WO2021219786A1

WO2021219786A1 - Anti-toxin secretory iga2 preparation

Info

Publication number: WO2021219786A1
Application number: PCT/EP2021/061264
Authority: WO
Inventors: Gottfried Himmler
Original assignee: Gottfried Himmler
Priority date: 2020-04-30
Filing date: 2021-04-29
Publication date: 2021-11-04

Abstract

A monoclonal secretory immunoglobulin A subtype 2 (SIgA2) preparation, which SIgA2 is specifically recognizing at least one target being toxin A and/or toxin B of Clostridium difficile.

Description

ANTI-TOXIN SECRETORY IGA2 PREPARATION

FIELD OF THE INVENTION

The invention refers to a monoclonal secretory immunoglobulin A subtype 2 (SlgA2) preparation, which SlgA2 is specifically recognizing at least one target being toxin A and/or toxin B of Clostridium difficile.

BACKGROUND OF THE INVENTION

The immune system of mammals is organized into discrete compartments and a complex organization of cells and effector molecules to keep an organism in homeostasis. An important part of the immune system is the antibody mediated immunity, which is also frequently utilized for therapeutic approaches.

Most immunoglobulins and antibodies in many species are similar in their overall structure, accounting for certain similarities in physiochemical features such as charge and solubility. Antibodies typically have a common core structure of two identical light chains, each about 24 kilodaltons, and two identical heavy chains of about 55-70 kilodaltons each. One light chain is attached to each heavy chain, and the two heavy chains are attached to each other. Both, the light and heavy chains, contain a series of repeating homologous units, each of about 110 amino acid residues in length which fold independently in a common globular motif, called an immunoglobulin (Ig) domain. The region of an antibody molecule formed by the association of the two heavy chains is hydrophobic. Because antibodies contain numerous cysteine residues, many disulfide bonds are contributing to the relative stability of the multichain antibody structure.

Antibody molecules can be divided into distinct classes and subclasses based on physiochemical characteristics such as size, charge and solubility, and on their behavior in binding to antigens. In humans, the classes of antibody molecules are IgA, IgD, IgE, IgG and IgM. Members of each class are said to be of the same isotype. IgA and IgG isotypes are further subdivided into subtypes called lgA1, lgA2 and lgG1 , lgG2, lgG3 and lgG4. The heavy chains of all antibody molecules in an isotype share extensive regions of amino acid sequence identity but differ from antibodies belonging to other isotypes or subtypes.

Sequence diversity in antibodies is predominantly found in three short stretches within the amino terminal domains of the heavy and light chains called variable (V) regions, respectively, to distinguish them from the more conserved constant (C) regions. V-regions of the heavy chains are denoted VH, and are mostly independent of the antibody class, V-regions of the light chains are denoted VL.

The antibody class is defined by the structure of the constant regions and therefore these regions are denoted according to the class with greek letters (e.g. Cgamma for IgG constant regions, Calpha for IgA constant regions, Cepsilon for IgE constant regions. Most antibodies can be cleaved by various proteolytic enzymes to give rise to specific fragments, e.g., Fab, F(ab)2, both containing the variable and antigen-binding regions and the Fc, comprising only constant regions.

Antibodies have been engineered in many ways to change physicochemical, technical and physiological properties including species, class, size, shape, valency, specificity etc.

In IgA antibodies, two antibody heavy chains (HCs) and two antibody light chains (LCs) are organized into two Fab regions (each comprising VH, CH1 (Calphal), VL, and CL domains), responsible for binding to antigen, linked via the hinge region to a single Fc region (comprising two CH2 - Calpha2 - and two CH3 - Calpha3- domains).

The interaction between chains is stabilized by disulphide bonds between the HCs and LCs within the Fab region and between the two HCs at the CH2 domains, and by close pairing of opposing domains: VH with VL, CH1 with CL, and one CH3 with the other one. Such pairing relies on an array of non-covalent interactions, chiefly hydrogen bonds and van der Waals between the domains involved. At the C-terminus of the IgA HC lies an 18 amino acid extension known as the tailpiece that allows for oligomer-formation. Dimeric or higher oligomeric IgA comprises a joining chain (JC), a 15 kDa protein.

SlgA is a polymeric antibody, typically composed of at least two IgA monomers (a dimeric IgA, dlgA), although higher order polymers have been reported, wherein monomers are linked through one JC and bound by one secretory component (SC). In the organism, SlgA assembly begins in plasma cells, which link two IgA monomers, each having two heavy chains (HC) and two light chains (LC), and one JC to form dimeric (d) IgA. The polymeric immunoglobulin receptor (plgR) binds dlgA on the basolateral surface of epithelial cells and transports it to the apical surface. Subsequently, the plgR ectodomain, called secretory component (SC), is proteolytically cleaved, releasing the SC-dlgA complex into the mucous lining of the gastrointestinal, urogenital and respiratory tracts, as well as into tears, saliva and milk where it is called SlgA. Usually, SlgA contains four antigen binding fragments (Fabs) and two Fc regions. Each Fab is a dimer of variable LC and constant LC domains (VL1-CL1) bound to variable HC and constant HC domains (VH1-CH1); each Fc is a dimer of two constant HC domains (CH), CH2-CH3. IgA CH3 domains have a unique C-terminal extension called a tailpiece (Tp), which is also found on IgM CH4, and is required for antibody oligomerization. In dlgA, the Tp is known to form inter-chain disulfide bonds with the JC, which is required for plgR binding. The plgR (and SC) contains five Ig-like domains (D1-D5), each having loops structurally similar to antibody complementarity determined regions (CDRs); D1 is necessary and sufficient for binding to dlgA, yet D5 is also known to bind through covalent and non-covalent interactions.

There are two closely related subclasses lgA1 and lgA2 in human SlgA. The major difference between human lgA1 and lgA2 subclasses occurs in the hinge region. The hinge region can be defined to be located between the conserved C-terminal Cysteine of the CH1 -domain and the most N-terminal Cysteine residue of the CH2- domain. The hinge region of human lgA1 is comprised of 20 residues and 5 O- glycosylation sites, while the hinge region of human lgA2 comprised of 7 residues and no sites of glycosylation.

It is presumed that lgA1 generally has a broader reach that is beneficial in antigen recognition with distantly spaced antigens, but at the cost of higher susceptibility for proteolysis. Because lgA2 lacks this extended hinge region, it may be less vulnerable to lgA1 bacterial proteases. Furthermore, post-translational glycosylation varies between IgA isotypes and sub-isotypes. lgA1 has two conserved N-linked glycosylation sites (Asn-263 and Asn-459), whereas lgA2 has either two (lgA2m1) or three (lgA2m2) additional N-linked glycosylation sites. lgA1 also harbors O-linked glycosylation in the hinge region, in contrast to lgA2. Mucosal SlgA comprises mostly dimeric IgA (two IgA monomers joined together through a JC), but can also comprise trimeric (three IgA monomers joined together through a JC) or tetrameric IgA (four IgA monomers joined together through a JC). It comprises predominantly the subclass lgA2, being found in at least the two allotypic forms, lgA2m(1) and lgA2m(2), lgA2m(1) being predominant in Europe and lgA2m(2) having the highest frequency in Africa. lgA2 can form dimers and higher-order oligomers, though there is a propensity for lgA2m(2) to form higher-order oligomers.

In serum, almost all IgA molecules are of subclass lgA1.

Polymeric IgA can be produced from plasma or by recombinant techniques in cells, microorganisms or transgenic organisms. Recombinant polymeric immunoglobulin can be produced by co-expression of heavy chain, light chain and J- chain in one cell. Subsequently, the polymeric immunoglobulin (e.g., dimeric IgA) can be complexed with recombinant or natural SC in vitro to form secretory immunoglobulin (Slg). Alternatively, the polymeric immunoglobulin can be used to form secretory immunoglobulins SlgA in the organism by utilizing the natural transcytosis mechanism. This is achieved by systemic application (e.g. intravenously) of the polymeric immunoglobulin. Another possibility to produce secretory immunoglobulin is the co-expression of heavy chain, light chain, J-chain and SC in one cell or organism.

W02013/150138A1 discloses a complex of SC and IgA or IgM, which SC is characterized by a Lewis-type N-glycosylation pattern.

WO201 3/174971 A1 discloses SlgA for use in secretory immunoglobulin deficiency treatment and prophylaxis.

Clostridium difficile ( C . difficile) is a gram-positive bacterium that causes gastrointestinal disease in humans. C. difficile is the most common cause of infectious diarrhea in hospital patients, and is one of the most common nosocomial infections.

Treatment with antibiotics such as ampicillin, amoxicillin, cephalosporins, and clindamycin that disrupt normal intestinal flora can allow colonization of the gut with C. difficile and lead to C. difficile disease. The onset of C. difficile disease typically occurs four to nine days after antibiotic treatment begins, but can also occur after discontinuation of antibiotic therapy. C. difficile can produce symptoms ranging from mild to severe diarrhea and colitis, including pseudomembranous colitis (PMC), a severe form of colitis characterized by abdominal pain, watery diarrhea, and systemic illness ( e.g ., fever, nausea). Relapsing disease can occur in up to 20% of patients treated for a first episode of disease, and those who relapse are at a greater risk for additional relapses.

C. difficile disease is believed to be caused by the actions of two exotoxins, toxin A and toxin B (TcdA and TcdB), on gut epithelium. Both toxins are high molecular weight proteins (280-300 kDa) that catalyze covalent modification of Rho proteins, small GTP-binding proteins involved in actin polymerisation, in host cells. Modification of Rho proteins by the toxins inactivates them, leading to depolymerization of actin filaments and cell death. Both toxins are lethal to mice when injected parenterally.

C. difficile disease can be diagnosed by assays that detect the presence or activity of toxin A or toxin B in stool samples, e.g., enzyme immunoassays. Cytotoxin assays can be used to detect toxin activity.

Antibiotics are the primary treatment option at present. Antibiotics least likely to cause C. difficile associated disease such as vancomycin and metronidazole are frequently used. Vancomycin resistance evolving in other microorganisms is a cause for concern in using this antibiotic for treatment, as it is the only effective treatment for infection with other microorganisms. Probiotic approaches, in which a subject is administered non-pathogenic microorganisms that presumably compete for niches with the pathogenic bacteria, are also used.

WO2013132054A discloses secretory immunoglobulin in the prevention of infection by Clostridium difficile, and exemplifies a secretory IgA preparation composed of a polyclonal IgA containing plasma fraction mixed with a secretory component, showing an about 2-fold improved effect against C. difficile toxin B when using the such secretory IgA preparation compared to such IgA containing plasma fraction without the secretory component.

Stubbe et al. (Journal of Immunology, 2000, 164: pages 1952-1960) describe polymeric IgA carrying variable domains of an IgG antibody directed against Clostridium difficile toxin A.

W02009156307A discloses SlgA preparations comprising at least one probiotic to treat bacterial infections such as a Clostridia infection.

There is a need for new anti-C. difficile immunoglobulin preparation to neutralize C. difficile toxin A and/or B. SUMMARY OF THE INVENTION

It is the objective of the present invention to provide for an immunoglobulin preparation which has improved activity to neutralize C. difficile toxin A and/or B, in particular for use as a pharmaceutical or to treat body or other surfaces potentially contaminated with C. difficile.

The objective is solved by the subject of the present invention and as further described herein.

The invention provides for a monoclonal secretory immunoglobulin A subtype 2 (SlgA2) preparation, which SlgA2 is specifically recognizing at least one target being toxin A and/or toxin B of Clostridium difficile.

According to a specific aspect, the SlgA2 comprises at least two antigen binding sites (valencies), such as two antigen-binding sites per antibody monomer, four antigen-binding sites in a dimeric immunoglobulin, six antigen-binding sites in a trimeric immunoglobulin, or eight antigen-binding sites in a tetrameric immunoglobulin.

The antigen-binding sites of a SlgA2 can be identical, or may differ to provide a multispecific SlgA2, wherein at least one of the antigen-binding sites has a specificity to bind a target toxin, and another antigen-binding site is different. Any different antigen-binding site may have a specificity to bind a target toxin as well, yet, at a different epitope. Alternatively, any different antigen-binding site may bind to any other target molecule (other than toxin A or B), or may be polyreactive.

According to a specific aspect, the SlgA2 is monospecific, or multispecific, such as e.g., bispecific, comprising: a) a recombinant IgA oligomer composed of at least two IgA monomers linked by a J-chain, wherein at least one of said IgA monomers is a target-specific IgA of the subtype 2 and comprises an antigen-binding site specifically recognizing one of said targets; and b) a recombinant secretory component (SC).

In a monospecific SlgA2, the target recognized by said target-specific IgA, or said at least two IgA monomers is any one of toxin A or toxin B, either targeting the same region, or the same epitope within one of toxin A or toxin B, or different regions or epitopes within one of toxin A or toxin B. Specifically, a target-specific IgA which is monospecific, is recognizing any one of toxin A or toxin B, either targeting the same region, or the same epitope within one of toxin A or toxin B, or different regions or epitopes within one of toxin A or toxin B.

In a bispecific or multispecific SlgA2, two different targets (or more) are recognized by said target-specific IgA, or said at least two IgA monomers, wherein at least one specificity is directed to any one of toxin A or toxin B, and at least another specificity is directed to the other of toxin A or toxin B, or to another target different from any of toxin A or toxin B.

Specifically, a target-specific IgA which is bispecific or multispecific, two (or more) different targets are recognized by said target-specific IgA, wherein one specificity is directed to any one of toxin A or toxin B, and another specificity is directed to the other of toxin A or toxin B, or to another target different from any of toxin A or toxin B.

Such other targets different from any of toxin A or toxin B can be of antigens of C. difficile, or C. difficile transferase (CDT; or binary toxin), such as surface antigens, adhesins (e.g., Cwp66, GroEL, and FbpA), C. difficile proteases or spores of C. difficile, in particular certain spore proteins such as CotE, CotA, and CotCB; exosporium protein CdeC; and cytosolic methyltransferase; C. difficile surface polysaccharides; or other microbial antigens, such as toxins or other antigens of any other bacteria.

Specific examples refer to SlgA2, wherein each IgA monomer comprised in the SlgA2 is a monospecific IgA.

Specific further examples refer to SlgA2, comprising at least one IgA monomer comprising two different antigen-binding sites, which can be monospecific or bispecific.

Preferred embodiments refer to anti-toxin B SlgA2, wherein all IgA monomers comprised in said SigA2 are target-specific IgA and monospecific to recognize toxin B.

Further preferred embodiments refer to anti-toxin A SlgA2, wherein all IgA monomers comprised in said SlgA2 are target-specific IgA and monospecific to recognize toxin A.

Specifically, the target toxins are of any naturally-occurring C. difficile strain or respective pathogen, such as any C. difficile strain from clade 1 to clade 5 or clade C- I carrying the PaLoc genome region; in particular, strains from clade 1 , clade 2 and clade 3, Clostridium difficile toxinotype I to XXXI, strains with genomes coding for at least parts of the genes tcdA or tcdB. e.g., VPI 10463 (ribotype 087), strains of ribotypes 001 , 012, 017, 027, and 53, as well as so-called hypervirulent ribotypes 027 and 078. Such strains are available from commercial sources e.g., at ATCC or tgcBIOMICS GmbH, Germany.

Specifically, a) said toxin B comprises or consists of SEQ ID NO:1 ; and b) said toxin A comprises or consists of SEQ ID NO:2.

