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, e.g. , The polypeptides shown in SEQ ID NO: 1-17. The binding affinity for ToxA or ToxB of C. difficile can be routinely measured by in vitro assays.

The antigen-binding polypeptide will preferably have a size which results in physicochemical properties which are particularly suitable for therapeutic and / or diagnostic applications, e.g. As a good solubility and tissue penetration, thermal stability and similar properties. Preferably, the antigen-binding polypeptide of the present invention will 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.

In a preferred embodiment, binding of the antigen-binding polypeptide to ToxA or ToxB results in the inhibition or reduction of the enzyme activity of the toxin. The enzyme activity, in particular the glycosyltransferase activity is z. B. reduced to about 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less of the activity of the unbound enzyme. In a preferred embodiment, the enzyme activity of the bound domain and / or the entire toxin is completely neutralized by binding the polypeptide of the invention.

VHH single domain antibodies are distinguished from conventional mammalian antibodies in particular by the fact that only three CDRs are required for the binding of the antigen. The invention therefore also relates to those antigen-binding polypeptides which comprise only the regions CDR1, CDR2 and CDR3 required for binding of the toxin of one of the polypeptides exemplified in SEQ ID NO: 1-17, the regions located between the CDR regions only show minor or even substantially no similarity to the corresponding regions of any of the polypeptides shown in SEQ ID NO: 1-17. The respective sequences of the CDR regions of the single domain antibodies shown in SEQ ID NOs: 1-17 are shown in U.S. Pat 2 and shown in SEQ ID NO: 18-68.

The invention provides, in another aspect, a nucleic acid molecule that encodes 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 genomic DNA or cDNA. Preferably, the polynucleotide encodes a polypeptide comprising one of the amino acid sequences shown in SEQ ID NOs: 1-17.

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 an origin of replication that allows the vector to replicate independently of the host cell. In addition, 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 be prokaryotic promoters (constitutive or regulatable, such as, for example, lac, trp, T3, T7 or gpt promoter), eukaryotic promoters (constitutive or regulatable, such as, for example, 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. Numerous prokaryotic and eukaryotic expression vectors suitable for the practice of the present invention are known to those skilled in the art. Examples are the prokaryotic vectors pHEN2, pGEM, pBluescript and pUC, which are regularly used for heterologous expression in E. coli. Examples of eukaryotic expression vectors include pcDNA3.1, pOG44, pSV2CAT Rc / CMV, Rc / RSV, GEM1 and the like.

The invention also relates to a host cell comprising a nucleic acid molecule encoding an antigen-binding polypeptide of the present invention, or an expression vector comprising such a nucleic acid molecule. In particular, it may be a prokaryotic or a eukaryotic host cell. Examples of host cells which can be used in the invention include prokaryotic host cells such as bacterial strains of Escherichia coli, Bacillus subtilis and the like. Suitable eukaryotic cells include yeast cells, insect cells, mammalian cells, plant cells, and the like. The antigen-binding polypeptide of the present invention is preferably expressed in bacterial cells, e.g. In 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 comprising: (a) cultivating a host cell as described above under conditions permitting the expression of a nucleic acid molecule encoding an antigen-binding polypeptide of the 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 ToxA or ToxB of C. difficile. Such a therapeutic treatment includes both the treatment of an acute disease state and the prophylactic treatment for the prevention of a disease.

The polypeptides of the present invention are also useful in the diagnosis of a disease caused by ToxA or ToxB of C. difficile. Diseases caused by these toxins include diarrhea, especially antibiotic-associated diarrhea (ADD), colitis, especially pseudomembranous colitis, enterocolitis or toxic megacolon. In a preferred embodiment, the antigen-binding polypeptides of the present invention are used for the therapeutic treatment of pseudomembranous colitis. The treatment comprises administering to a subject a polypeptide of the invention which has been detected to be infected with a ToxA and / or ToxB-expressing strain of C. difficile suspected of being infected with the causative agent or at risk is exposed to infect with the bacterial pathogen.

