Necrotic enteritis B-like toxin antibodies

Abstract

The present disclosure provides a binding protein (e.g., an antibody) comprising at least one antigen binding domain that binds to necrotizing enteritis B-like toxin (NetB), said antigen binding domain comprising a heavy chain variable region comprising three complementarity determining regions (CDRs), preferably wherein said binding protein binds to NetB at a KD of 250 pM or less at pH 7.4. The binding proteins can be used to treat or prevent Clostridium perfringens infection or necrotic enteritis. Nucleic acid molecules, expression vectors, host cells and compositions are also provided.

Description

Technical Field

[0001] The present invention generally relates to the field of binding proteins that bind to NetB (necrotic enteritis B-like toxin), in particular antibodies, in particular binding proteins and antibodies that bind to NetB produced by Clostridium perfringens. Such anti-NetB binding proteins and antibodies have therapeutic and protective uses, such as treating or preventing diseases caused by or associated with Clostridium perfringens NetB toxins, in particular necrotic enteritis (NE), e.g. reducing the incidence and severity thereof. Compositions, methods and kits based on the binding proteins and antibodies are also provided. Background Art

[0002] Necrotic enteritis (NE) is a life-threatening gastrointestinal disease that particularly affects animals and poultry, such as chickens. The disease is caused by Clostridium perfringens, a Gram-positive, anaerobic, spore-forming pathogen. Clostridium perfringens is ubiquitous in the environment, for example, commonly found in soil, garbage, dust, and is present at low levels in the intestines of many humans and animals (e.g., healthy chickens) (Hatheway, CL, Clin. Microbiol. Rev. 1990, 3, 66-98).

[0003] Avian NE is an intestinal disease characterized by necrotic lesions in the small intestinal mucosa (Kaldhusdal and Hofshagen, 1992, Poult. Sci., 71, 1145-1153). NE is a global problem for poultry, with an estimated annual cost of more than $6 billion in production losses and control measures to the international poultry industry (Cooper and Songer, 2009, Anaerobe, 15, 55-60; Wade et al., 2015, Veterinary Microbiology, 180, 299-303).

[0004] Necrotic enteritis occurs when large numbers of C. perfringens grow in the intestine and secrete toxins. These toxins can cause intestinal necrosis and lesions, hemorrhage, intestinal perforation, and ultimately death from septic shock. NE is a complex disease that requires a triggering factor that compromises intestinal integrity to promote the proliferation of C. perfringens and toxin production.

[0005] There are several different types of Clostridium perfringens that produce several toxins: alpha toxin, beta toxin, epsilon toxin, iota toxin, CPE toxin, and NetB toxin (Rood et al., 2018, Anaerobe, 53: 5–10). All strains of Clostridium perfringens produce an extracellular toxin, alpha toxin (Awad et al., 1995, Mol. Microbiol., 15, 191-202), but this toxin has been found not to be essential for NE disease (Keyburn et al., 2006, Infect. Immun., 74, 6496-6500). However, it is believed that NetB toxin (originally found in avian Clostridium perfringens type A strains, Keyburn et al., 2008, PLoS Pathog., 4, e6) is a key and possibly essential virulence factor for Clostridium perfringens strains to cause avian NE. Clostridium perfringens is the main causative agent of avian NE, which is usually caused by type A and type C strains of Clostridium perfringens (Songer 1996, Clin. Microbiol. Rev., 9, 216-234). Recently, a new classification scheme for Clostridium perfringens toxin types has been proposed, in which strains that express NetB and produce NE are currently classified as type G (Rood et al., 2018, Anaerobe, 53: 5–10).

[0006] Currently, the best treatment for NE in birds is antibiotics. Work has also been done to try to develop vaccines based on toxin molecules. This work has mainly focused on alpha toxin, but there has also been some research into vaccines based on NetB. However, to date, vaccines have proven only partially effective, and in addition, existing commercial vaccines use genetically modified Salmonella vectors, which pose potential safety issues for humans. Antibiotics have the disadvantage of creating selection pressure and leading to antibiotic resistance (in animals and humans).

[0007] There is therefore a clear need for alternative, preferably improved, treatment and prevention regimens for necrotic enteritis or infections caused by or associated with C. perfringens, and in particular infections caused by or associated with the NetB toxin of C. perfringens. Summary of the invention

[0008] The present invention provides such an alternative treatment or prevention regimen in the form of binding proteins and antibodies (e.g., antibody-based binding proteins) directed against NetB, which can be used to reduce or prevent necrotic enteritis (NE) or infections caused or associated with Clostridium perfringens or NetB toxins, particularly infections caused or associated with NetB toxins of Clostridium perfringens, such as NE. The anti-toxin antibody approach advantageously avoids the use of antibiotics and does not create selection pressure because the anti-toxin antibodies are not directed against the bacteria themselves but against the toxins produced by the bacteria.

[0009] Oral delivery of such biotherapeutics is a preferred option because it targets the site of infection. However, such delivery is often not possible due to degradation of these substances in the acidic and enzyme-rich environment of the gastrointestinal tract and stomach (or more specifically the crop, proventriculus and gizzard in avian species such as chickens).

[0010] The antibodies (monoclonal antibodies) of the present invention have been shown to be able to bind NetB with high affinity and also show good ability to inhibit NetB-induced toxicity. Advantageously, such antibodies also show these abilities under conditions found in the gastrointestinal tract and small intestine. Thus, such abilities are also shown at pH 6.0 and pH 7.4. These antibodies are also stable at lower pH, such as pH 2.5 or pH 1.8. In addition, these antibodies show resistance to certain proteases found in the gastrointestinal tract and small intestine, in particular chymotrypsin, pepsin and pancreatin. Such properties make these antibodies particularly suitable and advantageous for the described therapeutic uses.