Specifically, the target region of toxin B comprises or consists of any one or more of glucosyltransferase-domain (GTD), cysteine-autoprotease-domain (CPD), translocation-domain (TLD), pore forming region (PFR), receptor binding domain (receptor binding region), or C-terminal domain (CTD) with combined repetitive oligopeptide (CROP) subdomains, such regions and domains being described in Annu. Rev. Microbiol., 2017, Volume 71 , pages 281-307.

The target-specific IgA may specifically bind to and/or neutralize toxin B, and specifically binds to an epitope in a C-terminal portion of toxin B (e.g., between amino acids 1777-2366 of toxin B). Other particular antibodies or antigen binding portions thereof, can specifically bind to an epitope within amino acid residues 1-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 900-1000, 1100-1200, 1200- 1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1800-1900, 1900-200, 2100- 2200 or 2200-2366 of toxin B, or any internal, portion or range thereof.

Specific embodiments refer to target regions comprising or consisting of epitopes recognized by any one or more of the antibodies bezlotoxumab, PA-41 (Sequences SEQ ID NO:8 and 10 from patent US8986697), or R2 (Sequences SEQ ID NO:282 and 274 from patent US9399674)

Specifically, the epitope of antibody bezlotoxumab is characterized by comprising or residing in the N-terminal part of the Toxin B (TcdB) CROP domain, between residues 1874 and 1970 and between residues 2005 and 2102 of SEQ ID NO:1. Specifically, the epitope of antibody PA-41 is located within or comprises the amino acid residues 320 and 351 of SEQ ID NO:1 in the glucosyltransferase domain of Toxin B (TcdB).

Specifically, the epitope of antibody R2 is located outside of the carboxy terminal receptor binding domain of toxin B.

Specifically, the target region of toxin A comprises or consists of any one or more of domains glucosyltransferase-domain (GTD), cysteine-autoprotease-domain (CPD), translocation-domain (TLD), pore forming region (PFR), receptor binding domain (receptor binding region) or C-terminal domain (CTD) with combined repetitive oligopeptide (CROP) subdomains, such regions and domains being described in Nat Struct Mol Biol. 2019; vol. 26, pages 712-719..

The target-specific IgA may specifically bind to and/or neutralize toxin A, and specifically binds an epitope within the N-terminal half of toxin A, e.g., an epitope between amino acids 1-1256 of toxin A. In other embodiments, the antibodies or antigen binding portions thereof specifically bind to an epitope within the C-terminal receptor binding domain of toxin A, e.g., an epitope between amino acids 1853-2710 of toxin A, or an epitope between amino acids 659-1852, e.g., an epitope within amino acid residues 900-1852, 900-1200, or 920-1033 of toxin A. In other embodiments, the antibodies or antigen binding portions thereof specifically bind an epitope within amino acids 1-600, 400-600, or 415-540 of toxin A. Other particular antibodies or antigen binding portions thereof, can specifically bind to an epitope within amino acid residues 1-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 900-1000, 1100-1200, 1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1800-1900, 1900-200, 2100-2200 or 2200-2300, 2300-2400, 2400-2500, 2500-2600, 2600-2710 of toxin A, or any interval, portion or range thereof.

Specific embodiments refer to target regions comprising or consisting of epitopes recognized by any one or more of the antibodies actoxumab, PA-50 (sequences SEQ ID NO:6 and 7 from patent US8986697) and R1 (sequences SEQ ID NO:146 and 154 from patent US9399674).

Specifically, the epitope of antibody PA-50 is within the CROP-domain of Toxin A, including amino acids 2522 to 2685 of SEQ ID NO 2. Specifically, the epitope of antibody actoxumab is within the CROP domain of Toxin A (TcdA) and include residues between amino acids 2161 and 2437 of SEQ ID NO 2.

Specifically, antibody R1 recognizes an epitope within the carboxy terminal receptor binding domain of toxin A.

Specifically, the epitope of antibody PA-50 comprises amino acids sequence repeats in between amino acids 2449 and 2649 of SEQ ID NO:2.

Specifically, the target-specific IgA or SlgA2 comprises at least one antigen binding site of any one of the antibodies actoxumab, PA-50, R2, bezlotoxumab, PA- 41 or R1 , or which competitively binds to any one of the antibodies actoxumab, PA- 50, R1 , bezlotoxumab, PA-41 or R2.

Specifically, the SlgA2 is characterized by its functional activity as determined by its binding affinity to the target toxin, and/or to neutralize its toxic effect on animal cells, e.g. as determined in an in vitro or ex wVo assay.

The SlgA2 is considered functional, if specifically binding to the target toxin.

In preferred embodiments, SlgA2 is considered functional, if neutralizing the target toxin.

Exemplary binding tests employ native or recombinant C. difficile toxins A or B or toxoids with surface plasmon resonance setups, biolayer interferometry, homogeneous or heterogeneous and solid phase immunoassays.

Functional activity can be tested by in vitro cell killing assays, in a particular setup for toxin neutralization in which toxins are able to kill cells in a dose dependent manner. The antibodies are being added to the respective toxins in order to inhibit cell killing by either C. difficile toxin A or toxin B, or by both, e.g., in case of antibody mixtures. The readout can be the amount of release of certain substances from the cells or the cell shape, indicating cell death or cell disruption. Another type of in vitro toxin neutralization assay is the estimation of the integrity of cell layers before and after toxin (or toxin/antibody mixture) addition, as measured by transepithelial electric resistance, such as described in the examples provided herein.

Specifically, the SlgA2 preparation is a monoclonal one insofar as it comprises a complex consisting of monoclonal components and optionally additional synthetic components. In particular a monoclonal SlgA2 disclosed herein comprises or consists of at least two monoclonal lgA2 molecules associated with a monoclonal SC and a monoclonal (or synthetic) joining element such as a JC. To provide the monoclonal components of such monoclonal SlgA2, the respective amino acid sequences are produced as recombinant proteins or subunits, which are then assembled and optionally linked to obtain a recombinant SlgA2 complex. Respective coding nucleotide sequences ( e.g ., cDNA) may be employed to engineer a clone of a recombinant host cell expressing any of such sequences as a recombinant expression product in a host cell culture. The sequences preferably encode polypeptides of mammalian origin, chimerics, species-adapted sequences, or other naturally- occurring sequences which can be modified to produce functional variants. Respective sequence information may be derived from public databases, as appropriate.

Specifically, the lgA2 molecules comprised in such SlgA2 complex comprises two or more IgA molecules bound by a joining element, such as a J chain. For example, the IgA can be dimeric, trimeric or tetrameric IgA, joined by a JC.

According to a specific embodiment, the SlgA2 comprises dimeric lgA2, and a SC.

Specifically, the J-chain links the immunoglobulins of the SlgA2 via cysteine e.g., at the C-terminal tail of the immunoglobulins. For example, IgA dimers are produced by disulfide bonds between the monomers and the J-chain. Any suitable J- chain can be used, in particular naturally-occurring J-chains (including all isoforms), or functionally active variants thereof which are capable of linking IgA monomers, to produce the respective oligomers.

According to a specific aspect, the SlgA2 comprises the CDR-binding site of an anti-toxin A or anti-toxin B antibody of the IgG type, in particular selected from any such IgG antibodies that are being used or commercialized for the treatment of Clostridium difficile infection and pathogenesis. Any suitable monoclonal antibody (or antigen-binding fragments thereof including e.g., the variable domains, or Fab) of the anti-toxin A or anti-toxin B specificity can be used as a template (acting as “parent” antibody) to produce the SlgA2 with the same CDR-binding site, or with such CDR- binding site that is modified to a certain extent, to maintain or even improve its binding properties.

The CDR-binding site is typically composed of six CDR sequences, three of the heavy chain variable domain, and three of the light chain variable domain, which CDR sequences interact with the binding target. The suitable CDR sequences can be engineered into the variable domains of the SlgA2, and optionally varied for optimizing binding to the target toxin, such as by affinity maturation. Yet, it is preferred that any variation of a CDR sequence encompasses only one or two point mutations at selected position(s), such that the variant CDR sequence is functionally active in the respective CDR-binding site, to specifically recognize the target toxin, at least with the same, optimized, or improved binding affinity.

Specific point mutations are any of a deletion, insertion or substitution of only one or two amino acids.

The CDR-binding site, thus, may comprise the six identical CDR sequences of the respective CDR-binding site of an IgG antibody that has proven anti-toxin A or anti toxin B binding specificity and optionally proven toxin-neutralizing activity (which is then used as a “parent” antibody), or may comprise less than the six identical ones, e.g. only one, two, three, four, or five identical CDR sequences, and the other CDR sequences of the CDR-binding site may be CDR variants of the respective parent CDR sequences, or originate from another IgG antibody that binds to about the same epitope than the parent antibody.

Exemplary CDR-binding sites originate from well-known anti-toxin A or anti toxin B antibodies of the IgG type which can be used as parent antibodies to engineer the SlgA2. Exemplary parent antibodies are selected from the group consisting of actoxumab, PA-50, R1 , bezlotoxumab, PA-41 and R2.

According to a specific embodiment, the target-specific IgA or SlgA2 comprises at least one antigen-binding site which is a CDR-binding site specifically recognizing toxin A and comprises three VH-CDR sequences (VH-CDR1 , VH-CDR2 and VH- CDR3) of a variable heavy chain antibody domain (VH), and three VL-CDR sequences (VL-CDR1 , VL-CDR2 and VL-CDR3) of a variable light chain antibody domain (VL), wherein: A) (embodiment A refers to the six CDR sequences of actoxumab) a) VH-CDR1 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:3; b) VH-CDR2 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:4; c) VH-CDR3 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:5; d) VL-CDR1 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:6; e) VL-CDR2 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:7; and f) VL-CDR3 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:8; or B) (embodiment B refers to the six CDR sequences of antibody R1) a) VH-CDR1 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:9; b) VH-CDR2 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:10; c) VH-CDR3 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:11; d) VL-CDR1 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:12; e) VL-CDR2 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:13; and f) VL-CDR3 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:14; or C) (embodiment C refers to the six CDR sequences of antibody PA-50) a) VH-CDR1 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:15; b) VH-CDR2 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:16; c) VH-CDR3 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:17; d) VL-CDR1 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:18; e) VL-CDR2 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:19; and f) VL-CDR3 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:20; wherein the sequences are according to IMGT.

According to a specific aspect, the SlgA2 comprises a toxin A binding site which comprises the CDR sequences incorporated within the VH and VL domains, wherein a) said VH comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:21; and said VL comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:22; or b) said VH comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:23; and said VL comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:24; or c) said VH comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:25; and said VL comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:26.

According to a specific further embodiment, the target-specific IgA or SlgA2 comprises at least one antigen-binding site which is a CDR-binding site specifically recognizing toxin B and comprises three VH-CDR sequences (VH-CDR1, VH-CDR2 and VH-CDR3) of a variable heavy chain antibody domain (VH), and three VL-CDR sequences (VL-CDR1, VL-CDR2 and VL-CDR3) of a variable light chain antibody domain (VL), wherein:

A) (embodiment A refers to the six CDR sequences of bezlotoxumab) a) VH-CDR1 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:27; b) VH-CDR2 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:28; c) VH-CDR3 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:29; d) VL-CDR1 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NQ:30; e) VL-CDR2 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:31; and f) VL-CDR3 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:32; or B) (embodiment B refers to the six CDR sequences of antibody antibody R2) a) VH-CDR1 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:33; b) VH-CDR2 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:34; c) VH-CDR3 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:35; d) VL-CDR1 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:36; e) VL-CDR2 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:37; and f) VL-CDR3 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:38; or C) (embodiment C refers to the six CDR sequences of antibody antibody PA- 41) a) VH-CDR1 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:39; b) VH-CDR2 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:40; c) VH-CDR3 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:41; d) VL-CDR1 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:42; e) VL-CDR2 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:43; and f) VL-CDR3 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:44; wherein the sequences are according to IMGT. Specifically, the CDR sequences according to IMGT as referred to herein are understood as those amino acid sequences of an antibody as determined according to the IMGT system (The international ImMunoGeneTics, Lefranc et al., 1999, Nucleic Acids Res. 27: 209-212).

According to a specific aspect, the SlgA2 comprises a toxin B binding site which comprises the CDR sequences incorporated within the VH and VL domains, wherein a) said VH comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:45; and said VL comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:46; or b) said VH comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:47; and said VL comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:48; or c) said VH comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:49; and said VL comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:50.

Specifically, the CDR sequences provided herein have a certain sequence identity to a defined CDR sequence identified herein, herein referred to as “parent” CDR sequence. Such sequence identity can be at least any one of 80%, 85%, 90%, 95% or 100%, and particularly allows a) 0, 1 or 2 point mutations in a parent CDR sequence with a long length of at least 10 amino acids; b) 0, or 1 point mutation in a parent CDR sequence with a medium length of at least 5 amino acids; and c) 0 point mutation in a parent CDR sequence with a short length of less than 5 amino acids.

Specific embodiments are characterized by only 0, 1 or 2 point mutations in one, two three, four, five, or six CDR sequences of an antigen-binding site, preferably no more than 1 or 2 point mutations in each CDR sequence of one antigen-binding site.

In any case, such antigen-binding site comprising a limited number of point mutations in the CDR sequence(s) is still functional to specifically recognize and bind the target, though the affinity can be e.g. optimized or improved e.g., upon affinity maturation or otherwise mutagenesis or of any one or more of said CDR sequences. Specifically, the antigen-binding site comprises such optimized or affinity maturated CDR sequences as compared to the parent CDR sequences.

For the purpose of providing variants, such antibodies or antibody domains are herein referred to as parent ones. CDR or framework (FR) sequences can be used as parent CDR or parent framework sequences. It is well understood that any antibody sequence as described herein is considered a “parent” sequence which can be subject to variation e.g., by one or more point mutations.

According to a specific aspect, a functional variant antibody may be used which binds the same epitope as the parent antibody.

According to a further specific aspect, the functional variant antibody comprises the same binding site as the parent antibody.

Typically, such variant antibodies recognizing about the same epitope of a parent antibody, if competitively binding to the epitope recognized by the parent antibody. Competition of binding is preferably determined by competition ELISA analysis or by surface plasmon resonance (e.g.Biacore), biolayer-interferometry, BLI (e.g. ForteBio Octet) analysis, or isothermal titration calorimetry.

The antibody or the functional variant of any of the exemplified antibodies (which can be used as parent antibodies) that competitively binds to any of the parent antibodies is specifically characterized by a relative inhibition of binding to its target as determined by competition ELISA analysis or by surface plasmon resonance, Biacore, BLI or ForteBio analysis, which relative inhibition is preferably greater than 30%.

A specific antibody variant is e.g., a human or artificial variant of a parent antibody, wherein the parent CDR sequences are incorporated into human or artificial framework sequences (e.g., of non-human origin, such as human framework sequences including one or more point mutations), wherein optionally 1 , 2, 3, or 4 amino acid residues of each of the parent CDR sequences may be further mutated by introducing point mutations to improve the stability, specificity and affinity of the parent or humanized antibody.

According to a specific aspect, the CDR-binding site is comprised in VH and VL domains which comprises sequences originating from a mammal, such as a human being or non-human animal, or which are modified to resemble the VH and VL sequences of such animal, e.g. chimeric or species-adapted, such as humanized, caninized, equinized or felinized sequences. Accord ing to a specific aspect, the framework sequences include human, artificial or animal sequences. Specifically, the antibody comprises one or more constant domains, which are of a natural secretory immunoglobulin e.g., of an lgA2 or lgA1 type.