The invention accordingly also provides pharmaceutical compositions comprising antigen-binding polypeptides of the present invention. The antigen-binding polypeptide of the present invention is administered to the patient in a therapeutically effective amount, i. H. in an amount sufficient to substantially inhibit the toxin to which the antigen-binding polypeptide is directed. In a preferred embodiment of the invention, the administration of the antigen-binding polypeptide is in an amount that results in substantially complete inactivation of the toxin. Inactivation of the toxin can be monitored by improving one or more symptoms of the disease.

The exact amount of polypeptide that must be administered to achieve a therapeutic effect depends on several parameters. Factors relevant to the amount of the administered Polypeptides are, for. The type of administration of the polypeptide, the size of the particular polypeptide, the nature and severity of the disease, the history of the disease 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 and disclosure.

The polypeptide is preferably administered in an amount to a subject sufficient to achieve a plasma concentration of from about 0.05 μg / ml to about 150 μg / ml, preferably from about 0.1 to about 50 μg / ml, more preferably from about 1 μg / ml to about 20 μg / ml and normally from about 1 μg / ml to about 10 μg / ml, e.g. 5 μg / ml. The weekly dose may be from about 1 mg to about 500 mg polypeptide per m ² body surface area of the patient. The weekly dose of the polypeptide may / m ² range from about 10-300 mg, preferably from about 100-200 mg / m ^2, such as 150 mg / m ^2. Depending on the size of the polypeptide, 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, e.g. For oral administration as a tablet, capsule, powder, liquid or the like. However, it is preferred that the antigen-binding polypeptide be administered parentally by intravenous injection or intravenous infusion. The administration may be, for example, by intravenous infusion, e.g. Within 60 minutes, within 30 minutes, or within 15 minutes. Compositions suitable for administration by injection and / or infusion typically include solutions and dispersions as well as powders from which appropriate solutions and dispersions can be made. Such compositions will contain the immunologically active polypeptide and at least one 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 ^™ . 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 administration by injection or infusion should remain stable for a prolonged period after their preparation under storage conditions. The compositions may contain a preservative for this purpose. Suitable preservatives include chlorobutanol, phenol, ascorbic acid and thiomersal.

In one embodiment, 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. Sustained-release formulations may be present as semipermeable films of solid, hydrophobic polymer, e.g. In the form of microcapsules. The preparation of appropriate dosage forms and suitable auxiliaries are, for example, in "Remington: The Science and Practice of Pharmacy", Lippincott Williams &Wilkins; 21st edition (2005) described.

The polypeptides of the invention are further useful in the detection of ToxA and / or ToxB in biological samples, particularly in samples obtained from an individual who is to detect the presence of an infection with a strain of C. difficile, the ToxA and / or ToxB produces, such. As stool samples, tissue samples, etc. However, there may be other samples such. B. Water samples are tested for exposure to toxin.

• (a) ein wie oben beschriebenes und in den Ansprüchen definiertes Antigen-bindendes Polypeptid mit der biologischen Probe unter Bedingungen in Kontakt bringt, welche die Bildung von Komplexen aus dem Antigen-bindenden Polypeptid und dem Toxin ermöglichen; und
• (b) Komplexe aus dem Polypeptid und dem Toxin nachweist.

Therefore, the invention relates to a method for the detection of toxin A and / or toxin B of C. difficile in a biological sample in which

• (a) contacting an antigen-binding polypeptide as described above and defined in the claims with the biological sample under conditions which allow the formation of complexes of the antigen-binding polypeptide and the toxin; and
• (b) detecting complexes of the polypeptide and the toxin.

The method first comprises a step of contacting the antigen-binding polypeptide of the invention with the biological sample. This step is carried out under conditions suitable for the formation of binding complexes consisting of the antigen-binding polypeptide and the toxin to be detected. Conditions permitting the formation of binding complexes of antigen-binding polypeptides (particularly antibodies) and toxin are known in the art. Such conditions may be a temperature in the range of 4-37 ° C, a pH in the range of 6-8 and include an incubation period in the range of several minutes to several hours. It will be apparent to those skilled in the art that suitable binding conditions for a particular pair of antigen-binding polypeptide and toxin will depend on various factors, such as: The concentration of the antigen-binding polypeptide, the anticipated concentration of the toxin in the sample, and the like. The skilled artisan will be able to determine suitable conditions for binding between polypeptide and toxin by routine experimentation to optimize the formation of the binding complexes.