[0011] Thus, the inventors provide anti-NetB antibodies that are capable of binding to and inhibiting the activity or function of NetB, particularly NetB toxin produced by Clostridium perfringens. Such antibodies (or other binding proteins, for example, comprising a NetB antigen binding domain as described herein) can, for example, inhibit the toxic or cytotoxic ability of NetB, such as the ability of NetB to lyse or kill cells sensitive to NetB toxins, including the hemolytic activity of NetB, such as hemolytic activity against red blood cells. Such antibodies (or other binding proteins, for example, comprising a NetB antigen binding domain as described herein) can be conveniently and advantageously used to treat or prevent NE in avian species, particularly NE caused by or associated with Clostridium perfringens infection.

[0012] In one embodiment, the invention provides a binding protein (e.g., an antibody, e.g., a monoclonal antibody) that binds NetB (e.g., Clostridium perfringens NetB) for use in treating or preventing NE or infection caused by or associated with Clostridium perfringens in a subject (or group of subjects), in particular an infection caused by or associated with NetB toxin of Clostridium perfringens, such as NE. Preferably, the subject is an avian species, such as poultry.

[0013] Thus, specifically, the present invention provides a monoclonal antibody that binds NetB for use in treating or preventing NE or other infections in poultry caused by or associated with the NetB toxin of Clostridium perfringens. However, the antibodies (or binding proteins) of the present invention may be used to treat or prevent any pathology in any subject in which NetB plays a role, in which case binding and inhibition of this toxic protein would be a useful therapeutic approach.

[0014] As discussed elsewhere herein, preferred antibodies (or binding proteins) of the present invention and suitable for use in the methods of treatment described herein have high affinity for NetB, e.g., when measured by Biacore (the ability to bind NetB can be observed at both pH 6.0 and pH 7.4); the ability to reduce the toxic activity of NetB (this ability can be observed at both pH 6.0 and pH 7.4, and preferably at both 42°C and 37°C); resistance to various proteases, e.g., resistance to digestion with pepsin (e.g., at a pepsin concentration of 30 U / ml at a temperature of 37°C), resistance to digestion with chymotrypsin (e.g., at a chymotrypsin concentration of 1 μg / ml at a temperature of 37°C), and optionally resistance to digestion with pancreatin (e.g., at a pancreatin concentration of 0.5 mg / ml at a temperature of 37°C). Preferred antibodies (or binding proteins) also exhibit stability in water. To the best of the inventors' knowledge, no other anti-NetB antibodies have been disclosed that possess this advantageous combination of properties, and antibodies (or binding proteins) that possess one or more (preferably all) of these properties are preferred.

[0015] In another embodiment, the present invention provides a binding protein (e.g., an antibody) comprising at least one antigen binding domain that binds to necrotic enteritis B-like toxin (NetB) (e.g., NetB toxin produced by Clostridium perfringens), the antigen binding domain comprising a heavy chain variable region comprising three complementarity determining regions (CDRs), wherein the heavy chain variable region comprises:

[0016] (i) a variable heavy chain (VH) CDR1 comprising the amino acid sequence of GSIRSISFMN (SEQ ID NO: 2) or a sequence substantially homologous thereto, optionally wherein the substantially homologous sequence is a sequence containing 1, 2, 3 or 4 amino acid substitutions compared to the given CDR sequence,

[0017] (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence of INQIGNT (SEQ ID NO: 3) or a sequence substantially homologous thereto, optionally wherein the substantially homologous sequence is a sequence containing 1, 2 or 3 amino acid substitutions compared to the given CDR sequence, and

[0018] (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence of IVRGTVSPFQF (SEQ ID NO: 4) or a sequence substantially homologous thereto, optionally wherein the substantially homologous sequence is a sequence containing 1, 2, 3 or 4 amino acid substitutions compared to the given CDR sequence.

[0019] In another embodiment, the present invention provides a binding protein (e.g., an antibody) comprising at least one antigen binding domain that binds to necrotic enteritis B-like toxin (NetB) (e.g., NetB toxin produced by Clostridium perfringens), the antigen binding domain comprising a heavy chain variable region comprising three complementarity determining regions (CDRs), wherein the heavy chain variable region comprises:

[0020] (i) a variable heavy chain (VH) CDR1 comprising the amino acid sequence of GLTFSGYSLG (SEQ ID NO: 10) or a sequence substantially homologous thereto, optionally wherein the substantially homologous sequence is a sequence containing 1, 2, 3 or 4 amino acid substitutions compared to the given CDR sequence,

[0021] (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence of ISSSGMIT (SEQ ID NO: 11) or a sequence substantially homologous thereto, optionally wherein the substantially homologous sequence is a sequence containing 1, 2 or 3 amino acid substitutions compared to the given CDR sequence, and

[0022] (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence of RFPRPRSWLSTDSYNY (SEQ ID NO: 12) or a sequence substantially homologous thereto, optionally wherein the substantially homologous sequence is a sequence containing 1, 2, 3 or 4 amino acid substitutions compared to the given CDR sequence.