Specifically, the antibody comprised in the SlgA2 described herein is a humanized, chimeric (e.g., comprising variable domains originating from a non-human animal antibody and constant domains originating from human antibodies), or fully human antibody.

According to a specific aspect, the SlgA2 disclosed herein comprises at least one IgA molecule comprising: a) variable antibody domains comprising antibody framework sequences that are at least any one of 90%, 95%, or 100% identical to respective mammalian sequences, preferably of human, non-human primate, dog, horse, or cat, or of humanized, caninized, equinized, or felinized antibodies; and/or b) constant antibody domains comprising constant antibody domain sequences that are any one of 90%, 95%, or 100% identical to respective mammalian sequences, specifically Calpha sequences, preferably of human, non-human primate, dog, horse, or cat, or of humanized, caninized, equinized, or felinized antibodies; and/or c) a hinge of 6-12 amino acids length, in particular at least 6, 7 or 8, up to 12, 11 , 10 or 9 amino acids length, originating from a native lgA2 molecule which comprises no more than 0, 1 , 2, or 3 point mutations in such hinge of a native lgA2 molecule.

Specifically, the hinge is linking the Fab arms and the Fc part of an IgA monomer. The term “IgA molecule” is specifically understood to refer to an “IgA monomer”.

Certain hinge sequences may be originating from native lgA2, in particular lgA2m(1) or lgA2m(2), of human (e.g., SEQ ID NO:51, SEQ ID NO:83, SEQ ID NO:85) or non-human primate or ape origin, such as gorilla (SEQ ID NO:86), chimp (SEQ ID NO:85), gibbon (SEQ ID NO:87, or VLPPTPPHP (SEQ ID NO:88)) macaque (SETKPCL (SEQ ID NO:89)).

Certain hinge sequences may originate from non-primate native IgA sequences, such as canine, feline, porcine, bovine, ovine, murine, rat, opossum, possum, rabbit or equine (SEQ ID NO 52-82). Accord ing to a specific embodiment, the hinge comprises at least three, four or five proline residues.

Specific hinges may comprise or consist of an amino acid sequence identified as SEQ ID NO:84: XPPXXP

Wherein

X at position 1 is V or A;

X at position 4 is P or S;

X at position 5 is P or H.

Specific examples are selected from the group consisting of:

SEQ ID NO:85: VPPPPP; originating from human 2m(1), human 2m(2), or chimp;

SEQ ID NO:86: VPPSPP; originating from gorilla

SEQ ID NO:87: APPPHP; originating from gibbon

Specifically, the hinge comprises or consists of an amino acid sequence that has at least any one of 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to any one of SEQ ID NO:51-83 and SEQ ID NO:85-89. In particular, in a hinge with a length of at least 6 amino acids, such sequence identity allows 0, 1 or 2 point mutations.

According to a specific aspect, the SlgA2 comprises at least one IgA molecule that is modified to comprise at least one disulfide bridge linking an antibody heavy chain (HC) to an antibody light chain (LC), preferably wherein said modification is a point mutation. A specific mutation can be the substitution of the most C-terminal proline of the CH1 domain (Calphal) of IgA by an arginine in order to allow effective heavy and light chain disulfide linkage, such as a R101 P point mutation in the CH1 domain of the human alpha 2m(2) allotype.

According to a specific aspect, glycosylation sites may be at least partially of fully removed or changed. A specific mutation in the CH2 domain (Calpha2) of human lgA2 is the replacement of the amino acid sequence NIT by the sequence TLS in order to remove part of the lgA2 glycosylation and to stabilize the structure. Such mutations have been described e.g., in Cancer Res. 2016; volume 76, pages 403-417.

According to a specific aspect, the SlgA2 described herein comprises one, two or more full length lgA2 antibodies bound to or otherwise complexed with a SC, which are preferably stabilized by the SC. The preparation described herein may further comprise free, unbound SC besides the SC being complexed with immunoglobulin, such as to provide SlgA2 in a Slg preparation that optionally further comprises free SC to enhance its functional properties.

Specifically, the SlgA2 comprises an SC molecule attached to an immunoglobulin oligomer, such as an IgA-dimer, thereby providing SlgA, respectively, in order to provide a proteolytically or otherwise stable antibody preparation.

According to a specific aspect, the SlgA2 comprises a secretory component (SC) which comprises at least any one of 80%, 85%, 90%, 95%, or 100% sequence identity to a human, non-human primate, canine, equine, or feline SC, preferably at least any one of 80%, 85%, 90%, 95%, or 100% sequence identity to any one of SEQ ID NO: 90-93.

Specifically, the SlgA2 described herein comprises a combination of immunoglobulins and SC, wherein both may originate from the same or different species, or comprise sequences of the same or different species origin, or are characterized by a certain degree of sequence identity to such species.

Specifically, the SC can be produced by a recombinant host cell culture using a host cell line engineered to express the SC coding sequence.

Recombinant SC can be produced by engineering a recombinant host cell to express the complete gene of the transmembrane protein plgR, e.g. human plgR, and the expression product can be subsequently cleaved to release the extracellular part of the SC into the culture supernatant. An alternative method employs the nucleotide sequence encoding the extracellular domains of the SC to express the extracellular SC. The exemplary SC sequences provided herein identify the full-length extracellular SC, also referred to as soluble SC. Yet, the SC may be provided as a fragment of the full-length molecule e.g. comprising or consisting of at least any one of 60%, 70%, 80%, 85%, 90%, or 95% of the full-length extracellular SC molecule, which fragment particularly comprises at least the D1 domain, which is functional to bind to polymeric immunoglobulin thereby providing the Slg.

Suitable host cell lines and expression systems are well-known in the art. Host cell lines may be selected from the group consisting of mammalian cell lines, in particular human, or primate, or CHO cell lines, avian cell lines, bacteria, plant, yeast, insect, fungal, moss and archaea. Specifically, the SC comprises a Lewis-type N-glycosylation pattern and at least any one of 2, 3, 4, 5, 6, 7, 8, 9, or 10 mol non-core fucose per mol SC. In some embodiments, the SC comprises at least at least any one of 2, 3, 4, 5, 6, 7, 8, 9, or 10 mol sialyl Lewis x per mol SC. Such Lewis-type N-glycosylation pattern is specifically preferred when producing SC designed for treating human subjects or human tissue.

Specifically, the amount of Lewis epitopes amounts to at least 10% of the theoretical value, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% up to the theoretical value as calculated from the number of N-glycosylation sites on the SC.

Specifically, the SC comprises at least any one of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mol galactose per mol SC.

Specifically, the SC comprises at least any one of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mol sialic acid per mol SC.

A specifically preferred SC preparation comprises at least any one of 2, 3, 4, 5, or 6 mol sialyl-Lewis x epitopes per mol SC. Alternatively, the SC is non-sialylated, preferably comprising less than 0.1 mol sialic acid per mol SC. Preferably the SC contains either only asialylated glycans or fully sialylated glycans. Specifically, the SC is provided in the Slg preparation either as sialylated or asialylated protein.

According to a specific aspect, co-expressing the SC and autologous or heterologous N-glucosyltransferases and especially galactosyltransferases, sialyltransferases or fucosyltransferases may be desired, in particular when producing human SC, or an artificial SC comprising at least 80% sequence identity to human SC. A recombinant production host cell line can be used that co-expresses autologous or heterologous functional galactosyltransferases (such as e.g. alpha-1 , 4- galactosyltransferase) and/or sialyltransferases (such as e.g. alpha-2, 3- sialyltransferase) and/or fucosyltransferases (such as an alpha-1 , x- fucosyltransferase, wherein x is 2, 3 or 4) or a combination thereof. Thereby a recombinant SlgA can be produced with the desired N-glycosylation. A fucosylated SlgA can also be produced by enzymatic or chemical galactosylation, sialylation and/or fucosylation of the SC or the SlgA to obtain the desired glycosylation pattern.

According to a specific aspect, the SlgA2 described herein comprises a J-chain to assemble polymeric immunoglobulins. Specifically, a native J-chain (of the same species origin than the immunoglobulin), or a J-chain of any other species, or an artificial J-chain can be used.

Specifically, a J-chain comprising at least any one of 80%, 85%, 90%, 95%, 95%, or 100% sequence identity to any one of SEQ ID NO:94-97.

The recombinant immunoglobulin can be co-expressed together with the J- chain in the same recombinant host cell culture, thereby producing the joined polymeric immunoglobulins, e.g. as secreted molecule.

According to a specific embodiment, recombinant SC can be produced in a separate host cell culture, and then combined with the (polymeric) immunoglobulin(s) to produce the Slg. The Slg specifically comprises the SC being complexed to polymeric Ig, in particular at a ratio of 1 :1.

In a specific aspect, the SlgA2 preparation is provided for treating a subject in need thereof. Specifically, the treatment can be a surface treatment directed to an organ or other body surface treatment. The term “body” shall herein refer to a body of a human or non-human subject, or an organ thereof. The “surface” of the body is herein understood to be an outer surface which may get into contact with an antigen, such surfaces being present within an animal or on the outer side of the animal, e.g., potentially getting into contact with e.g. environmental antigens or microbiota antigens.

According to a specific aspect, the invention provides for a method for the surface treatment of a body using the preparation described herein e.g., by topically applying the preparation the surface of the body or biological membranes, in particular mucous or epithelial membranes, thereby binding the target toxin on contaminated surfaces and excluding such bound toxin from the surface or underlying tissue.

According to specific examples, the surface is treated with the preparation described herein, wherein an effective amount of SlgA2 is used to exclude the target toxin from the tissue by its binding (immunoreaction), or at least from its outer tissue surface. Specifically, the preparation is forming a barrier coating the surface which barrier is active to sustain contact with the surface for a duration of at least about any one of 5, 10, 15, 20, 25, or 30 minutes, or even longer.

Specifically, the SlgA2 described herein is provided in a formulation for topical use, preferably for intraoral (e.g., oral cavity, including peroral, buccal or sublingual use), ocular, otic, dermal, cutaneous, vaginal, intragastric, rectal use, or for application to the upper and lower respiratory tract (e.g., pulmonary, intranasal, bronchial, or respiratory use).

Specifically, the formulation is a topical preparation that is provided as a syrup, lozenge, tablet, chewing gum, spray, powder, instant powder, granules, capsules, pastes, cream, gel, drops, or food product, for example, including specific excipients or auxiliary means for providing the respective formulation. Such formulations can be produced by standard methods.

According to a specific aspect, there is provided a formulation in the form of a liquid, emulsion or suspension, slurry or in the dried form, preferably spray-dried or freeze-dried. The preferred preparation is in a ready-to-use, storage stable form, with a shelf life of at least one or two years. Preferred formulations are manufactured as a powder or granulate which can be formulated into a liquid instantly before use.

The preparation described herein is suitably provided for oral or mucosal use, including oral, nasal, vaginal, rectal use, e.g. to inhibit a pathogenic reaction, disease onset or disease progression. For certain medical indications the preparation may be provided in a form suitable for topical application, such as in a cream, spray or droplets.

Tablets preferably contain auxiliary additives such as fillers, binders, disintegrants, lubricants, flavors or the like). Granules may be produced using isomaltose. It is furthermore preferred to provide for a preparation formulated to act at the site of the mucosa, e.g. at mucosal sites (nose, mouth, eyes, esophagus, throat, lung, ears, gastric tract, vagina, penis, intestine, rectum and colon), e.g. locally without systemic action.

Specifically, the formulation may be provided as a dietary supplement, nutritional management food, food additive or medical food. The formulation specifically may be provided in the form of a dairy product, a protein bar, a nutritional bar, tablet, capsule, chewing gum, paste, powder, granules, suppository and syrup where the SlgA2 is provided as a supplement, or else as a synthetic formulation comprising the purified SlgA2 at a certain concentration.

The SlgA2 preparation described herein is preferably concentrated or enriched in SlgA2 or comprises the SlgA2 in the concentrated form, e.g. at least 18 mg SlgA2 per gram.

The SlgA2 preparation described herein may specifically be used in the manufacture of a medicament for therapeutic or prophylactic treatment of a disease condition and symptoms caused by C. difficile infection, such as Clostridium difficile associated disease (CDAD), abdominal cramping and tenderness, watery diarrhea 10 to 15 times a day, severe abdominal pain, rapid heart rate, fever, blood or pus in the stool, nausea, dehydration, loss of appetite, weight loss, swollen abdomen, kidney failure, or increased white blood cell count, in particular a disease onset or disease progression, or a relapse of disease.

In specific embodiments, a subject is treated which is a diseased subject or patient suffering from C. difficile caused disease upon getting in contact with the pathogen. The disease or symptoms thereof can be e.g., C. difficile- mediated colitis, antibiotic-associated colitis, toxic megacolon, or pseudomembranous colitis (PMC), diarrhea or a relapse of C. difficile- mediated disease.

According to a specific aspect, the invention provides for a pharmaceutical preparation comprising the preparation described herein and a pharmaceutically acceptable carrier.

Specifically, the pharmaceutical preparation is provided for medical use, in particular, for use in the prophylactic or therapeutic treatment of a disease condition caused by C. difficile, such as antibiotic-associated diarrhea.

According to a specific aspect, a subject is treated with a pharmaceutical preparation, e.g. to provide a single-dose of 10 mg to 10 g SlgA2 e.g., a preparation wherein the SlgA2 is contained in an amount of 10 mg to 10 g per administration unit, e.g., as the predominant immunoglobulin.

Doses are typically provided for single or for intermittent use at a specific time interval, e.g. for daily use or in longer intervals (at least every 2, 3, 4, 5 or 6 days, or at least weekly), such as in a retard or slow release formulation. A daily dose of 1 mg to 10 g SlgA2 is preferably provided in a formulation for use in humans. A specifically preferred preparation, e.g. for oral use, may contain about 500 mg SlgA2 e.g., ranging from 400-600 mg SlgA2. Such doses may be administered intermittently, e.g., every day, or every week, every two weeks, every three weeks, or every four weeks (e.g., such that the subject receives from about two to about twenty, or e.g., about six doses of the immunoglobulin). An initial higher loading dose, followed by one or more lower doses may be administered. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays. Doses may be applied in combination with antibiotics, e.g. upon the subject’s risk of pathogenic overgrowth of C. difficile, so to prevent a pathogen associated reaction.

Doses may be applied in acute phases, e.g. upon the subject’s contact with the C. difficile pathogen, so to prevent a pathogen associated reaction.

According to a specific aspect, the preparation comprises said SlgA2 as sole active component.

According to another specific aspect, the preparation comprises said SlgA2 as active component and optionally further comprises at least one further active component or supplement.

Specifically, a) said further active component is selected from the group consisting of immunoglobulins, such as another SlgA being different from said SlgA2, polyclonal immunoglobulin preparations, immunoglobulin fragments, alternative scaffold proteins such as designed ankyrin repeats, anticalins, nanobodies, antibodies derived from plasma or milk and colostrum, hyperimmune bovine colostrum, intravenous immunoglobuline (IVIG) and single domain antibodies; or active components selected from the group consisting of non-steroid anti-inflammation agents, steroids, antiphlogistiga, endolysins, bacteriocides, antibiotics (such as any one of the antibiotics referred to herein for antibiotic treatment), cholestyramine, tolevamer, calcium aluminosilicate anti-diarrheal, inositol hexakisphosphate analogs, vaccines, phage-based therapeutics, bile acid therapy, intestinal antibiotic inactivators, or enteroprotective agents; or b) said supplement is selected from the group consisting of probiotics, prebiotics, antibiotics, vitamins, non-toxigenic C. difficile strains, fecal microbiome transplant (FMT), Veillonella dispar cultures, and spore-forming commensal microorganisms.