Having given the antigen-binding polypeptide ample opportunity to bind to the toxin (ToxA or ToxB), the immune complexes consisting of toxin and the specific polypeptide bound thereto are detected. In this case, conventional methods can be used in the prior art. For example, the antigen-binding polypeptide of the invention may be provided with a detectable label prior to contacting the sample suspected of containing ToxA and / or ToxB. When marking it may be z. B. be a fluorescent acid, chemiluminescent or radioactive label. A label with enzymes, such as. As with horseradish peroxidase or alkaline phosphatase, is also possible. Another method that allows detection of the complexes is by recombinant expression of the antigen-binding polypeptides as fusion proteins that have one or more peptide epitopes that can be visualized using labeled antibodies.

• (a) ein wie oben beschriebenes und in den Ansprüchen definiertes Antigen-bindendes Polypeptid mit der biologischen Probe unter Bedingungen in Kontakt bringt, welche die Bildung von Komplexen aus dem Antigen-bindenden Polypeptid und dem Toxin ermöglichen; und
• (b) die Komplexe aus dem Antigen-bindenden Polypeptid und dem Toxin von der Probe abtrennt.

In a further aspect, the invention relates to a process for the purification and / or concentration of ToxA or ToxB of C. difficile, in which

• (a) contacting an antigen-binding polypeptide as described above and defined in the claims with the biological sample under conditions which allow the formation of complexes of the antigen-binding polypeptide and the toxin; and
• (b) separating the complexes of the antigen-binding polypeptide and the toxin from the sample.

The purification and / or concentration of the toxin is preferably carried out with antigen-binding polypeptides immobilized on a solid support, such as e.g. On a material suitable for use in chromatographic separation processes. Suitable columnar materials include Sepharose (e.g., Q-Sepharose, DEAE-Sepharose, SP-Sepharose) and the like. The toxin-binding polypeptides may also be immobilized on beads, e.g. B. on agarose or glass beads. Preferably, the beads are magnetizable, thereby facilitating the separation of the binding complexes that adhere to the beads after incubation in toxin-containing samples. Methods of coupling polypeptides to a solid support are well known in the art. For example, a method suitable for coupling may include immobilization of polypeptides coupled with biotin or derivatives of biotin to a support material previously coated with streptavidin or derivatives thereof. Such compounds are commercially available from various suppliers. Alternatively, the protein can be bound directly to the carrier by chemical reaction. The sample containing the toxin is passed through the appropriately prepared column so that the toxin is specifically adsorbed by the immobilized antigen-binding polypeptides. By using a suitable wash buffer, all non-specifically bound molecules are washed off the column while the toxin is retained in the column. In a final step, the toxin can be eluted from the column with a suitable elution buffer (eg, a high salt buffer) to obtain the purified and / or concentrated toxin. Alternatively, the toxin bound to the matrix or column can be detected by an immunological detection method.

In embodiments where the antigen-binding polypeptides are not coupled to a carrier, separation from the sample may be accomplished using fusion polypeptides having an affinity tag in addition to an antigen-binding polypeptide of the invention. Such fusion polypeptides can be purified efficiently with the aid of a material which has a particularly high affinity for the affinity tag used. For example, the affinity tag may have 6-12 histidine residues that specifically interact with a chelating nickel ion matrix. Alternatively, the affinity tag may also consist of a glutathione S-transferase which can be purified using a glutathione matrix. Numerous other affinity tags are known in the art which can be used in conjunction with the antigen-binding polypeptides of the invention. Examples of Affinity Tag and affinity ligand pairs include maltose binding protein (MBP) and maltose; Avidin and biotin; Streptag and streptavin or neutravidin.