[0023] In a preferred embodiment, the present invention provides a binding protein (e.g., an antibody) comprising at least one antigen binding domain that binds to a necrotic enteritis B-like toxin (NetB) (e.g., a NetB toxin produced by Clostridium perfringens), the antigen binding domain comprising a heavy chain variable region comprising three complementarity determining regions (CDRs), wherein the heavy chain variable region comprises:

[0024] (i) a variable heavy chain (VH) CDR1 comprising the GSIRSISFMN amino acid sequence (SEQ ID NO: 2),

[0025] (ii) a variable heavy chain (VH) CDR2 comprising an INQIGNT amino acid sequence (SEQ ID NO: 3), and

[0026] (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence of IVRGTVSPFQF (SEQ ID NO: 4).

[0027] In a preferred embodiment, the present invention provides a binding protein (e.g., an antibody) comprising at least one antigen binding domain that binds to a necrotic enteritis B-like toxin (NetB) (e.g., a NetB toxin produced by Clostridium perfringens), the antigen binding domain comprising a heavy chain variable region comprising three complementarity determining regions (CDRs), wherein the heavy chain variable region comprises:

[0028] (i) a variable heavy chain (VH) CDR1 comprising the amino acid sequence of GLTFSGYSLG (SEQ ID NO: 10),

[0029] (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence ISSSGMIT (SEQ ID NO: 11), and

[0030] (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence of RFPRPRSWLSTDSYNY (SEQ ID NO: 12).

[0031] Certain preferred embodiments of the present invention provide an antibody (or binding protein) that binds to necrotic enteritis B-like toxin (NetB) (e.g., NetB toxin produced by Clostridium perfringens), the antibody comprising a VH domain having an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 9, or a sequence substantially homologous thereto. In some embodiments, such an antibody (or binding protein) further comprises a VL domain comprising up to three light chain CDRs, and preferably comprising three light chain CDRs.

[0032] In a preferred embodiment, the present invention provides an antibody (or binding protein) that binds to a necrotic enteritis B-like toxin (NetB) (e.g., a NetB toxin produced by Clostridium perfringens), the antibody comprising a VH domain having an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 9, or a sequence having at least 60%, 65%, 70% or 75% sequence identity thereto, preferably a sequence having at least 80% sequence identity thereto (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity). In some embodiments, such an antibody (or binding protein) further comprises a VL domain comprising up to three light chain CDRs, and preferably comprising three light chain CDRs.

[0033] In a preferred embodiment, the present invention provides an antibody (or binding protein) that binds to a necrotic enteritis B-like toxin (NetB) (e.g., NetB toxin produced by Clostridium perfringens), the antibody comprising a VH domain having an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 9. In some embodiments, such an antibody (or binding protein) further comprises a VL domain comprising up to three light chain CDRs, and preferably comprising three light chain CDRs.

[0034] In some embodiments, the antibody (or binding protein) is a VHH antibody having three CDRs, or comprising a VH domain having three CDRs, but without a VL region or VL domain. Thus, in such embodiments, the antigen binding domain may comprise (or consist of) three CDR regions and optionally four FR regions. In such embodiments, an immunoglobulin or antibody constant region is optionally absent, for example, the CDR and optional FR regions may be the only antibody-derived sequences present. Thus, in some embodiments, the antibody (or binding protein) or its antigen binding domain comprises (only comprises) a single heavy chain variable region.

[0035] Other preferred embodiments are immunoglobulin (Ig) forms (e.g., IgG, IgA, IgD, IgE or IgM forms) of the various antibodies (or binding proteins) defined herein, including chicken IgY forms; or forms containing all or part of an immunoglobulin constant region (e.g., all or part of an IgG or other constant region), such as full-length Ig or IgG, IgA or IgY forms. In some embodiments, an IgY form is not used. Of course, it should be understood that complete IgG (and other) antibodies typically contain two identical or substantially identical heavy chains (with suitable variable and constant regions) and two identical or substantially identical light chains (with suitable variable and constant regions). In addition, full-length heavy chain-only antibodies may also be used, which typically contain two identical or substantially identical heavy chains (with suitable variable and constant regions), such as camelid antibodies (which typically have two identical or substantially identical heavy chains, which contain variable regions and CH2 and CH3 constant domains, particularly IgG constant domains, such as IgG2 or IgG3 constant domains). A preferred form containing a portion of an immunoglobulin constant region is a form containing an Fc region or domain, such as an Fc fusion. Such Fc regions or domains are known in the art and typically comprise the CH2 and CH3 domains of an antibody heavy chain (CH2, CH3, and CH4 domains in the case of IgY and IgE), which associate to form homodimers. Therefore, a configuration containing a CH2 and CH3 domain (or CH2, CH3, and CH4 domain) may be preferred, particularly an IgG, IgA, or IgY Fc region, and in some embodiments, no other antibody constant regions are present.

[0036] These regions may be derived from any suitable source or species, such as a source or species that is the same or different from the host species used to generate the antibody (e.g., by immunization), or a source or species that is the same or different from the source or species from which the antibody is derived or corresponds. In some embodiments, these regions may correspond to or be derived from an Fc region or domain of a species to which the antibody is to be administered (e.g., for treatment). Suitable species are defined elsewhere herein, such as avian species.