Any of the preparations or methods described herein may be used in combination with an additional therapeutic agent. In some embodiments, the additional therapeutic agent is selected from the group consisting of non-steroid anti inflammation agents, steroids, antiphlogistiga, endolysins, bacteriocides, antibiotics, cholestyramine, tolevamer, calcium aluminosilicate anti-diarrheal, inositol hexakisphosphate analogs, vaccines, phage-based therapeutics, bile acid therapy, intestinal antibiotic inactivators, enteroprotective agents.

Specifically, treatment can be combined with an antibiotic treatment, preferably wherein the pharmaceutical preparation is administered before, during or after said antibiotic treatment. Specifically, the SlgA preparation is combined with an antibiotic such as a beta lactam antibiotic, an aminoglycoside antibiotic, an ansamycin, a carbacephem, a carbapenem, a cephalosporin, a glycopeptide, a lincosamide, a lipopeptide, a macrolide, a monobactam, a nitrofuran, an oxazolidinone, a polypeptide, a sulfonamide, Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid, Pyrazinamide, Rifampicin, Rifabutin, Rifapentine, Streptomycin, Arsphenamine, Chloramphenicol, Fosfomycin, Mupirocin, Platensimycin, Quinupristin/Dalfopristin, Thiamphenicol, Tigecycline, Tinidazole, Trimethoprim, Teixobactin, Malacidins, Halicin, clindamycin, vancomycin, metronidazole, fusidic acid, thiopeptides, fidaxomicin, quinolons, tetracyclins, omadacycline, rifamycin, kibdelomycin, oxazolidinone, ketolides, thiazolides, amixicile, teicoplanin, ramoplanin, oritavancin, lantibiotics, capuramycin, surotomycin, thuricin, endolysin, avidocin CD, cadazolid, ramizol, defensins, ridinilazole, medium- chain fatty acids, phages, berberine, lactoferrin.

In particular, a combination therapy is provided which includes treatment with the preparation described herein and standard therapy of a C. difficile caused disease.

Further described herein are isolated nucleic acid molecules encoding the SlgA2 described herein, in particular, the IgA antibody, SC and joining elements. Such nucleic acid sequences or cDNAs can be derived from the specific amino acid sequences provided herein.

Further described herein are expression cassettes or vectors comprising a coding sequence to express the respective polypeptides or proteins.

Further described herein are host cells comprising the expression cassettes or vectors.

The invention further provides for a method of producing the SlgA2 preparation, in particular in a formulation for topical use, wherein the host cells are cultivated or maintained under conditions to produce said SlgA2 or its components, the immunoglobulins and SC, and optionally joining elements, and complexing said components thereby obtaining the SlgA2, and formulating said SlgA2 to produce the SlgA2 preparation.

FIGURES

Figure 1 : Sequences provided herein.

DETAILED DESCRIPTION

Unless indicated or defined otherwise, all terms used herein have their usual meaning in the art, which will be clear to the skilled person. Reference is for example made to the standard handbooks, such as Sambrook et al, "Molecular Cloning: A Laboratory Manual" (2nd Ed.), Vols. 1 -3, Cold Spring Harbor Laboratory Press (1989); Lewin, "Genes IV", Oxford University Press, New York, (1990), and Janeway et al., "Immunobiology" (5th Ed., or more recent editions), Garland Science, New York, 2001.

The subject matter of the claims specifically refers to artificial products or methods employing or producing such artificial products, which may be variants of native (wild-type) products. Though there can be a certain degree of sequence identity to the native structure, it is well understood that the materials, methods and uses of the invention, e.g., specifically referring to isolated nucleic acid sequences, amino acid sequences, expression constructs, transformed host cells and recombinant proteins, are “man-made” or synthetic, and are therefore not considered as a result of “laws of nature”.

The terms “comprise”, “contain”, “have” and “include” as used herein can be used synonymously and shall be understood as an open definition, allowing further members or parts or elements. “Consisting” is considered as a closest definition without further elements of the consisting definition feature. Thus “comprising” is broader and contains the “consisting” definition.

The term “about” as used herein refers to the same value or a value differing by +/- 10% or +/-5% of the given value.

The term “antibody” (the terms “antibody” and “immunoglobulin” being interchangeably used) herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

The term “antibody” as used herein shall refer to proteins that consist of or comprise constant and variable antibody domains of the heavy and/or light chains of immunoglobulins, with or without a linker sequence. Antibody domains may be of native structure or modified by mutagenesis or derivatization e.g., to modify the antigen binding properties or any other property, such as stability or functional properties, such as binding to the Fc receptors Fcalpha receptor, polyimmunoglobulin receptor, DC-SIGN, FcpR, FcRn and/or Fc gamma receptor.

The antibody as used herein has a specific binding site to bind one or more antigens or one or more epitopes of such antigens, specifically comprising one or more CDR-binding sites of a pair of variable antibody domains, namely a VL/VH domain pair, and constant antibody domains.

The term “full length antibody” as used herein refers to an antibody having a structure substantially similar to a native antibody structure, or having heavy chains that contain an Fc region, or at least most of the Fc domain. This phrase is used herein to emphasize that a particular antibody molecule is not an antibody fragment.

The term “oligomeric antibody” also referred to as “polymeric antibody” as used herein refers to a molecule or molecule complex comprising at least two antibody monomers, wherein the monomers can be identical or different. A polymeric immunoglobulin comprises the association of at least 2, 3, 4, 5 or even a higher number up to 10 immunoglobulin molecules, for example, a dimeric (e.g., IgA dimer), trimeric, or tetrameric immunoglobulin, or even higher polymers or aggregates. Polymeric immunoglobulins may comprise the immunoglobulin molecules associated with each other by covalent bonding, or other interactions, like electrostatic, hydrophobic, ionic interactions or affinity binding with or without J-chain.

The term “antibody” shall apply to antibodies of animal origin, including human species, such as mammalian, including human, murine, rabbit, goat, lama, cow and horse, or avian, such as hen, which term shall particularly include recombinant antibodies which are based on a sequence of animal origin e.g., human sequences.

The term “antibody” further applies to chimeric antibodies with sequences of origin of different species, such as sequences of murine and human origin. The term “chimeric” as used with respect to an antibody refers to those antibodies wherein one portion of each of the amino acid sequences of heavy and light chains is homologous to corresponding sequences in antibodies derived from a particular species, while the remaining segment of the chain is homologous to corresponding sequences in another species or class. Typically, the variable region of both light and heavy chains mimics the variable regions of antibodies derived from one species of mammals, while the constant portions are homologous to sequences of antibodies derived from another. For example, the variable region can be derived from presently known sources using readily available B-cells or hybridomas from non human host organisms in combination with constant regions derived from, for example, human cell preparations.

The term “antibody” may further apply to humanized antibodies.

The term “humanized” as used with respect to an antibody refers to a molecule having an antigen binding site that is substantially derived from an immunoglobulin from a non-human species, wherein the remaining immunoglobulin structure of the molecule is based upon the structure and/or sequence of a human immunoglobulin. The antigen binding site may either comprise complete variable domains fused onto constant domains or only the complementarity determining regions (CDR) grafted onto appropriate framework regions in the variable domains. Antigen-binding sites may be wild-type or modified e.g., by one or more amino acid substitutions, preferably modified to resemble human immunoglobulins more closely. Some forms of humanized anti bodies preserve all CDR sequences (for example a humanized mouse antibody which contains all six CDRs from the mouse antibody). Other forms have one or more CDRs which are altered with respect to the original antibody.

The term “caninized”, “equinized”, or “felinized” is understood in the same way as “humanized” yet employing the immunoglobulin structure based upon the structure and/or sequence of a canine, equine, and feline immunoglobulin, respectively, thereby producing species-adapted sequences.

The term “antibody” further applies to human antibodies.

The term “human” as used with respect to an antibody, is understood to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibody of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs. Human antibodies include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin genes or derived from human B cells by immunoglobulin gene cloning and recombinant antibody expression or from immortalized human B cell lines.

The term “fully human antibody” as used herein refers to a human antibody, which is composed of only human parts, in particular human CDR, human FR, and human constant regions, each originating from a human source, e.g. cells expressing human antibody sequences, libraries displaying human antibody sequences, or genes encoding human antibody sequences. Fully human antibodies may be naturally- occurring antibodies or artificial antibodies, which are understood as being composed of parts, each obtained from a different origin, thus, not occurring in nature. Exemplary artificial fully human antibodies are human switch variants of human antibodies, wherein at least one constant region is obtained from a human antibody of a different isotype.

The term “antibody” as used herein specifically applies to antibodies of any isotype or subclass. Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be assigned to the major classes of antibodies IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses, e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 , and lgA2.

The term further applies to monoclonal or polyclonal antibodies, specifically a recombinant antibody, which term includes all antibodies and antibody structures that are prepared, expressed, created or isolated by recombinant means, such as anti bodies originating from animals e.g., mammalians including human, that comprises genes or sequences from different origin e.g., murine, chimeric, humanized antibodies, or hybridoma derived antibodies. Further examples refer to antibodies isolated from a host cell transformed to express the antibody, or antibodies isolated from a recombinant, combinatorial library of antibodies or antibody domains, or antibodies prepared, expressed, created or isolated by any other means that involve splicing or fusing antibody gene sequences to other DNA sequences.

It is understood that the term “antibody” also refers to derivatives of an antibody, in particular functionally active derivatives. An antibody derivative is understood as any combination of one or more antibody domains or antibodies and / or a fusion protein, in which any domain of the antibody may be fused at any position of one or more other proteins, such as other antibodies e.g., a binding structure comprising CDR loops, a receptor polypeptide, but also ligands, scaffold proteins, enzymes, toxins and the like. A derivative of the antibody may be obtained by association or binding to other substances by various chemical techniques such as covalent coupling, electrostatic interaction, di-sulfide bonding etc. The other substances bound to the antibody may be lipids, carbohydrates, nucleic acids, organic and inorganic molecules or any combination thereof (e.g., PEG, prodrugs or drugs). In a specific embodiment, the antibody is a derivative comprising an additional tag allowing specific interaction with a biologically acceptable compound. There is not a specific limitation with respect to the tag usable in the present invention, as far as it has no or tolerable negative impact on the binding of the antibody to its target. Examples of suitable tags include His-tag, Myc-tag, FLAG-tag, Strep-tag, Calmodulin-tag, GST-tag, MBP-tag, and S-tag. In another specific embodiment, the antibody is a derivative comprising a label. The term “label” as used herein refers to a detectable compound or composition which is conjugated directly or indirectly to the antibody so as to generate a “labeled” antibody. The label may be detectable by itself e.g., radioisotope labels or fluorescent labels, or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.

Antibodies derived from a parent antibody or antibody sequence, such as a parent CDR or FR sequence, are herein particularly understood as mutants or variants obtained by e.g., in silico or recombinant engineering or else by chemical derivatization or synthesis.

It is understood that the term “antibody” also refers to functionally active variants of a parent antibody, including antibodies with a functionally active CDR-binding site. Antibody variants may comprise a modified sequence e.g., in a constant domain to engineer the antibody stability, effector function or half-life, or in a variable domain to improve antigen-binding properties e.g., by affinity maturation techniques available in the art.

The term “variant” shall particularly refer to polypeptides or proteins obtained by mutagenesis methods, in particular to delete, exchange, introduce inserts into a specific amino acid sequence or region or chemically derivatize an amino acid sequence. Any of the known mutagenesis methods may be employed, including point mutations at desired positions e.g., obtained by randomization techniques. In some cases, positions are chosen randomly e.g., with either any of the possible amino acids or a selection of preferred amino acids to randomize the antibody sequences. The term “mutagenesis” refers to any art recognized technique for altering a polynucleotide or polypeptide sequence. Preferred types of mutagenesis include error prone PCR mutagenesis, saturation mutagenesis, or other site directed mutagenesis.

A point mutation is particularly understood as the engineering of a polynucleotide that results in the expression of an amino acid sequence that differs from the non-engineered amino acid sequence in the substitution or exchange, deletion or insertion of one or more single (non-consecutive) or doublets of amino acids for different amino acids.

Conservative substitutions are those that take place within a family of amino acids that are related in their side chains and chemical properties. Examples of such families are amino acids with basic side chains, with acidic side chains, with non-polar aliphatic side chains, with non-polar aromatic side chains, with uncharged polar side chains, with small side chains, with large side chains, etc.

Preferred point mutations refer to the exchange of amino acids of the same polarity and/or charge. In this regard, amino acids refer to twenty naturally-occurring amino acids encoded by sixty-four triplet codons. These 20 amino acids can be split into those that have neutral charges, positive charges, and negative charges

An “affinity-matured” antibody is one with one or more alterations in one or more CDRs and/or framework regions which result in optimization or an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s).

Affinity-matured antibodies are produced by procedures known in the art. For example, methods of preparing and/or using affinity maturation libraries may be employed in order to generate affinity matured antibodies. Exemplary such affinity maturation methods and uses, such as random mutagenesis, bacterial mutator strains passaging, site-directed mutagenesis, mutational hotspots targeting, parsimonious mutagenesis, antibody shuffling, light chain shuffling, heavy chain shuffling.

Affinity matured antibodies may exhibit a several log-fold greater affinity than a parent antibody. A preferred affinity matured variant of an antibody may exhibit at least a 2-fold increase in affinity of binding, preferably at least a 5, preferably at least 10, preferably at least 50, or preferably at least 100-fold increase.

Single parent antibodies may be subject to affinity maturation. Alternatively, pools of antibodies with similar binding affinity to the target antigen may be considered as parent structures that are varied to obtain affinity matured single antibodies or affinity matured pools of such antibodies. The affinity maturation may be employed in the course of the selection campaigns employing respective libraries of parent molecules. Preferred antibodies have a binding affinity with a KD of less than 10E-6 M. Preferred affinity-matured antibodies will have an affinity with a KD of less than any one of 10E-7, 10E-8 or 10E-9 M.

Affinity matured antibodies are specifically characterized by binding to the same epitope (or substantially the same epitope) as the parent antibody.

An “antibody that binds to the same epitope” as a reference (parent) antibody refers to an antibody that contacts an overlapping set of amino acid residues of the antigen as compared to the reference antibody or blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more in some embodiments, the set of amino acid residues contacted by the antibody may be completely overlapping or partially overlapping with the set of amino acid residues contacted by the reference antibody.

Use of the term “having the same specificity”, “having the same binding site” or “binding the same epitope” indicates that antibodies exhibit the same or essentially the same, i.e. similar immunoreaction (binding) characteristics and compete for binding to a pre-selected target binding sequence. The relative specificity of an antibody molecule for a particular target can be relatively determined by competition assays e.g., as described in Harlow, et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988).