Affinity tags can, for. B. by ligation of a DNA coding for an antigen-binding polypeptide according to the invention, be prepared with a coding for the affinity tag sequence. In cases where the affinity tag is not a peptide, the affinity tag can be linked to the protein by chemical coupling reactions Antigen-binding polypeptide can be conjugated. For example, 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. B. BirA from E. coli (see, for example Smith et al. (1998) Nucleic Acids Research 26: 1414-1420 ; Recket et al. (1999) Protein Science 8: 921-929 ). 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 of which is called AviTag ^™ ( Tirat et al. (2006), Int. J. Biol. Macromol., 39 (1-3): 66-76 ) and the 76 amino acid BioEase ^™ sequence ( Tirat et al. (2006) , supra), all of which are biotinylated by the biotin protein ligase BirA on a central lysine residue. Also suitable are strep tags such as Streptag II (SA-WSHPQFEK) and One-STrEP-tag (SA-WSHPQFEK (GGGS) 2GGSAWSHPQFEK) (IBA BioTAGnology, IBA GmbH, Germany).

In addition, the antigen-binding polypeptides of the present invention may also be coupled with enzymes (such as alkaline phosphatase) or with recognition sequences for enzymes (such as the above BirA biotinylation sequences). Also, a coupling of various antigen-binding polypeptides, such as. VHHs directed against different epitopes of the same target antigen (ToxA or ToxB) is possible to enhance the efficiency of binding to the target antigen in this way. For this purpose, for example, 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 can bind four molecules of D-biotin. Such multimerized, single-domain VHH tetravalent antibodies have an increased affinity for the toxins. For multimerization streptavidin-coated beads, z. As magnetic or Sepharose beads used. Suitable beads are z. B. Strep-Tactin Magnetic Nanobeads (IBA BioTAGnology, IBA GmbH, Germany). In addition, coupling of ToxA or ToxB directed VHH domains to VHH domains directed against albumin or against a transcytotic receptor is also useful. Of course, the VHH single domain antibodies of the present invention may also be coupled to immunoglobulins, e.g. To the Fc region of an immunoglobulin.

• (a) ein wie oben beschriebenes und in den Ansprüchen definiertes Antigen-bindendes Polypeptid mit der biologischen Probe eines Patienten unter Bedingungen in Kontakt bringt, die die Bildung von Komplexen aus dem Antigen-bindenden Polypeptid und dem Toxin ermöglichen; und
• (b) Komplexe aus dem Polypeptid und dem Toxin nachweist; wobei der Nachweis der Komplexe in Schritt (b) darauf hinweist, dass der Patient an einer durch C. difficile vermittelten Erkrankung leidet.

In a preferred embodiment of the present invention there is provided a method of diagnosing a disease caused by ToxA and / or ToxB of Clostridium difficile, comprising:

• (a) contacting an antigen-binding polypeptide as described above and defined in the claims with the biological sample of a patient under conditions which allow the formation of complexes of the antigen-binding polypeptide and the toxin; and
• (b) detecting complexes of the polypeptide and the toxin; wherein the detection of the complexes in step (b) indicates that the patient is suffering from a C. difficile mediated disease.

The disease may be diarrhea, especially antibiotic-associated diarrhea (ADD), colitis, especially pseudomembranous colitis, enterocolitis, or a toxic megacolon. In a preferred embodiment, the antigen-binding polypeptides of the present invention are used for the therapeutic treatment of pseudomembranous colitis.

In a particularly preferred embodiment, the disease caused by C. difficile is pseudomembranous colitis. By using magnetic beads coated with streptavidin-biotin-VHH complexes, as described above, particularly effective accumulation of toxins from biological samples is possible.

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 Clostridium difficile.

Finally, the present invention also provides a kit for the detection of C. difficile, which kit comprises an antigen-binding polypeptide as described above. The kit may further comprise other reagents and means necessary for detection, e.g. B. Reagents for the visualization of a marker.

BRIEF DESCRIPTION OF THE FIGURES

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 region 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.

2 shows the amino acid sequences of VHHs selected by ToxA-CP and ToxB-GT. 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. Marked in bold letters are cysteine residues found in the VHHs.

3A shows the specificity of the selected VHHs in an ELISA analysis in bivalent format against the CP domain of ToxA (ToxA-CPD), ToxA 1-809, ToxA holotoxin, ToxB 1-807, ToxB holotoxin, the catalytic domain of CDT (CDTa) and milk powder (MPBS). 3B shows the specificity of the selected VHHs in an ELISA analysis in bivalent format against ToxB-GT, ToxB 1-807, ToxA 1-809, the catalytic domain of CDT (CDTa) and milk powder (MPBS). coVHH: Control VHH.