[0037] Since such Fc regions are homodimeric (or form homodimers), they can be conveniently used to dimerize two polypeptide chains. Therefore, by connecting or fusing one or more single domain antibodies (e.g., VHH antibodies) of the present invention to each chain of the Fc region, when the two chains of the Fc region dimerize, it can be used to provide multiple copies of the single domain antibodies (e.g., VHH antibodies) of the present invention in a single construct or molecule. If more than one single domain antibody (e.g., VHH antibody) of the present invention is sequentially connected or fused to each chain of the Fc region, these antibodies can be the same antibody (e.g., two or more copies of the same VHH can be provided on each chain) or different antibodies. Therefore, for example, Fc fusions can be used to provide such a construct, which contains more than one copy of the same single domain antibody of the present invention, or more than one copy of different single domain antibodies of the present invention. Since such constructs typically contain more than one copy of the same antibody of the invention (e.g., more than one copy of one single domain antibody or VHH antibody, or more than one copy of multiple different single domain antibodies or VHH antibodies), such constructs may show improved NetB binding, e.g. due to avidity effects.

[0038] Preferred are binding proteins, such as antibodies, based on the Net 83 or Net 14 antibody sequences listed in Table A or Table B, respectively. The present invention is exemplified by monoclonal antibodies, which are VHH antibodies (single domain antibodies), whose sequences are shown in Table A or Table B herein. The VH CDR domains and VH domains of each of these VHH antibodies are shown in Table A or Table B herein. Antibodies (or binding proteins) comprising these VH CDR domains or VH domain groups, or antibodies (or binding proteins) comprising these FR domain groups, or antibodies (or binding proteins) comprising such domains (or sequences substantially homologous thereto) containing IgG / Fc configurations are preferred embodiments of the present invention.

[0039] Some examples of substantially homologous sequences are sequences that have at least 60% or 65% identity to the disclosed amino acid sequences. In certain embodiments, the antibodies (or binding proteins) of the invention or their antigen binding domains comprise one or at least one heavy chain variable region that comprises an amino acid sequence region that has at least 60%, 65%, 70% or 75%, more preferably at least 80%, more preferably at least 85%, 86%, 87%, 88% or 89%, more preferably at least 90%, 91%, 92%, 93%, 94% or 95%, and most preferably at least 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 9.

[0040] Other preferred examples of substantially homologous sequences are sequences containing conservative amino acid substitutions of the disclosed amino acid sequences.

[0041] Other preferred examples of substantially homologous sequences are sequences containing one, two, three or four, preferably one, two or three, preferably one or two (more preferably one) altered amino acids in one or more of the disclosed CDR regions or one or more of the disclosed FR regions. Such alterations may be conservative or non-conservative amino acid substitutions, or a mixture of the two.

[0042] Other preferred examples of "substantially homologous" sequences are sequences having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% amino acid sequence identity with the amino acid sequence of one or more of the CDR regions disclosed in Table A or Table B or one or more of the FR regions disclosed. Thus, in some embodiments, a "substantially homologous" CDR sequence may be a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity with a given CDR sequence described herein.

[0043] In some embodiments, in an antibody having a sequence that is "substantially homologous" to a given sequence, or in an antibody having a certain degree of sequence identity to a given sequence, the altered amino acid residue is not in a CDR region. For example, in some embodiments, in an antibody whose VH domain has a certain degree of sequence identity to a given VH domain sequence of a particular antibody of the invention (e.g., Net 14 or Net 83), the altered (or variant) residue is not in a CDR region. Thus, in some embodiments, in an antibody having a sequence that is "substantially homologous" to a given sequence, or in an antibody having a certain degree of sequence identity to a given sequence, the altered amino acid residue is in one or more framework regions.

[0044] As will be apparent from elsewhere herein, in other embodiments, in antibodies having a "substantially homologous" sequence compared to a given sequence, or in antibodies having a certain degree of sequence identity compared to a given sequence, the amino acid residues that are altered may be in the CDR regions.

[0045] In some embodiments, in antibodies having a "substantially homologous" sequence compared to a given sequence, or in antibodies having a certain degree of sequence identity compared to a given sequence, three VH CDR amino acid sequences (i.e., all three VH CDR sequences taken together) are collectively considered to be the complete (or full) CDR complement of the antibody, and the amino acid sequence of the complete CDR complement of the antibody is at least 70%, preferably at least 80%, or at least 90%, or at least 95% identical to the corresponding complete (or full) CDR complement of a given starting (or reference) antibody. The starting (or reference) antibody may have the CDR sequences of the Net 14 or Net83 antibodies of the present invention.

[0046] The altered residues may be conservative or non-conservative amino acid substitutions, or a mixture of both.

[0047] In such embodiments, preferred alterations are conservative amino acid substitutions.

[0048] In all embodiments, the binding protein (e.g., antibody) containing the substantially homologous sequence retains the ability to bind NetB, such as the NetB toxin produced by Clostridium perfringens or the NetB of SEQ ID NO: 17. Preferably, the binding protein (e.g., antibody) containing the substantially homologous sequence retains one or more (preferably all) of the other properties described herein associated with the Net 83 or Net 14 antibodies described herein.

[0049] Further examples of substantially homologous amino acid sequences according to the invention are described elsewhere herein.

[0050] The CDRs of the antibodies (or binding proteins) of the invention are preferably separated by suitable framework regions, such as those found in naturally occurring antibodies and / or effective engineered antibodies. H (e.g. VHH), V L The individual CDR sequences are preferably provided within or incorporated into a suitable framework or scaffold to achieve antigen (here NetB) binding. Such framework sequences or regions may correspond to naturally occurring framework regions FR1, FR2, FR3 and / or FR4 (as appropriate) to form a suitable scaffold, or may correspond to a consensus framework region, which is identified, for example, by comparing various naturally occurring framework regions. Alternatively, non-antibody scaffolds or frameworks, such as T cell receptor frameworks, may be used.