The term “compete”, as used herein with regard to an antibody, means that a first antibody binds to an epitope in a manner sufficiently similar to the binding of a second antibody, such that the result of binding of the first antibody with its cognate epitope is detectably decreased in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. The alternative, where the binding of the second antibody to its epitope is also detectably decreased in the presence of the first antibody, can, but need not be the case. That is, a first antibody can inhibit the binding of a second antibody to its epitope without that second antibody inhibiting the binding of the first antibody to its respective epitope. However, where each antibody detectably inhibits the binding of the other antibody with its cognate epitope, whether to the same, greater, or lesser extent, the antibodies are said to “compete” with each other for binding of their respective epitope(s). Antibodies that compete with any of the exemplified antibodies for binding the target antigen are particularly encompassed by the present invention.

“Competitively binding” or “competition” herein means a greater relative inhibition than about 30%, e.g., as determined by competition ELISA analysis or by ForteBio or BLI analysis. It may be desirable to set a higher threshold of relative inhibition as criteria of what is a suitable level of competition in a particular context e.g., where the competition analysis is used to select or screen for new antibodies designed with the intended function of the binding of the antigen. Thus, for example, it is possible to set criteria for the competitive binding, wherein at least 40% relative inhibition is detected, or at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or even at least 100%, before an antibody is considered sufficiently competitive.

An “neutralizing” antibody is one which neutralizes i.e. inhibits or reduces the biological activity of the antigen it binds. Certain lgA2 molecules or SlgA2 as described herein are neutralizing a target toxin and substantially or completely inhibit its toxic effect, as determined in a suitable toxin neutralization assay.

In some instances, the activity may be ... In some embodiments, an antibody can inhibit a biological activity, such as a toxic effect, of the antigen it binds, by at least about 1 %, about 5%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%.

The term “antigen-binding site” or “binding site” refers to the part of an antibody that participates in the target antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable (“V”) regions of the heavy (“H”) and/or light (“L”) chains, or the variable domains thereof. Three highly divergent stretches within the V regions of the heavy and light chains, referred to as “hypervariable regions”, are interposed between more conserved flanking stretches known as framework regions, The antigen-binding site provides for a surface that is complementary to the three-dimensional surface of a bound epitope or antigen, and the hypervariable regions are referred to as “complementarity-determining regions”, or “CDRs.” The antigen-binding site incorporated in the CDRs and herein also called “CDR binding site”.

The term “antigen” as used herein interchangeably with the term “target” or “target antigen” shall refer to a whole target molecule or a fragment of such molecule recognized by an antibody binding site. Specifically, substructures of an antigen e.g., a polypeptide or carbohydrate structure, generally referred to as “epitopes” e.g., B-cell epitopes or T-cell epitope, which are immunologically relevant, may be recognized by such binding site.

The term “toxin” is herein understood to refer to antigens to which an animal cell, tissue or a human or non-human animal respond with cell necrosis, tissue disintegration and disease symptoms, respectively.

Herein, the terms “toxin A” and “toxin B” shall refer to the respective toxins of Clostridium difficile, in particular exotoxins.

The term "toxin A" particularly refers to the toxin A protein encoded by C. difficile. The amino acid sequence of C. difficile toxin A (TcdA) is herein identified as SEQ ID NO:2.

The term "toxin B" particularly refers to the toxin B protein encoded by C. difficile.

The amino acid sequence of C. difficile toxin B (TcdB) is herein identified as SEQ ID NO:1.

The term “epitope” as used herein shall in particular refer to a molecular structure which may completely make up a specific binding partner or be part of a specific binding partner to a binding site of an antibody. An epitope may either be composed of a carbohydrate, a peptidic structure, a fatty acid, an organic, biochemical or inorganic substance or derivatives thereof and any combinations thereof. If an epitope is comprised in a peptidic structure, such as a peptide, a polypeptide or a protein, it will usually include at least 3 amino acids, preferably 5 to 40 amino acids, and more preferably between about 10-20 amino acids. Epitopes can be either linear or conformational epitopes. A linear epitope is comprised of a single segment of a primary sequence of a polypeptide or carbohydrate chain. Linear epitopes can be contiguous or overlapping. Conformational epitopes are comprised of amino acids or carbohydrates brought together by folding the polypeptide to form a tertiary structure and the amino acids are not necessarily adjacent to one another in the linear sequence. Specifically, and with regard to polypeptide antigens a conformational or discontinuous epitope is characterized by the presence of two or more discrete amino acid residues, separated in the primary sequence, but assembling to a consistent structure on the surface of the molecule when the polypeptide folds into the native protein/antigen.

The term “functionally active variants” of a parent molecule is herein understood in the following way. Functionally active variants may be obtained by changing the sequence as provided herein or the glycosylation pattern of a polypeptide or protein, and are characterized by having a biological activity about the same or similar to that displayed by the respective sequence or glycosylation pattern, from which the variant is derived. Typically, a series or a library of variants are produced and then screened to select functionally active variants with improved characteristics. Typically, the functional activity of variants is proven, if they exhibit substantially the same functional activity or substantially the same biological activity as the comparable (parent or non- modified) molecule.

The term “substantially the same” with regard to binding a target antigen or biological activity as used herein refers to the activity being at least 20%, at least 50%, at least 75%, at least 90% e.g., at least 100%, or at least 125%, or at least 150%, or at least 175%, or e.g., up to 200%, or even a higher activity of the comparable or parent binding molecule.

In specific cases, a functionally active variant can be used comprising one or more point mutations, such as insertion, deletion or substitution of one amino acid at a certain position, including e.g. conservative amino acid substitutions. Conservative substitutions are those that take place within a family of amino acids that are related in their side chains and chemical properties. Examples of such families are amino acids with basic side chains, with acidic side chains, with non-polar aliphatic side chains, with non-polar aromatic side chains, with uncharged polar side chains, with small side chains, with large side chains etc.

A variant of a polypeptide (or the respective coding nucleotide sequence) is considered functionally active in the context of the Slg preparation described herein, if the activity of the variant polypeptide when used in the Slg, amounts to at least any one of 50%, 60%, 70%, 80%, 90%, 95%, or 100% or higher when compared to the Slg comprising the unmodified polypeptide (without sequence alteration).

In some cases, a functionally active variant of an antibody is used, such as variants further described herein, in particular affinity-matured variants. Such variants are considered functionally active, if comprising a functionally active CDR-binding site with a specificity to bind the target molecule.

In some cases, a functionally active variant of an SC is used. Such variants are considered functionally active with respect of the Slg integrity, if capable of being bound (in particular covalently bound) to an oligomeric immunoglobulin thereby obtaining Slg, which can be determined by e.g., reducing and non-reducing gel- electrophoresis. SC (including functional variants thereof) is also functional to mediate binding to receptors which may influence biological activity of the molecule. The binding to the respective receptor can be measured e.g., by ELISA, immuno-cyto- fluorometry, or surface plasmon techniques. Functionally active variants are binding in a similar way as naturally-occurring SC in such assays.

Variants of a SC may comprise additional N-glycosylation sites, and/or comprise a different glycosylation pattern, such as Lewis-type glycosylation or other non-core fucosylation, as further described herein. This can be achieved by genetic engineering techniques as well as chemical and enzymatic means.

A functionally active Slg variant may be obtained by exchange of domains or components between Slg from different species, or the use of chimeric sequences e.g. J-chain from horse may be combined with Ig from dog and this molecule may be complexed with secretory component of human origin. Exemplary immunoglobulins comprise human or canine antibody sequences. Functionality of chimeric Slg can be proven, upon complexing such antibodies with an SC of another species, such as human antibody in complex with canine SC, and canine antibody in complex with human SC. All kinds of combinations of the various polypeptide chains that make up Slg can be employed. The various chains may be recombinant and/or non recombinant molecules, they may be chimeric (such as e.g. mouse/human heavy and light chains) or otherwise modified.

The Slg described herein is specifically provided as an isolated compound.

The term “isolated” or “isolation” as used herein shall refer to such compound that has been sufficiently separated from the environment with which it would naturally be associated, so as to exist in “purified” or “substantially pure” form. “Isolated” does not necessarily mean the exclusion of artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with the fundamental activity, and that may be present, for example, due to incomplete purification.

Isolated immunoglobulins and respective preparations described herein are particularly non-naturally occurring, e.g., as provided in a combination preparation with another antibody, which combination (in certain ratios and concentrations) does not occur in nature, or comprising a recombinant component, such as an artificial variant of a parent antibody or a parent SC, chimerics, or species adapted variants.

Specifically, an isolated Slg is considered free or substantially free of material with which they are naturally associated such as other compounds with which they are found in their natural environment, or the environment in which they are prepared (e.g. cell culture) when such preparation is by recombinant DNA technology practiced in vitro or in vivo. Isolated compounds can be formulated with diluents or adjuvants and still for practical purposes be isolated - for example, the polypeptides can be mixed with pharmaceutically acceptable carriers or excipients when used in diagnosis or therapy.

The term “isolated” as used herein is specifically meant to include recombinant polypeptides or proteins obtained from cell culture, such as produced by cultivating recombinant host cells that have been transformed with artificial nucleic acid constructs encoding the antibodies, or those chemically synthesized.

“Percent (%) amino acid sequence identity” with respect to the polypeptide sequences described herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific polypeptide sequence, after aligning the sequence and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

The term “recombinant” as used herein shall mean “being prepared by or the result of genetic engineering”. A recombinant host specifically comprises an expression vector or cloning vector, or it has been genetically engineered to contain a recombinant nucleic acid sequence, in particular employing nucleotide sequence foreign to the host. A recombinant protein is produced by expressing a respective recombinant nucleic acid in a host. The term “recombinant antibody”, as used herein, includes antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or trans-chromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library or library of antigen-binding sequences of an antibody, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant antibodies comprise antibodies engineered to include rearrangements and mutations which occur, for example, during antibody maturation. In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Maniatis, Fritsch & Sambrook, "Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, (1982).

The term “secretory immunoglobulin” or Slg as used herein means an immunoglobulin molecule to which secretory component (SC) is bound. The Ig molecule can be an IgA or IgM, and may comprise constant or variable domains or respective sequences of IgG, IgD or IgE. IgA can be lgA1 or lgA2. In certain aspects, components of Slg are described which may be an immunoglobulin (antibody), SC, and optionally one or more J-chains, which are interlinked or otherwise associated to form the Slg. Herein the SlgA2 described herein is also referred to as Slg or SlgA.

In secretory immunoglobulins (Slg) as described herein, oligomeric (such as dimeric) IgA is bound to or associated with SC, thereby obtaining IgA complexed with SC, such complex being also referred to as SlgA. SlgA comprising one, two or more molecules of the lgA2 subtype, is herein referred to as SlgA2.

SlgA can be determined in a sample of the Slg preparation described herein, or in a biological sample (e.g. to determine the recovery of the Slg upon administration or application), such as saliva, nasal mucus, gastric juice, faeces, bronchial lavage, by standard immunoassay techniques or by molecular biology techniques. Immunological detection of SlgA can be performed by ELISA, RIA, by fluorescence based immune assays, time-resolved fluorometry, precipitation assays, nepheliometric assays, surface plasmon resonance-based assays and similar setups with and without labels. SC bound to the immunoglobulin can be determined to discriminate SlgA from IgA.

The term “Secretory Component” or “SC” as used herein shall refer to a SC comprising or consisting of the native amino acid sequence as secreted by a mammary gland of an animal, including humans, or variants thereof, including functional variants, which SC can be in complex with an immunoglobulin, e.g. in the form of a secretory immune complex, e.g. mediated by the J-chain (or variants thereof) or other structures of immunoglobulins that bind specifically to the polyimmunoglobulin receptor (plgR). An artificial SC can be produced synthetically or by recombinant expression techniques.

In many cases, the SC sequence is provided as a sequence of the complete polyimmunoglobulin receptor (plgR), yet, for the purpose of the Slg described herein, only the extracellular part of these sequences are relevant, e.g., the sequence of exemplary mammalian SC as provided in SEQ ID NO:90-93.

In certain cases, functionally active variants of a naturally-occurring SC may be used. The functionally active variant may be obtained by sequence alterations in the amino acid or the nucleotide sequence, wherein the sequence alterations retain a function of the unaltered amino acid or the nucleotide sequence, when used in combination of the invention. Such sequence alterations can include, but are not limited to, (conservative) substitutions, additions, deletions, mutations and insertions.

A functionally active SC variant may also be obtained by exchange of domains between SC from different species, or by deletion or addition of domains. Changing of the natural order of the domains (e.g. 1-2-3-4-5 for mammalian SC) may also result in a functionally active variant (e.g. 1-4-3-2-5).

According to a specific aspect, the Slg is employed as immune complex with SC comprising a human-like N-glycosylation pattern. Specifically, the Slg is provided with high galactosylation and sialylation of the SC. The glycan pattern found on recombinant SC is dependent on the host species, the host organism, the tissue of origin and the physiological state of the genetically engineered cell. Suitable production systems would employ recombinant cell cultures which provide for the desired glycosylation pattern with peripheral or antennary, such as outer arm fucosylation, galactosylation or sialylation as determined by suitable analytical means, such as electrophoretic, chromatographic, mass spectroscopic, chemical and enzymatic techniques or combinations thereof.

"Specific” binding, recognizing or targeting as used herein, means that the binder e.g., antibody or antigen-binding portion thereof, such as variable domains comprising a CDR-binding site, exhibits appreciable affinity for the target antigen or a respective epitope in a heterogeneous population of molecules. Thus, under designated conditions (e.g., immunoassay), a binder specifically binds to the target antigen and does not bind in a significant amount to other molecules present in a sample. The specific binding means that binding is selective in terms of target identity, high, medium or low binding affinity or avidity, as selected. Selective binding is usually achieved if the binding constant or binding dynamics is at least 10-fold different (understood as at least 1 log difference), preferably the difference is at least 100-fold (understood as at least 2 logs difference), and more preferred a least 1000-fold (understood as at least 3 logs difference) as compared to another target.

The term “subject” as used herein shall refer to a warm-blooded mammalian, particularly a human being or a non-human animal, including e.g., dogs, cats, rabbits, horses, cattle, and pigs. In particular the treatment and medical use described herein applies to a subject in need of prophylaxis or therapy of a disease condition associated with C. difficile infection. Specifically, the treatment may be by interfering with the pathogenesis of a disease condition where a C. difficile pathogen or toxin is a causal agent of the condition. The subject may be a patient at risk of such disease condition or suffering from disease.

The term “patient” includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment. The term “patient” as used herein always includes healthy subjects. The term “treatment” is thus meant to include both prophylactic and therapeutic treatment.

Specifically, the term “prophylaxis” refers to preventive measures which is intended to encompass prevention of the onset of pathogenesis or prophylactic measures to reduce the risk of pathogenesis.

The term “therapy” as used herein with respect to treating subjects refers to medical management of a subject with the intent to cure, ameliorate, stabilize, reduce the incidence or prevent a disease, pathological condition, or disorder, which individually or together are understood as “disease condition”. The term includes active treatment, directed specifically toward the improvement of a disease condition, prophylaxis directed specifically toward the prevention of a disease condition, and also includes causal treatment directed toward removal of the cause of the associated disease condition. In addition, this term includes palliative treatment designed for the relief of symptoms rather than the curing of the disease condition, and further curing a disease condition directed to minimizing or partially or completely inhibiting the development of the associated disease condition, and supportive treatment employed to supplement another specific therapy directed toward the improvement of the associated disease condition.