4A Figure 10 shows the dose-dependent binding of the selected anti-ToxA VHHs in an ELISA analysis in monovalent format against ToxA 1-809. 4B Figure 10 shows the dose-dependent binding of the selected anti-ToxB VHHs in an ELISA analysis in monovalent format against ToxB-GT. 4C Figure 10 shows the dose-dependent binding of the selected VHH S + 12 in an ELISA analysis in monovalent format against ToxA 1-809 and ToxB 1-507. coVHH: Control VHH.

5 shows the result of in vitro cleavage analysis. The toxin A directed VHHs L-10 and L-11 dose-dependently inhibit the cysteine protease activity, ie the autocatalytic cleavage of ToxA.

EXAMPLES

Example 1 Induction of ToxA- and ToxB-Specific Heavy Chain Antibodies by Immunization

For the induction of heavy chain antibodies directed against ToxA and ToxB, three llamas (designated Lamas # 5, #s 5026 and 5037) were immunized with polypeptides derived from the ToxA, ToxE and CDT toxins of C Difficile are derived. Lama # 5 received a ToxA-derived polypeptide, Lama # 5026 received a ToxB-derived polypeptide, and Lama # 5037 each received a polypeptide derived from ToxA, ToxB, and CDT. For the immunization with ToxA, the cysteine protease domain of ToxA (SEQ ID NO: 71 = amino acids 547-779 of SEQ ID NO: 69, 25 kDa) and for the immunization with ToxB the glycosyltransferase domain of ToxB (SEQ ID NO: 72 = amino acids 1-546 of SEQ ID NO: 70, 60 kDa). In each case, 400 μg of the ToxA or ToxB fragments or 50 μg of the CDT subunit CDTa were used for the primary immunization.

The toxin fragments were added to 500 μl of the Specol adjuvant (Cedi Diagnostics, Lelystad, The Netherlands) in 400 μl PBS and sonicated three times at 4 ° C. The injections were made subcutaneously in the larynx. Immunization of the llamas was enhanced by several consecutive injections of the antigens. Llamas # 5 and # 5026 were injected twice more, and Lama # 5037 was injected with the antigens three more times. Finally, Llamas # 5 and 5026, 11 days after the 3rd injection, Lama # 5037 ten days after the 4th injection, was bled 20-100 ml of blood for phage display library preparation (See Example 2).

The successful induction of the humoral immune response was confirmed by a serum titer ELISA. Overnight, the wells of an ELISA plate were coated at 4 ° C with 100 ng of antigen per well and then blocked for 2 h at room temperature (RT) with 5% milk powder in PBS. As antigens, the fragments were used, which were already used for immunization. The sera was used at a 1: 1000 dilution in PBS. To detect the bound antibodies, a polyclonal rabbit anti-llama IgG serum was used (1: 1000). As a secondary antibody, a sheep anti-rabbit peroxidase (PO) 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 preimmune sera were compared with the llamas' immune sera after the third (Lamas No. 5, 5026) and fourth injection (Lama No. 5037). The preimmune sera showed no reactivity against the antigens tested. The immune serum of the lama 5026 showed after the Immunizations no strong reactivity against ToxB. The llama immunized with three antigens showed high reactivity for all toxin antigens.

Example 2: Selection and sequence analysis of ToxA and ToxB specific VHHs from phage display libraries of the immunized llamas

(r, m, s und w bezeichnen Positionen mit variablen Nukleotiden: r = a oder g; m = a oder c; s = c oder g; w = a oder t.)For the preparation of a phage display library, RNA was first isolated from the peripheral blood lymphocytes of the llamas after the 3rd or 4th injection and transcribed into cDNA by means of reverse transcription. For reverse transcription, random hexamers (RH6) were used, which bind to the RNA at different positions. From this cDNA, the region encoding the VHHs of the heavy chain antibodies was amplified by PCR with the specific and partially degenerate oligonucleotides SHFmu / SHRmu and LHFmu / LHRmu:

(r, m, s and w denote positions with variable nucleotides: r = a or g, m = a or c, s = c or g, w = a or t.)