[0051] Suitable sequences that can be used for framework regions are well known and documented in the art, and any of these sequences can be used. Preferred sequences for the framework region are one or more of the framework regions that make up the VHH antibody framework region of the present invention, preferably one or more of the framework regions of the Net 83 or Net 14 VHH antibody framework region disclosed in Table A or Table B, or framework regions that are substantially homologous thereto, and in particular framework regions that allow the antigen specificity to be maintained, such as framework regions that enable the 3D structure of the antibody to be substantially identical or identical.

[0052] In certain preferred embodiments, all four variable heavy chain (SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8) framework regions (FR) (as the case may be) or FR regions substantially homologous thereto are present in the antibodies (or binding proteins) of the invention.

[0053] In other preferred embodiments, all four variable heavy chain (SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16) framework regions (FR) (as the case may be) or FR regions substantially homologous thereto are present in the antibodies (or binding proteins) of the invention.

[0054] The CDR sequences of certain antibodies of the invention are listed herein in Table A and Table B. In some other embodiments, the CDR sequences of the antibodies of the invention may be CDR sequences in the VH and VL domains of the antibodies of the invention identified using any suitable method (or tool), such as those well known to Kabat (e.g., Kabat, et al., "Sequences of Proteins of Immunological Interest", 5th ed., National Institutes of Health, Public Health Service, Bethesda, Maryland, 647-669, 1991) or Chothia (e.g., Chothia C et al., (1989) Nature, 342: 877-883 or Al-Lazikani et al., (1997) JMB 273, 927-948), or identified using the IMGT numbering scheme (e.g., Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); www.imgt.org)). The IMGT numbering scheme has been used to identify the CDR sequences in Table A and Table B.

[0055] As described above, the present invention provides binding proteins (e.g., antibodies), or binding proteins comprising an antigen binding domain of an antibody, which binds (or specifically recognizes or specifically binds) NetB, such as the NetB toxin produced by Clostridium perfringens. Preferred binding proteins of the present invention are antibodies, and in particular VHH antibodies. However, the embodiments relating to antibodies (e.g., VHH antibodies) as described herein are equally applicable to other types of binding proteins, and vice versa, after necessary modifications. Therefore, other binding proteins may comprise an antibody of the present invention, or may comprise an antigen binding domain of an antibody of the present invention, such as three CDR regions (CDR1, CDR2, and CDR3), or three CDR regions (CDR1, CDR2, and CDR3) and four FR regions (FR1, FR2, FR3, and FR4). Multispecific antibodies, such as bispecific antibodies or trispecific antibodies, incorporating antibodies of the present invention are also provided.

[0056] Preferred binding proteins are any single polypeptide chain that can bind (e.g., specifically bind) NetB (e.g., NetB toxin produced by Clostridium perfringens). Suitable types of binding proteins that can be used in the present invention are known in the art. For example, in some embodiments, immunoglobulin-based polypeptides are used, which typically include CDR regions (and optionally FR regions or immunoglobulin-based frameworks), such that the CDR regions (and optionally FR regions) of the antibodies of the present invention can be grafted onto a suitable backbone or framework, such as an immunoglobulin backbone.

[0057] However, in other embodiments, non-immunoglobulin-based scaffolds (e.g., single-chain binding scaffolds) can be used, onto which suitable CDRs that mediate target antigen binding can be grafted. For example, the CDR regions (and optionally the FR regions) of an antibody of the invention can be grafted onto a suitable non-immunoglobulin scaffold and can be selected for the ability to specifically bind to NetB (e.g., NetB toxin produced by Clostridium perfringens). Such molecules are also referred to as antibody mimetics (or antibody mimetics). Examples of suitable non-immunoglobulin-based single-chain binding proteins are known and described in the art, and include fibronectin (or fibronectin-based molecules), e.g., based on the tenth module of the fibronectin type III domain, such as Adnectin (e.g., from Compound Therapeutics, Inc., Waltham, Massachusetts); affimers (e.g., from Avacta); ankyrin repeat proteins or DARPins (e.g., from Molecular Partners AG, Zurich, Switzerland); lipocalins, such as anticalins (e.g., from Pieris Proteolab AG, Freising, Germany); human A domains (e.g., Avimers); Staphylococcal protein A (e.g., from Affibody AG, Sweden); thioredoxins; and molecules based on gamma-B-crystallin or ubiquitin, such as affilin (e.g., from Scil Proteins GmbH, Halle, Germany).

[0058] Preferred non-antibody binding proteins (or binding portions) of the invention have the ability to bind to the same epitope as an anti-NetB antibody of the invention, and such binding proteins (or binding portions) can be selected, for example, by competition assays (such as those described elsewhere herein), e.g., using an antibody of the invention as a reference antibody.

[0059] NetB is a known toxin produced by Clostridium perfringens. NetB has been identified as a type I pore-forming protein of 130 kDa, 322 amino acids in length, with a signal peptide of 30 amino acids in length. This toxin forms pores that disrupt the phospholipid membrane bilayer of human and animal cells, leading to ion influx and resulting in cell lysis. NetB has high hemolytic activity against avian erythrocytes.