The term “therapeutically effective amount”, used herein interchangeably with any of the term “effective amount” of a compound e.g., an Slg described herein, is a quantity or activity sufficient to, when administered to the subject effect beneficial or desired results, including clinical results, and, as such, an effective amount or synonym thereof depends upon the context in which it is being applied.

An effective amount is intended to mean that amount of a compound that is sufficient to treat, prevent or inhibit such diseases or disorder. In the context of disease, therapeutically effective amounts of the antibody as described herein are specifically used to treat, modulate, attenuate, reverse, or affect a disease or condition that benefits from binding or neutralizing C. difficile toxin A and/or B. The amount of the compound that will correspond to such an effective amount will vary depending on various factors, such as the given drug or compound, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject or host being treated, and the like, but can nevertheless be routinely determined by one skilled in the art.

A treatment or prevention regime of a subject with a therapeutically effective amount of the Slg described herein may consist of a single administration, or alternatively comprise a series of applications. For example, the antibody may be administered at least once a month, or at least once a week. However, in certain cases of an acute phase, the Slg may be administered to the subject from about one time per week to about a daily administration for a given treatment. The length of the treatment period depends on a variety of factors, such as the severity of the disease, either acute or chronic disease, the age of the patient, and the concentration of the Slg. It will also be appreciated that the effective dosage used for the treatment or prophylaxis may increase or decrease over the course of a particular treatment or prophylaxis regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art.

Specific pharmaceutical compositions described herein comprise the Slg and a pharmaceutically acceptable carrier or excipient. A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

Specific pharmaceutical compositions described herein can be administered by the topical route e.g. onto biological membranes, including e.g., mucosa or skin. Pharmaceutical carriers suitable for facilitating such means of administration are well known in the art.

Pharmaceutically acceptable carriers generally include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible with an antibody or related composition or combination preparation described herein. Further examples of pharmaceutically acceptable carriers include sterile water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations of any thereof.

In one such aspect, an antibody can be combined with one or more carriers appropriate a desired route of administration, Slg may be e.g., admixed with any of lactose, sucrose, starch, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine, polyvinyl alcohol, and optionally further tableted or encapsulated for conventional administration. Alternatively, an antibody may be dissolved in saline, water, polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidal solutions, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers. Other carriers, adjuvants, and modes of administration are well known in the pharmaceutical arts. A carrier may include a controlled release material or time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art. Additional pharmaceutically acceptable carriers are known in the art and described in, e.g., Remington: The Science and Practice of Pharmacy, 22^nd revised edition (Allen Jr, LV, ed., Pharmaceutical Press, 2012). Liquid formulations can be solutions, emulsions or suspensions and can include excipients such as suspending agents, solubilizers, surfactants, preservatives, and chelating agents.

Pharmaceutical compositions are contemplated wherein Slg (in particular SlgA2) as described herein and one or more further therapeutically active agents are formulated.

Stable formulations of the Slg (in particular SlgA2) as described herein are prepared for storage by mixing said antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers, e.g., in the form of lyophilized formulations or aqueous solutions.

Once Slg with the desired binding properties are identified, such Slg or one or more of its components can be produced by methods well-known in the art, including, for example, hybridoma techniques or recombinant DNA technology. Recombinant production methods typically employ cell cultures and fermentation techniques such as batch, fed batch or continuous or perfusion cell culture.

According to a specific aspect, there are provided isolated nucleic acid molecules comprising a sequence that codes for a recombinant polypeptide or protein as described herein, such as an antibody, SC, or J-chain. Such encoding nucleic acid molecule is, for example, in the form of DNA, RNA, or a hybrid thereof, and may include non-naturally-occurring bases, a modified backbone, e.g., a phosphorothioate backbone that promotes stability of the nucleic acid, or both. The nucleic acid advantageously may be incorporated in one or more expression cassettes, vectors or plasmids, comprising features that promote desired expression, replication, and/or selection in target host cell(s). Examples of such features include an origin of replication component, a selection gene component, a promoter component, an enhancer element component, a polyadenylation sequence component, a termination component, and the like, numerous suitable examples of which are known.

The present disclosure specifically refers to the recombinant DNA constructs comprising one or more of the nucleotide sequences described herein. These recombinant constructs are used in connection with a vector, such as a plasmid, phagemid, phage or viral vector, into which a DNA molecule encoding any disclosed antibody is inserted.

Recombinant polypeptides or proteins can, for example, be produced by identifying or isolating the DNA (e.g., cDNA) encoding the required amino acid sequences and transfecting a recombinant host cell with the coding sequences for expression, using well known recombinant expression vectors or expression cassette(s) comprising the coding nucleotide sequences. Recombinant host cells can be prokaryotic and eukaryotic cells, e.g., including animal or human cell lines in cell cultures.

According to a specific aspect, the nucleotide sequence may be used for genetic manipulation to obtain polypeptides or proteins containing artificial sequences, e.g., to improve the affinity, or other characteristics of an antibody. For example, the constant region may be engineered to more nearly resemble human constant regions to avoid immune response, if the antibody is used in clinical trials and treatments in humans. It may be desirable to genetically manipulate an antibody sequence to obtain greater affinity to the respective target and greater efficacy against the target. It will be apparent to one of skill in the art that one or more polynucleotide changes can be made to elements of Slg, such that the Slg still maintains its binding ability to the target toxins.

Isolation and purification methods used for obtaining an Slg described herein (or isolated compounds thereof) may utilize differences in solubility, such as salting out and solvent precipitation, differences in molecular weight, such as ultrafiltration and gel electrophoresis, differences in electric charge, such as ion-exchange chromatography, or may utilize specific affinities, such as affinity chromatography, or may utilize differences in hydrophobicity, such as reverse phase high performance liquid chromatography, or utilize differences in isoelectric point, such as isoelectric focussing. Specific purification steps that are preferably employed to separate any SC or immunoglobulin polypeptides alone or in complex with other compounds are ultrafiltration techniques with molecular cutoffs between 100 kDa and 500 kDa and precipitation techniques such as precipitation with salts such as ammonium sulfate or organic solvents.

The isolated and purified products can be identified and analysed by conventional methods such as Western blotting or assay of its activity, e.g. by its ability to bind to other components of the Slg complex, such as SC, IgA, dimeric IgA or the J-chain, or by detection with specific antisera.

The structure of the purified compound can be defined by amino acid analysis, amino-terminal analysis, primary structure analysis, glycoanalysis and the like. It is preferred that the products are obtainable in large amounts and in specific cases with a high purity, thus meeting the necessary requirements for being used as active ingredient in pharmaceutical compositions.

The Slg preparation described herein can be stabilized through the content of the SC, thereby providing an increased thermostability, pH stability and/or protease stability of the immunoglobulins, resulting in an increased recovery or prolonged half- life upon application or administration. The stabilising effect is particularly important for the mucosal recovery. Upon administration of the Slg, the immunoactivity of the Slg binding the target toxin may be determined by immunological techniques such as ELISA. An increased level of secretory immunoglobulins (free or bound to its target) indicates an increased recovery.

The Slg recovery is typically measured as mucosal Slg level as compared to a normal value of Slg level, a reference. The normal value of Slg level at a specific site may be the value of the average of a population or the value of the individual or a group of individuals before treatment. Such preparations of the invention are preferred which provide for a (maximal) increase in Slg, an Slg recovery of at least 50%, preferably at least 100%, more preferably 500%.

The foregoing description will be more fully understood with reference to the following examples. Such examples are, however, merely representative of methods of practicing one or more embodiments of the present invention and should not be read as limiting the scope of invention.

EXAMPLES

Example 1 : Production of a dimeric human IgA, specific for Clostridium difficile toxin A (TcdA), with a short, IgA hinge region

This example describes the construction of the proteins and the respective gene constructs and their cloning into expression vectors for mammalian cells as well as the expression and purification of the antibodies. The example describes the construction of a dimeric IgA based on the variable region of an anti-TcdA antibody, R1 .

1.1. Design of proteins and cloning of respective genes

1.1.1. R1-lgA HC, Human IgA heavy chain

The source of the antibody variable domain (VH) is a human antibody protein. (Sequence SEQ ID NO:146 from patent US9399674). Protein sequence of human IgA constant heavy chain (lgA1) with a shortened hinge sequence of lgA2 is used. The resulting mature heavy chain protein sequence is identified as SEQ ID NO:98. For the construction of the expression vector, a signal peptide (SEQ ID NO:99) is added to N- terminus of the SEQ ID NO:98. The protein sequence is reverse translated to a DNA sequence utilizing codons optimized for eukaryotic cells. In order to facilitate cloning into the mammalian expression vector pcDNA3.4 the DNA sequence is provided with a Kozak sequence at the 5-prime end, just before the start codon and a stop codon at the 3-prime end. The resulting gene is obtained by GeneArt (a service by ThermoFisher Scientific). This gene is cloned into the mammalian expression vector by AT-cloning (ThermoFisher, cat.no. A14697). All detailed steps for amplification, cloning, screening for orientation in the vector, plasmid purification from bacteria and quality control are described in the manual of the kit manufacturer. Plasmid purification for transfection is performed with PureLink Expi Endotoxin-Free Mega Plasmid Purification Kit (Thermo Fisher Scientific, cat.no. A31232). The resulting plasmid is sequence controlled and named pR1-hlgA-HC.

1.1.2. R1- LC, Human kappa light chain

The antibody variable domain (VL) sequence used is from R1 antibody protein (Sequence SEQ ID NO:154 from patent US9399674). Protein sequences of human constant kappa chains are obtained from UniProt or NCBI. The resulting mature light chain protein sequence is identified as SEQ ID NO:100. For the construction of the expression vector, a signal peptide (SEQ ID NO:101) is added to N-terminus of the SEQ ID NO:98. The protein sequence is reverse translated to a DNA sequence utilizing codons optimized for eukaryotic cells. In order to facilitate cloning into the vector pcDNA3.4 the DNA sequence is provided with a Kozak sequence at the 5-prime end, just before the start codon and a stop codon at the 3-prime end. The resulting gene is obtained by GeneArt (a service by ThermoFisher). This gene is cloned into the mammalian expression vector by AT-cloning (ThermoFisher, cat.no. A14697). All detailed steps for amplification, transformation, cloning, screening for orientation in the vector, plasmid purification from bacteria and quality control are described in the manual of the kit manufacturer. Plasmid purification for transfection is performed with PureLink Expi Endotoxin-Free Mega Plasmid Purification Kit (Thermo Fisher Scientific, cat.no. A31232). The resulting plasmid is pR1-k-LC.

1 .1 .3. Human J-chain

Protein sequences of human J-chain sequences are obtained from UniProt or NCBI. The resulting mature J-chain protein sequence is identified as SEQ ID NO:94. For the construction of the expression vector, a signal peptide (SEQ ID NO: 101) is added to N-terminus of the SEQ ID NO:94. The protein sequence is reverse translated to a DNA sequence utilizing codons optimized for eukaryotic cells. In order to facilitate cloning into the vector pcDNA3.4 the DNA sequence is provided with a Kozak sequence at the 5-prime end, just before the start codon and a stop codon at the 3- prime end. The resulting gene is obtained by GeneArt (a service by ThermoFisher). This gene is cloned into the mammalian expression vector by AT-cloning (ThermoFisher, cat.no. A14697). All detailed steps for amplification, transformation, cloning, screening for orientation in the vector, plasmid purification from bacteria and quality control are described in the manual of the kit manufacturer. Plasmid purification for transfection is performed with PureLink Expi Endotoxin-Free Mega Plasmid Purification Kit (Thermo Fisher Scientific, cat.no. A31232). The resulting plasmid phJC is sequence controlled.

1.1.4. Transfection, expression and purification

For transfection and expression, the ExpiCHO system is utilized (Thermo Fisher, Cat. no. A29133). ExpiCHO cells are transiently transfected at the 30 mL scale with 6 pg each of DNA of pR1-hlgA-HC and pR1-k-LC and 18 pg of phJC. All procedures are performed as described by the ExpiCHO System provider. The dimeric IgA from clarified supernatants is affinity purified in batch with Protein L (GE Healthcare). Proteins are washed with phosphate-buffered saline (PBS), eluted with 50 mM phosphoric acid pH 3.0, and neutralized with 20x PBS, pH 11. For subsequent size exclusion chromatography (SEC), HiLoad Superdex 200 pg column is used to separate dimeric IgA from monomeric IgA. The dimeric IgA is eluted earlier in the chromatography process and easily separated from monomeric IgA and other smaller protein parts. The purified R1-hdlgA protein is stored in phosphate buffered saline, pH7.2.

Example 2: Production human Secretory Component:

The protein of human Secretory Component used for this experiment is described by SEQ ID NO:90. For the construction of the expression vector, a signal peptide (SEQ ID NO:102) is added to N-terminus of the SEQ ID NO:90. The resulting protein sequence is reverse translated to a DNA sequence utilizing codons optimized for eukaryotic cells. In order to facilitate cloning into the vector pcDNA3.4, the DNA sequence is provided with a Kozak sequence at the 5-prime end, just before the start codon and a stop codon at the 3-prime end. The resulting gene is obtained by GeneArt (a service by ThermoFisher). This gene is cloned into the mammalian expression vector by AT-cloning (ThermoFisher, cat.no. A14697). All detailed steps for amplification, transformation, cloning, screening for orientation in the vector, plasmid purification from bacteria and quality control are described in the manual of the kit manufacturer. Plasmid purification for transfection is performed with PureLink Expi Endotoxin-Free Mega Plasmid Purification Kit (Thermo Fisher Scientific, cat.no. A31232). The resulting plasmid phSC is DNA-sequence controlled. For transfection and expression, the ExpiCHO system is utilized (Thermo Fisher, Cat. no. A29133). ExpiCHO cells are transiently transfected at the 30 ml_ scale with 30 pg of DNA of purified phSC. All procedures are performed as described by the ExpiCHO System provider. The supernatant of the recombinant ExpiCHO is checked for the approximately 80 kDa hSC protein content and quality controlled by SDS-PAGE and analytical SEC and dialyzed against PBS.

Example 3: Production of a dimeric human lqA2 specific for Clostridium difficile toxin B (TcdB) This example describes the construction of the proteins and the respective gene constructs and their cloning into expression vectors for mammalian cells as well as the expression and purification of the antibodies. The example describes the construction of a dimeric IgA based on the variable region of the anti-TcdB antibody bezlotoxumab (CDB1), binding to the CROP region of the toxin.

3.1. Design of proteins and cloning of respective genes

3.1.1. CDB1-lgA2 HC, Human lgA2 heavy chain

Antibody variable domain (VH) sequence used is from bezlotoxumab antibody protein (IMGT chain ID no. 9608H). Protein sequences of human lgA2 constant heavy chains are obtained from public sources, UniProt or NCBI. For this example, a modified human lgA2 heavy chain constant sequence is used. It bears intentional mutations over the wild type sequences in order to stabilize the light chain-heavy chain disulfide, to favor dimerization, and to reduce glycosylation complexity. The resulting mature heavy chain protein sequence is identified as SEQ ID NO:103. For the construction of the expression vector, a signal peptide (SEQ ID NO:102) is added to N-terminus of the SEQ ID NO:103. The protein sequence is reverse translated to a DNA sequence utilizing codons optimized for eukaryotic cells. In order to facilitate cloning into the mammalian expression vector pcDNA3.4 the DNA sequence is provided with a Kozak sequence at the 5-prime end, just before the start codon and a stop codon at the 3-prime end. The resulting gene is obtained by GeneArt (a service by ThermoFisher Scientific). This gene is cloned into the mammalian expression vector by AT-cloning (ThermoFisher, cat.no. A14697). All detailed steps for amplification, cloning, screening for orientation in the vector, plasmid purification from bacteria and quality control are described in the manual of the kit manufacturer. Plasmid purification for transfection is performed with PureLink Expi Endotoxin-Free Mega Plasmid Purification Kit (Thermo Fisher Scientific, cat.no. A31232). The resulting plasmid is sequence controlled and named pCDB1-hlgA-HC.