The amplified VHH fragments were cloned into the phagemid vector pHEN2 after digestion with the restriction endonucleases SfiI and NotI ( 1 ). The ligation products were transformed into E. coli to give a VHH fragment-expressing phage library.

Single domain specific antibodies were selected from the phage display libraries by panning, whereby the single domain antibodies bound via their VHHs to the immobilized antigens (ToxA, ToxB) which had previously been immobilized in the wells of an ELISA plate. For biotin panning, the ToxA and ToxB fragments described above were biotinylated with the EZ-Link sulfo-NHS biotinylation kit Thermo Scientific (Fisher Scientific, Schwerte Germany). The biotin is able to bind to streptavidin-agarose. 20 μl of streptavidin agarose were incubated with 2 μg of the biotinylated antigens and then incubated with 10 μl of the phage library. After washing, the phages were competitively eluted with 2 μg of the unconjugated antigen, and the supernatant was used for reinfection of E. coli with the phage. The libraries were subjected to 2 to 3 such selection cycles. After each round, 12 clones were sequenced to verify the success of the selection. The sequence of the final selected clones is in 2 listed.

DNA sequence analysis from the randomly selected clones confirmed the successful cloning of a broad library VHH repertoire. The sequencing also showed that the CDR3 of VHHs is 6-21 amino acids in length. All clones have the canonical cysteine pair that forms between the FR1 and the FR3 (see 2 ). Some of the sequenced clones also had an additional cysteine pair between CDR2 and CDR3.

Example 3: Recombinant Production of VHH Single Domain Antibodies in E. coli

The VHHs identified as described above were produced recombinantly in E. coli. Soluble single-domain 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, lacIq, 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. The transformed cells of E. coli were dissolved in 100 ml 2 × YT (BD Difco, Heidelberg, 16 g / l tryptone, 5 g / l NaCl, 10 g / l yeast extract) were cultured with 100 μg / ml carbenicillin to an optical density of 0.5. Protein expression was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) and terminated by centrifugation after three hours. By osmotic shock, the outer cell wall was broken. The VHHs were purified via their histidine tag by metal ion affinity chromatography at a yield of between 0.5 mg / L to 1 mg / L. The VHHs were then dialysed against PBS and stored after determination of the protein concentration at 4 ° C. The success of the production and purification was confirmed by SDS-PAGE analysis.

Example 4: Affinity analysis of the selected VHHs

The binding of the recombinantly produced VHHs to ToxA and ToxB was tested by ELISA ( 3 ). Single-domain antibodies were detected by peroxidase-conjugated antibodies against their C-terminal myc-tag. As antigens, 500 ng of ToxA-CPD (the toxA cysteine protease domain), ToxB-GT (ToxB's glucosyltransferase domain), ToxA 1-809 (a fragment of toxin A, the glucosyltransferase domain and the cysteine protease domain from ToxA), ToxB 1-807 (a fragment of toxin B comprising the Glucosyltransferase domain and the cysteine protease domain of ToxB), CDTa (the catalytic domain of CDT) or milk powder (MPBS) overnight at 4 ° C immobilized in the wells of a microtiter / ELISA plate. After blocking the free binding sites with 5% milk powder / PBS for 2 h at room temperature, VHH anti-Myc antibody complexes (compare Example 3) were incubated in the wells for 1 hour. For this purpose, the VHHs (100 ng) were first preincubated with a mouse anti-c-Myc antibody (700 ng) for 10 min at RT, resulting in VHH-anti-Myc complexes with bivalent properties, and then in the ELISA plate given. As a tertiary antibody, 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. As controls milk powder (MPBS), CDTa and the CDTa specific VHH single domain antibody (1 + 8) were used. The VHHs L-10a (# 2150, SEQ ID NO: 1), L-11a (# 2147, SEQ ID NO: 6), and L-14 (# 2145, SEQ ID NO: 8) showed specific reactions with ToxA- CPD and with ToxA 1-809 ( 3A ). VHHs L-7.II (# 2971, SEQ ID NO: 15), S + 12 (# 2967, SEQ ID NO: 16), L-14 (# 2966, SEQ ID NO: 17), and L-7c. I (# 2954, SEQ ID NO: 11) show specific binding to ToxB-GT and to ToxB 1-807 ( 3B ). VHH 5 + 12 cross-reacted with ToxA 1-809, but not with CDTa or MBPS ( 3B ).