[0060] Of particular relevance to the present invention, NetB can be produced by Clostridium perfringens, and it is believed that NetB plays a critical role in the ability of this pathogen to cause disease in animals, particularly avian species. In this regard, NetB, particularly NetB produced by Clostridium perfringens, is believed to be a major pathogenic agent of necrotic enteritis (NE) disease in animals, particularly avian species such as chickens, turkeys, etc. Therefore, the ability of the binding proteins or antibodies of the present invention to bind NetB, and the ability to inhibit, reduce or otherwise neutralize the toxic activity of NetB is important and preferred.

[0061] Thus, the binding protein or antibody of the present invention binds or is capable of binding (or specifically binds) NetB. In a preferred embodiment of the present invention, the binding protein or antibody of the present invention binds or is capable of binding (or specifically binds) NetB produced by Clostridium perfringens. In a preferred embodiment of the present invention, the binding protein or antibody of the present invention can inhibit, reduce or otherwise neutralize the toxic activity of NetB.

[0062] NetB to which the antibody of the present invention binds or is capable of binding (or specifically recognizes or specifically binds) may be from (or may correspond to) any strain or subtype of Clostridium perfringens, in particular a type A strain (also referred to as a type G strain) of Clostridium perfringens.

[0063] The binding proteins and antibodies of the invention may bind to any suitable form of NetB, in particular a toxic form of NetB. Thus, such forms may include full-length NetB, or non-full-length forms of NetB, such as truncated forms or fragments (functional fragments) of NetB, or other variant forms of NetB that, for example, retain their toxicity capabilities. Preferred and convenient forms of NetB to which the binding proteins and antibodies of the invention may bind include recombinant NetB (e.g., a recombinant form of Clostridium perfringens NetB), or a native or natural form of NetB (e.g., a secreted or soluble form of NetB (e.g., produced by Clostridium perfringens)), or a form of NetB present on the surface of a cell (cell surface NetB), for example, in a cell in which NetB has formed polypores.

[0064] Suitable cell types in which NetB can form pores are well known to those skilled in the art and include intestinal cells.

[0065] To assess the binding ability of binding proteins and antibodies, exemplary forms of NetB (e.g., recombinant NetB) that can be used herein are full-length NetB (with or without a signal peptide, conveniently without a signal peptide), preferably full-length NetB corresponding to the (toxic) NetB protein produced by Clostridium perfringens, or an inactive mutant form of NetB, such as a toxoid mutant form, e.g., the W262A mutant form well known and described in the art.

[0066] The sequence of NetB is well known and described in the art and can be obtained, for example, from various sequence databases (eg Uniprot).For ease of reference, the Uniprot number for NetB of Clostridium perfringens is A8ULG6 or D5K6C0 or EU143239.

[0067] Preferred and exemplary NetB molecules have or comprise (or consist of) the sequence EU143239 or SEQ ID NO: 17 (signal peptide removed). Thus, preferred binding proteins or antibodies of the invention bind or are capable of binding (or specifically bind) to SEQ ID NO: 17, or a sequence substantially homologous thereto (e.g., having at least 80% identity thereto), or a fragment thereof, such as a biologically active fragment. Preferred biologically active fragments of NetB have or retain its toxic activity. However, some non-toxic or inactive forms (e.g., mutant or variant forms) of NetB are known in the art, such as toxoid mutant forms of NetB, and in some embodiments, the binding proteins or antibodies of the invention may also bind to these NetB forms. A specific example of an inactive form of NetB is the W262A mutant form (without signal peptide numbering, SEQ ID NO: 18), which can be used to immunize animals, such as in a vaccination regimen.

[0068] Methods of assessing binding (or binding ability) to an appropriate NetB form are well known to those skilled in the art, and any suitable method may be used.

[0069] Convenient and suitable methods for assessing binding include in vitro binding assays (such as ELISA assays) to assess binding of the antibody to an immobilized antigen (such as an immobilized form of NetB as described above, e.g., monomeric NetB, e.g., comprising SEQ ID NO: 17 or a mutant form thereof, e.g., a W262A mutant form). Those skilled in the art will be familiar with ELISA assays and will be able to readily establish appropriate conditions to assess the ability of a binding protein or antibody to bind NetB in such an assay. A particularly preferred ELISA assay (binding ELISA) is described in the Examples section.

[0070] In certain embodiments, the binding protein or antibody of the invention binds NetB (e.g., recombinant NetB or NetB corresponding to NetB of Clostridium perfringens, e.g., NetB having SEQ ID NO: 17), and this binding ability can be determined by a surface plasmon resonance (SPR) assay (e.g., a BIACore assay). Suitable SPR assays are known in the art. In certain preferred SPR assays, a suitable form of NetB (e.g., NetB, e.g., monomeric NetB having SEQ ID NO: 17) or a mutant form thereof (e.g., a W262A mutant form) is captured (or immobilized) on a solid support (e.g., a sensor chip), e.g., by amine coupling (e.g., immobilizing about 350 response units (RU) NetB), and then various concentrations (e.g., serial dilutions, e.g., two-fold or three-fold serial dilutions) of the binding protein or antibody to be tested are injected. The antibody concentration is typically selected within a range that does not saturate the chip and allows the SPR / Biacore software to perform a robust fit. The preferred injection concentrations and flow rates are described in the Examples section.

[0071] Such SPR assays can also be conveniently used to measure the binding kinetics of antibody-antigen interactions, such as determining association rate (ka), dissociation rate (kd) and affinity (KD). In certain embodiments, the measurement can be carried out in a suitable buffer at 25°C, pH 7.4, such as a standard HEPES-EDTA buffer, such as HBS-EP (sold by GE Healthcare LifeSciences, 0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.0005% surfactant P20). The kinetic parameters can be determined or calculated by any suitable model or software, such as by assuming a 1: 1 interaction to fit the sensorgram experimental data, such as using the single-cycle kinetics predefined evaluation method of the Biacore Insight evaluation software. The Examples section herein describes a particularly preferred SPR assay.