3.1.2. CDB1- LC, Human kappa light chain

Antibody variable domain (VL) sequence used is from bezlotoxumab antibody protein (IMGT chain ID no. 9608L). The resulting mature light chain protein sequence is identified as SEQ ID NO: 104. For the construction of the expression vector, a signal peptide (SEQ ID NQ:101) is added to N-terminus of the SEQ ID NQ:104. The protein sequence is reverse translated to a DNA sequence utilizing codons optimized for eukaryotic cells. In order to facilitate cloning into the vector pcDNA3.4 the DNA sequence is provided with a Kozak sequence at the 5-prime end, just before the start codon and a stop codon at the 3-prime end. The resulting gene is obtained by GeneArt (a service by ThermoFisher). This gene is cloned into the mammalian expression vector by AT-cloning (ThermoFisher, cat.no. A14697). All detailed steps for amplification, transformation, cloning, screening for orientation in the vector, plasmid purification from bacteria and quality control are described in the manual of the kit manufacturer. Plasmid purification for transfection is performed with PureLink Expi Endotoxin-Free Mega Plasmid Purification Kit (Thermo Fisher Scientific, cat.no. A31232). The resulting plasmid is pCDB1-k-LC.

3.1.3. Transfection, expression and purification

For transfection and expression, the ExpiCHO system is utilized (Thermo Fisher, Cat. no. A29133). ExpiCHO cells are transiently transfected at the 30 ml_ scale with 6 pg each of DNA of pCDB1-hlgA-HC and pCDB1-k-LC and 18 pg of phJC (described in example 1). All procedures are performed as described by the ExpiCHO System provider. The dimeric IgA from clarified supernatants is affinity purified in batch with Protein L (GE Healthcare). Proteins are washed with phosphate-buffered saline (PBS), eluted with 50 mM phosphoric acid pH 3.0, and neutralized with 20x PBS, pH 11. For subsequent size exclusion chromatography (SEC), HiLoad Superdex 200 pg column is used to separate dimeric IgA from monomeric IgA. The dimeric IgA is eluted earlier in the chromatography process and easily separated from monomeric IgA and other smaller protein parts. The purified CDB1-hdlgA protein is stored in phosphate buffered saline, pH7.2.

Example 4: Production of a dimeric human lqA2 specific for Clostridium difficile toxin A (Ted A)

This example describes the construction of the proteins and the respective gene constructs and their cloning into expression vectors for mammalian cells as well as the expression and purification of the antibodies. The example describes the construction of a dimeric IgA based on the variable region of the anti-TcdA antibody actoxumab (CDA1).

4.1. Design of proteins and cloning of respective genes

4.1.1. CDA1-lgA2 HC, Human lgA2 heavy chain

Antibody variable domain (VH) sequence used is from actoxumab (CDA1; IMGT chain ID no. 9568H) antibody protein. Protein sequences of human lgA2 constant heavy chains are obtained from public sources, UniProt or NCBI. For this example, a modified human lgA2 heavy chain constant sequence is used. It bears intentional mutations over the wild type sequences in order to stabilize the light chain- heavy chain disulfide, to favor dimerization, and to reduce glycosylation complexity. The resulting mature heavy chain protein sequence is identified as SEQ ID NO:105. For the construction of the expression vector, a signal peptide (SEQ ID NO: 102) is added to N-terminus of the SEQ ID NO:105. The protein sequence is reverse translated to a DNA sequence utilizing codons optimized for eukaryotic cells. In order to facilitate cloning into the mammalian expression vector pcDNA3.4 the DNA sequence is provided with a Kozak sequence at the 5-prime end, just before the start codon and a stop codon at the 3-prime end. The resulting gene is obtained by GeneArt (a service by ThermoFisher Scientific). This gene is cloned into the mammalian expression vector by AT-cloning (ThermoFisher, cat.no. A14697). All detailed steps for amplification, cloning, screening for orientation in the vector, plasmid purification from bacteria and quality control are described in the manual of the kit manufacturer. Plasmid purification for transfection is performed with PureLink Expi Endotoxin-Free Mega Plasmid Purification Kit (Thermo Fisher Scientific, cat.no. A31232). The resulting plasmid is sequence controlled and named pCDA1-hlgA-HC.

4.1.2. CDA1- LC, Human kappa light chain Antibody variable domain (VL) sequence used is from actoxumab antibody protein (IMGT chain ID no. 9568H). The resulting mature light chain protein sequence is identified as SEQ ID NO:106. For the construction of the expression vector, a signal peptide (SEQ ID NO:101) is added to N-terminus of the SEQ ID NO:106. The protein sequence is reverse translated to a DNA sequence utilizing codons optimized for eukaryotic cells. In order to facilitate cloning into the vector pcDNA3.4 the DNA sequence is provided with a Kozak sequence at the 5-prime end, just before the start codon and a stop codon at the 3-prime end. The resulting gene is obtained by GeneArt (a service by ThermoFisher). This gene is cloned into the mammalian expression vector by AT-cloning (ThermoFisher, cat.no. A14697). All detailed steps for amplification, transformation, cloning, screening for orientation in the vector, plasmid purification from bacteria and quality control are described in the manual of the kit manufacturer. Plasmid purification for transfection is performed with PureLink Expi Endotoxin-Free Mega Plasmid Purification Kit (Thermo Fisher Scientific, cat.no. A31232). The resulting plasmid is pCDA1-k-LC.

4.2. Transfection, expression and purification

For transfection and expression, the ExpiCHO system is utilized (Thermo Fisher, Cat. no. A29133). ExpiCHO cells are transiently transfected at the 30 ml_ scale with 6 pg each of DNA of pCDA1-hlgA-HC and pCDA1-k-LC and 18 pg of phJC. All procedures are performed as described by the ExpiCHO System provider. The dimeric IgA from clarified supernatants is affinity purified in batch with Protein L (GE Healthcare). Proteins are washed with phosphate-buffered saline (PBS), eluted with 50 mM phosphoric acid pH 3.0, and neutralized with 20x PBS, pH 11. For subsequent size exclusion chromatography (SEC), HiLoad Superdex 200 pg column is used to separate dimeric IgA from monomeric IgA. The dimeric IgA is eluted earlier in the chromatography process and easily separated from monomeric IgA and other smaller protein parts. The purified CDA1-hdlgA protein is stored in phosphate buffered saline, pH7.2.

Example 5: Production of SlqA The production of SlgA from dimeric IgA is performed by mixing and incubating hSC from example 2 and the respective dimeric IgA preparations from examples 1,3 and 4 (R1-hdlgA, CDB1-hdlgA and CDA1-hdlgA, respectively) with subsequent size exclusion chromatography: 8 mg of hdlgA is combined with 2 mg of hSC from example 2 at a combined concentration of 1 mg/ml PBS. The complex formation reaction is performed for 3 hours at room temperature. The mixture is then subjected to SEC with a Superdex 200 Increase 10/300 GL (GE Healthcare) in PBS in order to separate SlgA from unbound hSC and other impurities. The hSIgA peak is collected and checked for purity and concentration by SDS-PAGE and analytical HPLC. These SlgA preparations are named R1-SlgA2, CDB1-SlgA2 and CDA1-SlgA2 respectively.

Example 6: Production of PA41-cdlqA, a chimeric canine dimeric IqA specific for TcdB

This example describes the construction of the proteins and the respective gene constructs and their cloning into expression vectors for mammalian cells as well as the expression and purification of the antibody. The example describes the construction of a dimeric IgA based on the variable region of the anti-TcdB antibody PA-41 , binding to the glucosyltransferase domain of the toxin.

6.1. Design of proteins and cloning of respective genes

6.1.1. PA41-clgA HC, chimeric canine anti — TcdB heavy chain

The antibody variable domain (VH) sequence used is from PA-41 antibody protein (sequence SEQ ID NO:8 from patent US8986697). A canine IgA heavy chain constant sequence is fused to the VH domain. The resulting mature heavy chain protein sequence is identified as SEQ ID NO:107. For the construction of the expression vector, a signal peptide (SEQ ID NO:102) is added to N-terminus of the SEQ ID NO: 107. The protein sequence is reverse translated to a DNA sequence utilizing codons optimized for eukaryotic cells. In order to facilitate cloning into the vector pcDNA3.4 the DNA sequence is provided with a Kozak sequence at the 5-prime end, just before the start codon and a stop codon at the 3-prime end. The resulting gene is obtained by GeneArt (a service by ThermoFisher). This gene is cloned into the mammalian expression vector by AT-cloning (ThermoFisher, cat.no. A14697). All detailed steps for amplification, cloning, screening for orientation in the vector, plasmid purification from bacteria and quality control are described in the manual of the kit manufacturer. Plasmid purification for transfection is performed with PureLink Expi Endotoxin-Free Mega Plasmid Purification Kit (Thermo Fisher Scientific, cat.no. A31232). The resulting plasmid is sequence controlled and named pPA41-clgA-HC.

6.1.2. PA41-ck LC, chimeric canine anti-TcdB light chain

Antibody variable domain (VL) sequence used is from PA-41 antibody protein (sequence SEQ ID NO:10 from patent US8986697). The protein sequence of a canine constant kappa chain is fused to the variable domain. The resulting mature light chain protein sequence is identified as SEQ ID NO:108. For the construction of the expression vector, a signal peptide (SEQ ID NO:101) is added to N-terminus of the SEQ ID NO: 108. The protein sequence is reverse translated to a DNA sequence utilizing codons optimized for eukaryotic cells. In order to facilitate cloning into the vector pcDNA3.4 the DNA sequence is provided with a Kozak sequence at the 5-prime end, just before the start codon and a stop codon at the 3-prime end. The resulting gene is obtained by GeneArt (a service by ThermoFisher). This gene is cloned into the mammalian expression vector by AT-cloning (ThermoFisher, cat.no. A14697). All detailed steps for amplification, transformation, cloning, screening for orientation in the vector, plasmid purification from bacteria and quality control are described in the manual of the kit manufacturer. Plasmid purification for transfection is performed with PureLink Expi Endotoxin-Free Mega Plasmid Purification Kit (Thermo Fisher Scientific, cat.no. A31232). The resulting plasmid is pPA41-ck-LC.

6.1.3. Canine J-chain

Protein sequences of canine J-chain are obtained from UniProt or NCBI. The resulting mature canine J-chain protein sequence is identified as SEQ ID NO:95. For the construction of the expression vector, a signal peptide (SEQ ID NO:101) is added to N-terminus of the SEQ ID NO:95. The protein sequence is reverse translated to a DNA sequence utilizing codons optimized for eukaryotic cells. In order to facilitate cloning into the vector pcDNA3.4 the DNA sequence is provided with a Kozak sequence at the 5-prime end, just before the start codon and a stop codon at the 3- prime end. The resulting gene is obtained by GeneArt (a service by ThermoFisher). This gene is cloned into the mammalian expression vector by AT-cloning (ThermoFisher, cat.no. A14697). All detailed steps for amplification, transformation, cloning, screening for orientation in the vector, plasmid purification from bacteria and quality control are described in the manual of the kit manufacturer. Plasmid purification for transfection is performed with PureLink Expi Endotoxin-Free Mega Plasmid Purification Kit (Thermo Fisher Scientific, cat.no. A31232). The resulting plasmid pcJC is sequence controlled.

6.2. Transfection, expression and purification

For transfection and expression, the ExpiCHO system is utilized (Thermo Fisher, Cat. no. A29133). ExpiCHO cells are transiently transfected at the 30 ml_ scale with 6 pg each of DNA of pPA41-clgA-HC and pPA41-ck-LC and 18 pg of pcJC. All procedures are performed as described by the ExpiCHO System provider. The dimeric IgA is affinity purified in batch with Protein L (GE Healthcare). Proteins are washed with phosphate-buffered saline (PBS), eluted with 50 mM phosphoric acid pH 3.0, and neutralized with 20x PBS, pH 11. For subsequent SEC, HiLoad Superdex 200 pg column is used to separate dimeric IgA from monomeric IgA. The purified dlgA protein, PA41-cdlgA, is stored in phosphate buffered saline, pH7.2.

Example 7: Production of cSC, a recombinant canine Secretory Component

The protein of canine Secretory Component used for this experiment is described by SEQ ID NO:91. For the construction of the expression vector, a signal peptide (SEQ ID NO:102) is added to N-terminus of the SEQ ID NO: 91. The resulting protein sequence is reverse translated to a DNA sequence utilizing codons optimized for eukaryotic cells. In order to facilitate cloning into the vector pcDNA3.4 the DNA sequence is provided with a Kozak sequence at the 5-prime end, just before the start codon and a stop codon at the 3-prime end. The resulting gene is obtained by GeneArt (a service by ThermoFisher). This gene is cloned into the mammalian expression vector by AT-cloning (ThermoFisher, cat.no. A14697). All detailed steps for amplification, transformation, cloning, screening for orientation in the vector, plasmid purification from bacteria and quality control are described in the manual of the kit manufacturer. Plasmid purification for transfection is performed with PureLink Expi Endotoxin-Free Mega Plasmid Purification Kit (Thermo Fisher Scientific, cat.no. A31232). The resulting plasmid pcSC is DNA-sequence controlled. For transfection and expression, the ExpiCHO system is utilized (Thermo Fisher, Cat. no. A29133). ExpiCHO cells are transiently transfected at the 30 ml_ scale with 30 pg of DNA of purified plasmid pcSC. All procedures are performed as described by the ExpiCHO System provider. The supernatant of the recombinant ExpiCHO is checked for cSC protein content and quality by SDS-Page and dialyzed against PBS.

Example 8: Production of PA41-cSlqA, a canine Secretory IgA specific for Ted B

The production of SlgA from dimeric IgA is performed by mixing and incubating cSC from example 6 and PA41-cdlgA from example 5 with a subsequent size exclusion chromatography step: 8 mg of PA41-cdlgA from example 5 is combined with 2 mg of cSC from example 6 at a combined concentration of 1 mg/ml PBS. The complex-formation reaction is performed for 3 hours at room temperature. The mixture is then subjected to SEC with a Superdex 200 Increase 10/300 GL (GE Healthcare) in PBS in order to separate SlgA from unbound SC and other impurities. The larger Ll- cSIgA peak is collected and checked for purity and concentration by SDS-PAGE and analytical HPLC.

Example 9: in vitro qalactosylation and sialylation of a SlgA

Galactosylation and sialylation can be performed in a single reaction in vitro as described in Biochemistry (2001) volume 40, pages 8868-8876, the description in this example is an optimized procedure.