The concentration-dependent binding of VHHs to ToxA 1-809 (300 ng / well) and to ToxB 1-546 (230 ng / well) was analogously tested with monovalent VHHs ( 4 ). For this purpose, the above-described ELISA was carried out, wherein the VHHs were not pre-incubated with anti-Myc antibodies. The VHHs (1 μg to 3 ng) were incubated in 1: 3,3 dilution steps for one hour in the wells of the ELISA plate. The wells were washed 2 times. To detect the bound VHHs, a mouse anti-c-Myc antibody was used (700 ng / well). The animal antibody used was a sheep anti-mouse IgG peroxidase (PO) conjugate (1: 5000). Detection was as described above. The VHHs L-10a (# 2150, SEQ ID NO: 1), L-11a (# 2147, SEQ ID NO: 6), and L-14 (# 2145, SEQ ID NO: 8) bound to ToxA 1 concentration-dependently. 809 ( 4A ). VHHs L-7.II (# 2971, SEQ ID NO: 15), S + 12 (# 2967, SEQ ID NO: 16), L-14 (# 2966, SEQ ID NO: 17), and L-7c. I (# 2954, SEQ ID NO: 11) bound to ToxB-GT (depending on concentration) ( 4B ). In addition, VHHs L-7.II and S + 12 also bound to ToxA-1-800 (depending on the concentration). 4C ). In the bivalent ELISA test format, as in 3B shown, S + 12 reacted significantly more strongly with ToxA 1-809 than in the monovalent assay format.

Example 5: Inhibition of Cysteine Protease Activity by ToxA-Specific VHHs

Immunization of the llamas was performed with the Cysteine Protease Domain (CPD) of ToxA. ToxA CPD is activated by inositol hexakisphosphate (InsP6). InsP6 is only present in eukaryotes, so that ToxA is cleaved only on contact with eukaryotes. In the presence of InsP6, which occurs only in the host cell, an autocatalytic processing of the toxin is initiated. By CPD, the enzymatically active N-terminus is cleaved from the C-terminal part and released into the cytosol ( Reineke et al. (2007) Nature, 446 (7134): 415-419 ).

The toxins ToxA 1-809 or ToxB 1-807 (1 μg) were preincubated with the anti-ToxA specific VHHs (from 0.08 μg to 1.25 μg) for 1 h at RT. The autoproteolytic processing of the toxins was induced by addition of 100 μM InsP6 and the batch was induced in the absence or presence of the VHHs for 1 h at 37 ° C. The protease activity was then visualized by SDS-PAGE ( 5 ). There was no autocatalytic cleavage in the control without the addition of InsP6 ("-"), and two fragments in the gel when added to InsP6 ("+").

The anti-ToxA VHHs L-10a and L-11a dose-dependently blocked the autocatalytic cleavage of ToxA. Control VHHs (coVHH) showed no inhibition of the autocatalytic cleavage of ToxA even at high dose. None of the anti-ToxA VHHs blocked the autocatalytic cleavage of ToxB.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

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Claims (20)