[0072] Thus, in particularly preferred embodiments, the binding proteins or antibodies of the invention bind to NetB (e.g., recombinant NetB or NetB corresponding to NetB of Clostridium perfringens, e.g., NetB having SEQ ID NO: 17), and this binding ability can be determined by a surface plasmon resonance (SPR) assay (e.g., a BIACore assay) (as determined in the evaluation). Preferred SPR assays and conditions for such measurements are described elsewhere herein. Exemplary binding affinities are also described elsewhere herein.

[0073] In certain preferred embodiments, when the antibodies of the invention are in VHH configuration, they have a high binding affinity for NetB (e.g., recombinant NetB or NetB corresponding to Clostridium perfringens NetB, e.g., NetB having SEQ ID NO: 17), e.g., when the K is determined in an SPR assay. D When K D (equilibrium dissociation constant) in the range of 1 nM or 500 pM or lower (better).

[0074] Thus, preferably, when the antibody of the invention is in VHH configuration, it has binding affinity for NetB (e.g., recombinant NetB or NetB corresponding to NetB of Clostridium perfringens, e.g., NetB having SEQ ID NO: 17), e.g., when K is determined in an SPR assay. D When the corresponding K D Less than 10 nM, less than 1 nM (or less than 1000 pM), less than 900 pM, less than 800 pM, less than 700 pM, less than 600 pM, less than 500 pM, less than 450 pM, less than 400 pM, less than 350 pM, less than 300 pM, less than 250 pM, less than 225 pM, less than 200 pM, less than 180 pM, less than 160 pM, less than 150 pM, less than 140 pM, less than 130 pM, less than 120 pM, less than 110 pM, less than 100 pM, less than 90 pM, less than 80 pM, less than 70 pM or less than 60 pM. Specific exemplary binding affinities are disclosed in the Examples. For example, at pH 7.4, the Net 83 antibody exemplified by the present invention exhibited a binding affinity of 213 pM, and the Net 14 antibody of the present invention exhibited a binding affinity of 95 pM. Thus, preferred binding proteins or antibodies of the invention comprise an antigen binding domain or at least one antigen binding domain that binds NetB with a KD of 250 pM or less (or less than 250 pM) at pH 7.4. Additional exemplary KDs are provided above. In other words, the binding proteins (e.g., antibodies) of the invention bind NetB with a KD of 250 pM or less at pH 7.4.

[0075] Therefore, in one embodiment, the present invention provides a binding protein (e.g., an antibody) comprising one (or at least one) antigen binding domain that binds to necrotic enteritis B-like toxin (NetB), wherein the antigen binding domain comprises one (or at least one) heavy chain variable region comprising three complementarity determining regions (CDRs), preferably wherein the binding protein (or antigen binding domain) binds to NetB with a KD of 250 pM or less at pH 7.4.

[0076] An exemplary NetB format that can be used to assess such binding affinities is recombinant NetB, such as a recombinant NetB having the sequence of SEQ ID NO: 17. Suitable exemplary formats are described in the Examples section. Thus, when or if these sequences are used to assay the antibodies of the invention, for example in an SPR assay, the above binding affinities can be observed. Preferred SPR assays and conditions (e.g., immobilized NetB concentration, buffer, and antibody concentration) for such measurements are described elsewhere herein, including in the Examples section. An exemplary pH used is pH 7.4.

[0077] For example, as described above, the antibodies (or binding proteins) of the invention are capable of binding to NetB at neutral pH or pH levels found in neutral biological fluids (such as serum or blood). Thus, for example, as described above, the antibodies of the invention are capable of binding to NetB at a pH of or about 7.4.

[0078] Advantageously, for example, as described above, the antibodies (or binding proteins) of the invention are also capable of binding to NetB at low pH or lower, such as the pH levels found in the gastrointestinal tract or small intestine. For example, the pH levels of the gastrointestinal tract or small intestine are believed to range from about 5.7 to 6.5. Thus, for example, as described above, the antibodies of the invention are capable of binding to NetB at a pH of or about 6.0, for example with a binding affinity as described above. This property is advantageous because it means that the antibodies of the invention can continue to bind to the NetB target antigen under conditions found in the gastrointestinal tract or small intestine, which is a key site of action for NetB toxins, such as when such toxins are produced by Clostridium perfringens. This property is also advantageous for oral administration or other routes of administration that deliver the antibodies to the gastrointestinal tract or small intestine.

[0079] In a preferred embodiment of the invention, the antibody (or binding protein) shows similar or improved binding levels at pH 6.0 to pH 7.4.

[0080] In addition to being able to bind NetB, the antibodies (or binding proteins) of the invention can also inhibit (or reduce or neutralize or block) the toxic (or cytotoxic) effect or activity of a NetB toxin (e.g., a NetB toxin produced by Clostridium perfringens). For example, the antibodies (or binding proteins) of the invention can inhibit (or reduce or neutralize or block) the ability of NetB to cause lysis or hemolysis of cells (e.g., suitable target cells, such as red blood cells).