Materials: Bovine alpha-1 , 4-galactosyltransferase (b1 ,4GT; cat.no. G5507 Sigma-Aldrich), Uridine 5'-diphosphogalactose disodium salt (UDP-Gal; cat no. U4500 Sigma-Aldrich), recombinant rat liver alpha-2, 3-sialyltransferase (a2,3ST; cat.no. S7435 Sigma Aldrich) and CMP-Sialic Acid, Disodium Salt - CAS 3063-71-6 - Calbiochem (CMP-Sia; cat.no. 233264 Merck-Millipore) and other chemicals can be obtained from Merck KGA (Darmstadt). NAP-5 and protein L columns can be obtained from Cytiva (VWR International GmbH).

Procedure: SlgA is brought into either 100 mM sodium cacodylate (20840 Sigma-Aldrich) or 50 mM MES (M3058 Sigma Aldrich) buffer (pH 6.4) (10 mg in 1.0 ml_ of buffer) using NAP-5 columns according to the manufacturer. To this solution are added 100 milliunits each of b-1,4GT and a-2,3ST and 5 micromol each of UDP-Gal, CMP-Sia, and MnCte (glycosylation mix).

The mixture is incubated at 37 °C. After 6 h, another aliquot of glycosylation mix is added and the mixture incubated for an additional 6 h at 37 °C. The reglycosylated SlgA is purified on a HiTrap protein L column (Cytiva, 17547815) and used for further analysis. It can be shown that a higher sialic acid content of SlgA enhances the TcdA binding and neutralizing capabilities of SlgA. It even confers TcdA binding to SlgA specific for TcdB.

Example 10: Cytoxicitv and toxin neutralization assays

10.1 Vero cells based assay

Material: Toxins TcdA and TcdB can be purchased from List Biological Laboratories, Inc. (Campbell, CA) and tgcBIOMICS GmbH (Mainz, Germany).

Vero cells (African green monkey kidney; ATCC CCL-81) are cultured in Eagle's minimum essential medium (EMEM) (ATCC 30-2003) supplemented with 10% heat- inactivated fetal bovine sera (FBS; HyClone) and 100 units/ml of penicillin- streptomycin (Invitrogen) in 15mL petri dishes. At the time of confluency, cells are washed with phosphate buffered saline, PBS, and the adherent culture is released by treating with 1mL of 0.25% Trypsin-EDTA (Thermo; 25200-056). Cells are seeded at a density of 1.5x10⁴ cells/mL in a 96-well plate in a final volume of 0.1 mL of medium. Solutions of the toxins are prepared in Vero cell complete medium (Eagle’s minimum essential medium [EMEM] plus 5% heat-inactivated FBS).

A dose response curve is established with technical triplicate for each treatment of cells with the respective toxin, in the range 0-64ng/mL for TcdA (BioTrend; LL-152C) and 0-640pg/mL for TcdB (BioTrend; LL-155L). The cells are incubated with the respective toxin for 3 days at 37°C and then washed twice with minimum essential medium (MEM) (Invitrogen) without phenol red, L-glutamine, or FBS. Next, 100 microL of the MEM and 10 microL of alamarBlue (Invitrogen) are added to each well. The plates are gently mixed and incubated at 37°C for 4 h before reading fluorescence at 560 to 590 nm with a cutoff at 590 nm. Resazurin, the active ingredient of alamarBlue, is a nontoxic cell-permeable blue compound. As only living cells are able to reduce resazurin to a red fluorescent compound, the viable cell number is directly proportional to red fluorescence. The fluorescence results are plotted over antibody concentration.

For neutralization experiments, solutions of the toxins are prepared in Vero cell complete medium (Eagle’s minimum essential medium [EMEM] plus 5% heat- inactivated FBS) and used at a final concentration of 4-times the 50% maximum cytopathic concentration (MC50). MC50 here is defined as the lowest concentration of toxin inducing 50% maximum cytopathic response (as derived from the dose response curve established above).

To assess antibody toxin neutralizing activity, 2-fold dilutions of the antibody preparations are prepared in Vero cell medium and added to a 96-well plate. An equal volume of 8-fold MC50 C. difficile toxin A or toxin B solution and individual dilutions of the antibody solutions are combined in a new 96-well plate and incubated at 37°C with 5% C02 and humidity for 1 h. It is suggested to use at least four orders of magnitude molar excess of antibodies over toxin, even at the lowest concentration of antibody assessed. After 1 h, complete Vero cell medium is removed from 96-well plates containing the Vero cell monolayer, and 100 microL of antibody-toxin mixture is added to the wells. The plates are incubated for 3 days at 37°C supplied with 5% C02 and humidity. After this incubation, the cells are washed twice with minimum essential medium (MEM) (Invitrogen) that do not contain phenol red, L-glutamine, or FBS. Next, 100 microL of the MEM and 10 microL of alamarBlue (Invitrogen) are added to each well. The plates are gently mixed and incubated at 37°C for 4 h before reading fluorescence at 560 to 590 nm with a cutoff at 590 nm.

The fluorescence results are plotted over antibody concentration. The NT50, which is defined as the lowest concentration of antibody that resulted in 50% neutralization of cytotoxicity, can be calculated for each antibody using e.g. GraphPad Prism (GraphPad Software, Inc., La Jolla, CA). The controls for each assay are treatment with toxin A or B alone and treatment with medium alone.

10.2. Transepithelial electrical resistance T84 cell-based toxin neutralization assay.

The transepithelial electrical resistance (TEER) assay uses a polarized monolayer of the human colonic adenocarcinoma cell line T84 and is designed to mimic the human colon in vitro. The assay measures changes in the electrical resistance across the monolayer of T84 cells postexposure to purified C. difficile toxin A or toxin B.

In order to induce the polarization of T84 human colonic carcinoma derived cells (ATCC CCL-248), the cells are seeded into 0.4-micrometer polyester transwell plates (Costar) at a seeding density of 3.6x10⁵ cells/cm². The cells are maintained at 37°C with 5% C02 in 10% heat-inactivated FBS in Dulbecco’s modified Eagle medium (DMEM)-F12 culture medium for 10 to 12 days until stable transepithelial resistance is achieved.

Transepithelial resistance can be measured using a Millipore Millicell ERS-2 V- Ohm meter. Medium is replaced in both the upper and lower compartments daily from day 6 and on the day of assay.

For potency testing of antibodies, either toxin A or toxin B is combined with antibody at a 1 :1 ratio by volume and incubated at 37°C with gentle rocking for 30 min before being added to polarized T84 cells. For TcdA TEER assays, toxin-only or toxin- antibody mixtures are added to the upper compartment of the transwell, exposing only the apical surface of T84 cells to toxin. A final concentration of about 0.6 nM toxin A maybe used as the challenge dose. The chosen dose is equivalent to 6 times the challenge dose required to produce a loss of transepithelial electrical resistance of 50% (6xTEER50). The apical surface of T84 is less sensitive to toxin B than the basolateral surface; therefore, TcdB TEER assays are performed by adding toxin B or toxin-antibody combinations to the lower compartment, exposing the basolateral surface to toxin. The controls consist of at least one well per plate of toxin challenged without antibody and one well containing medium only. Medium is removed from the appropriate compartment, and the toxin-antibody mixture is added to the well. After preparation of the sample, transepithelial resistance is measured immediately (TO) before sample addition and then after 2.5 to 6 h (T150-T360) of incubation at 37°C and 5% C02.

Percent TEER loss can be calculated for each sample using the equation [(T0- T150)/T0] x 100% - (%TEER loss in negative well). The percent protection for antibody was calculated for each treatment using the equation:

(% TEER loss in toxin-only challenge)-(% TEER loss in antibody neutralized toxin challenge)

NT50 is defined as the lowest concentration of antibody conferring 50% protection. The percent completeness of protection represents the proportion of toxin- induced damage that is prevented by the highest concentration of antibody. Antibody concentrations are increased until no further protection is observed. It can be shown in such assays that the SlgA versions of the antibodies have a more than 10-fold higher toxin neutralization capacity as compared to the respective dimeric IgA antibodies. In vitro galactosylated and sialylated SlgA antibodies show an enhanced neutralization of TcdA, independent of the antibody specificity.

Example 11 : Prophylactic treatment of mice challenged with Clostridium difficile spores

This example is to show that SlgA against Clostridium difficile toxins can be used to prevent C. diff. induced symptoms and mortality. CDB1-SlgA2 and CDA1-SlgA2 antibodies are prepared according to example

5, and mixed 1:1 to prepare a SlgA mixture. This SlgA mixture is diluted with phosphate-buffered saline (PBS) and water to yield a final total concentration of SlgA of 70 microgram per ml (35 microgram CDB1-SlgA2 and 35 microgram CDA1-SlgA2 per ml) in a half-concentrated PBS (0.5xPBS). 12-week-old female C57BL/6 mice are employed for the experiment. All mice are housed in a pathogen-free facility in 2 groups of 10 mice under the same conditions. Standard food, bedding, and cages are autoclaved, 0.5xPBS and antibody preparation in 0.5xPBS is filtered with a 0.2 micrometer sterile filter. The antibody treated mice receive the SlgA mixture (210 pg SlgA/mouse/day in 3ml 0.5xPBS) over the entire test duration (from day -3 to day 7), whereas the control group receives the same amount of 0.5xPBS, without SlgA. On day -1 all mice including the control group receive a single dose of clindamycin (10 mg/kg) intraperitoneally; on day 0 all mice are infected by gavage with C. diff. spores (each mouse approximately 10,000 spores) from strain VP110463 (ATCC no. 43255) according to results of preliminary experiments. With this experiment it can be shown that more mice in the SlgA group survive the experiment as compared to the control.

Claims

1. A monoclonal secretory immunoglobulin A subtype 2 (SlgA2) preparation, which SlgA2 is specifically recognizing at least one target being toxin A and/or toxin B of Clostridium difficile.

2. The preparation of claim 1 , wherein a) said toxin B comprises or consists of SEQ ID NO:1 ; and b) said toxin A comprises or consists of SEQ ID NO:2.

3. The preparation of claim 1 or 2, wherein said SlgA2 is monospecific or bispecific, comprising: a) a recombinant IgA oligomer composed of at least two IgA monomers linked by a J-chain, wherein at least one of said IgA monomers is a target-specific IgA of the subtype 2 and comprises an antigen-binding site specifically recognizing one of said targets; and b) a recombinant secretory component (SC).

4. The preparation of claim 3, wherein the antigen-binding site is a CDR-binding site specifically recognizing toxin B and comprises three VH-CDR sequences (VH- CDR1 , VH-CDR2 and VH-CDR3) of a variable heavy chain antibody domain (VH), and three VL-CDR sequences (VL-CDR1 , VL-CDR2 and VL-CDR3) of a variable light chain antibody domain (VL), wherein:

A) a) VH-CDR1 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:27; b) VH-CDR2 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:28; c) VH-CDR3 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:29; d) VL-CDR1 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NQ:30; e) VL-CDR2 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:31; and f) VL-CDR3 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:32; or B) a) VH-CDR1 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:33; b) VH-CDR2 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:34; c) VH-CDR3 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:35; d) VL-CDR1 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:36; e) VL-CDR2 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:37; and f) VL-CDR3 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:38; or C) a) VH-CDR1 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:39; b) VH-CDR2 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:40; c) VH-CDR3 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:41; d) VL-CDR1 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:42; e) VL-CDR2 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:43; and f) VL-CDR3 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:44; wherein the sequences are according to IMGT.

5. The preparation of claim 4, wherein a) said VH comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:45; and said VL comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:46; or b) said VH comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:47; and said VL comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:48; or c) said VH comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:49; and said VL comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:50.

6. The preparation of claim 3, wherein the antigen-binding site is a CDR-binding site specifically recognizing toxin A and comprises three VH-CDR sequences (VH- CDR1 , VH-CDR2 and VH-CDR3) of a variable heavy chain antibody domain (VH), and three VL-CDR sequences (VL-CDR1 , VL-CDR2 and VL-CDR3) of a variable light chain antibody domain (VL), wherein:

A) a) VH-CDR1 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:3; b) VH-CDR2 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:4; c) VH-CDR3 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:5; d) VL-CDR1 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:6; e) VL-CDR2 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:7; and f) VL-CDR3 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:8; or B) a) VH-CDR1 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:9; b) VH-CDR2 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:10; c) VH-CDR3 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:11; d) VL-CDR1 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:12; e) VL-CDR2 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:13; and f) VL-CDR3 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:14; or C) a) VH-CDR1 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:15; b) VH-CDR2 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:16; c) VH-CDR3 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:17; d) VL-CDR1 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:18; e) VL-CDR2 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:19; and f) VL-CDR3 comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:20; wherein the sequences are according to IMGT.

7. The preparation of claim 6, wherein a) said VH comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:21; and said VL comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:22; or b) said VH comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:23; and said VL comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:24; or c) said VH comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:25; and said VL comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO:26.

8. The preparation of any one of claims 1 to 7, wherein said SlgA2 comprises at least one IgA molecule comprising: a) variable antibody domains comprising antibody framework sequences that are at least 90% identical to respective mammalian sequences, preferably of human, non-human primate, dog, horse, or cat, or of humanized, caninized, equinized, or felinized antibodies; and/or b) constant antibody domains comprising constant antibody domain sequences that are at least 90% identical to respective mammalian sequences, preferably of human, non-human primate, dog, horse, or cat, or of humanized, caninized, equinized, or felinized antibodies; and/or c) hinge of 6-12 amino acids length originating from a native lgA2 molecule which comprises no more than 0, 1 , or 2 point mutations in such hinge of a native lgA2 molecule.

9. The preparation of any one of claims 1 to 8 wherein said SlgA2 comprises at least one IgA molecule that is modified by a R101 P point mutation in the CH1 domain of the alpha 2m(2) allotype.

10. The preparation of any one of claims 1 to 9, wherein said SlgA2 comprises a secretory component (SC) which comprises at least 80% sequence identity to a human, non-human primate, canine, equine, or feline SC, preferably at least 80% sequence identity to any one of SEQ ID NO:90-93.

11. The preparation of claim 10, wherein said SC is human SC comprising at least 2 mol galactose or at least 2 mol sialylic acid per mol SC.

12. The preparation of any one of claims 1 to 11 , comprising said SlgA2 as active component and further comprising at least one or more further active components or supplements.

13. The preparation of claim 12, wherein a) said further active component is selected from the group consisting of immunoglobulins, non-steroid anti-inflammation agents, steroids, antiphlogistiga, endolysins, bacteriocides, antibiotics, cholestyramine, tolevamer, calcium aluminosilicate anti-diarrheal, inositol hexakisphosphate analogs, vaccines, phage- based therapeutics, bile acid therapy, intestinal antibiotic inactivators, and enteroprotective agents; b) said supplement is selected from the group consisting of probiotics, prebiotics, antibiotics, vitamins, non-toxigenic C. difficile strains, fecal microbiome transplant, Veillonella dispar cultures, and spore-forming commensal microorganisms.

14. The preparation of any one of claims 1 to 13, comprising said SlgA2 in a formulation for topical use, preferably intraoral, ocular, otic, dermal, cutaneous, vaginal, intragastric, rectal use, or for application to the upper and lower respiratory tract.

15. A method for the surface treatment of a body using the preparation of any one of claims 1 to 14.

16. A pharmaceutical preparation comprising the preparation of any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

17. The pharmaceutical preparation of claim 16, for use in the prophylactic or therapeutic treatment of a disease condition caused by C. difficile, such as antibiotic- associated diarrhea.

18. The pharmaceutical preparation for use according to claim 17, wherein treatment is combined with an antibiotic treatment, preferably wherein the pharmaceutical preparation is administered before, during or after said antibiotic treatment.