An antigen-binding polypeptide comprising the CDR1, CDR2, and CDR3 region of a VHH domain of a camelid heavy chain antibody, characterized in that the polypeptide is specifically in the region of amino acids 1-800 of a toxin having glycosyltransferase activity of Clostridium difficile binds. An antigen-binding polypeptide according to claim 1, characterized in that the CDR regions are derived from a VHH domain of a lamase The antigen-binding polypeptide according to claim 1 or 2, characterized in that the polypeptide binds specifically in the region of amino acids 543-800 of toxin A of Clostridium difficile. An antigen-binding polypeptide according to any one of claims 1 or 2, characterized in that the polypeptide binds specifically to the region of amino acids 1-544 of toxin B of Clostridium difficile. An antigen-binding polypeptide according to claim 3, characterized in that the polypeptide: (a) the CDR sequences shown in SEQ ID NOs: 18-20; (b) the CDR sequences shown in SEQ ID NOs: 21-23; (c) the CDR sequences shown in SEQ ID NOs: 24-26; (d) the CDR sequences shown in SEQ ID NOs: 27-29; (e) the CDR sequences shown in SEQ ID NOs: 30-32; (f) the CDR sequences shown in SEQ ID NOs: 33-35; (g) the CDR sequences shown in SEQ ID NOS: 36-38; (h) the CDR sequences shown in SEQ ID NOS: 39-41; or CDR sequences other than the CDR sequences depicted in (a) - (h) in at most one amino acid per CDR. An antigen-binding polypeptide according to claim 4, characterized in that the polypeptide: (a) the CDR sequences shown in SEQ ID NO: 42-44; (b) the CDR sequences shown in SEQ ID NOs: 45-47; (c) the CDR sequences shown in SEQ ID NOs: 48-50; (d) the CDR sequences shown in SEQ ID NOs: 51-53; (e) the CDR sequences shown in SEQ ID NOs: 54-56; (f) the CDR sequences shown in SEQ ID NOs: 57-59; (g) the CDR sequences shown in SEQ ID NOs: 60-62; (h) the CDR sequences shown in SEQ ID NOs: 63-65; (i) the CDR sequences shown in SEQ ID NOs: 66-68; or CDR sequences which differ from the CDR sequences depicted in (a) - (i) in at most one amino acid per CDR. The antigen-binding polypeptide of claim 3, characterized in that the polypeptide comprises an amino acid sequence selected from the group consisting of: (a) the amino acid sequences of SEQ ID NO: 1-8; and (b) an amino acid sequence having at least 80% or more sequence identity to any of the amino acid sequences of SEQ ID NOs: 1-8. The antigen-binding polypeptide of claim 4, characterized in that the polypeptide comprises an amino acid sequence selected from the group consisting of: (a) the amino acid sequences of SEQ ID NO: 9-17; and (b) an amino acid sequence having at least 80% or more sequence identity to any of the amino acid sequences of SEQ ID NOS: 9-17. An antigen-binding polypeptide according to any one of claims 1-8 for use in a method for the therapeutic treatment or diagnosis of a disease caused by Clostridium difficile. Nucleic acid molecule encoding an antigen-binding polypeptide according to any one of claims 1-8. An expression vector comprising a nucleic acid molecule according to claim 10. A host cell, characterized in that the host cell comprises a nucleic acid molecule according to claim 10 or an expression vector according to claim 11. A process for the recombinant production of an antigen binding polypeptide according to any one of claims 1-8 comprising: (a) cultivating a host cell according to claim 12 under conditions permitting the expression of a nucleic acid molecule according to claim 10; and (b) isolating the antigen-binding polypeptide from the culture. A pharmaceutical composition comprising an antigen-binding polypeptide according to any one of claims 1-8. A method of detecting a glycosyltransferase activity-containing toxin of Clostridium difficile in a biological sample comprising: (a) contacting an antigen-binding polypeptide according to any one of claims 1-8 with the biological sample under conditions which. allow the formation of complexes of the antigen-binding polypeptide and the toxin; and (b) detecting the complexes of the antigen-binding polypeptide and the toxin. A process for the purification and / or concentration of a glycosyltransferase activity-containing toxin of Clostridium difficile, in which (a) contacting an antigen-binding polypeptide of any one of claims 1-8 with the biological sample under conditions which allow the formation of complexes of the antigen-binding polypeptide and the toxin; and (b) separating the complexes of the antigen-binding polypeptide and the toxin from the sample. A method for the diagnosis of a Clostridium difficile-mediated disease in which (a) contacting an antigen-binding polypeptide of any one of claims 1-8 with the biological sample of a patient under conditions which permit the formation of complexes of the antigen-binding polypeptide and the toxin; and (b) detecting complexes of the polypeptide and the toxin; wherein the detection of the complexes in step (b) indicates that the patient is suffering from a disease caused by Clostridium difficile. Use of an antigen-binding polypeptide according to any one of claims 1-8 for the therapeutic treatment or diagnosis of a disease caused by Clostridium difficile. A method according to claim 17 or a use according to claim 18, characterized in that the disease is colitis. A kit for the detection of Clostridium difficile, characterized in that the kit comprises an antigen-binding polypeptide according to any one of claims 1-8.

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