[0081] Preferably, the inhibition or reduction is a measurable inhibition or reduction, more preferably a significant inhibition or reduction, such as a statistically significant inhibition or reduction, such as a probability value ≤ 0.05 or < 0.05. In certain embodiments, the binding protein or antibody of the present invention can inhibit or reduce (or neutralize) the ability of NetB (e.g., NetB produced by Clostridium perfringens) to cause cell hemolysis, and the degree of inhibition or reduction (or neutralization) can reach at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95% or at least 98%. Preferred antibodies of the present invention have the ability to almost completely inhibit NetB toxicity (e.g., NetB-induced hemolysis), for example, at least 90% inhibition can be observed. Typically, this percentage of inhibition (and other percentage inhibition levels as described herein) is compared (or relatively compared) to an appropriate control assay or control level (e.g., a control assay or control level in the absence of a binding protein or antibody (anti-NetB antibody), such as a negative control or background level or assay). Thus, a 0% inhibition (control) level (or conversely, a 100% or maximal lysis level) is generally the level at which no binding protein or antibody (anti-NetB antibody) is present.

[0082] Such ability to inhibit (or reduce or neutralize) the toxic effects or activity of NetB toxins, or the ability to inhibit NetB-induced toxicity (e.g., NetB toxins produced by Clostridium perfringens), etc., can be determined or tested in any suitable assay, examples of which can be readily derived by those skilled in the art. Suitable assays can be, for example, in vitro assays, for example involving the use of suitable NetB toxin target cells, and measuring the effect of the antibodies (or binding proteins) of the invention in inhibiting (or reducing or neutralizing) the toxic effects or activity of NetB toxins on such cells.

[0083] Particularly useful assays may involve measuring the ability of an antibody (or binding protein) of the invention to inhibit (or reduce or neutralize) the hemolytic effect of NetB toxin on a target cell (such as erythrocytes, particularly chicken erythrocytes). Such assays may also be referred to as NetB inhibition assays or erythrocyte lysis assays, such as chicken erythrocyte lysis assays. In such assays, suitable cells (e.g., erythrocytes, such as chicken erythrocytes) may be contacted with NetB at a level that results in hemolysis, such as complete / maximal hemolysis of the cells, or submaximal hemolysis (e.g., 80% lysis or an EC of NetB). 80 The ability of the binding proteins or antibodies of the invention to inhibit or reduce such hemolysis can then be readily analyzed, for example by comparison (or relative comparison) with a 100% hemolysis level set in a control assay. 50 For the avoidance of doubt, the term IC as used herein 50Values ​​refer to the concentration of binding protein or antibody required to inhibit 50% of the toxic / cytotoxic activity of NetB present.A suitable and exemplary hemolytic assay using chicken erythrocytes is described in the Examples section.

[0084] Any suitable concentration of binding protein or antibody can be used to inhibit or reduce toxicity or hemolysis. Exemplary antibodies of the invention have the ability to cause inhibition, e.g., levels of inhibition as outlined herein, when the antibodies, particularly VHH antibodies, are used at a concentration of at least 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0 μg / ml, e.g., up to 1.0, 1.5, 1.8, 2.0, 3.0, 4.0 or 5.0 μg / ml, or between 0.3, 0.4 or 0.5 and 1.0, 1.5, 1.8 or 2.0 μg / ml, or between 0.3, 0.4 or 0.5 and 3.0, 4.0 or 5.0 μg / ml.

[0085] It is noteworthy that the binding proteins or antibodies of the present invention target the NetB toxin itself, rather than the entity that produces NetB, such as an infectious entity such as Clostridium perfringens. This provides an important advantage, namely the ability to inhibit or reduce diseases or symptoms caused by infection with any entity that produces NetB (e.g., NetB as described elsewhere herein) or any form of NetB (or other protein) that can be bound by the antibodies (or binding proteins) of the present invention. In this way, the antibodies of the present invention, etc. can provide methods for treating diseases caused by a variety of Clostridium perfringens strains and other infectious sources that produce NetB or similar toxins, provided that these infectious sources use such toxins to cause disease (in other words, NetB or similar toxins are the causative agent of the disease). Therefore, the antibodies of the present invention, etc. have a wide range of uses and can be used, for example, to treat or prevent any disease in which NetB (or a similar toxin to which the antibodies of the present invention can bind) is a causative agent. The antibodies of the present invention can also be used to treat early stage diseases because the antibodies target the toxic agents produced by Clostridium perfringens rather than Clostridium perfringens itself. Therefore, compared with various previous methods (such as vaccination), the antibodies of the present invention provide important advantages and flexibility.

[0086] In certain embodiments, the antibodies (or binding proteins) of the invention have an IC of 300 nM or less, 280 nM or less, 260 nM or less, 250 nM or less, 240 nM or less, 220 nM or less, 210 nM or less, 200 nM or less, 190 nM or less, 180 nM or less, 170 nM or less, 160 nM or less, 150 nM or less, 140 nM or less, 130 nM or less, 120 nM or less, 110 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 35 nM or less, 30 nM or less, 25 nM or less, or 20 nM or less. 50 (For example, for inhibiting or reducing or neutralizing NetB, such as inhibiting NetB hemolysis of target cells (such as red blood cells, preferably chicken red blood cells)). In some embodiments, IC 50 20nM or 30nM to 300nM, 250nM or 200nM; or 20nM or 30nM to 150nM; or 20nM or 30nM to 140nM; or 20nM or 30nM to 130nM; or 20nM or 30nM to 120nM; or 20nM or 30nM to 110nM; or 20nM or 30nM to 100nM; or 20nM or 30nM to 90nM; or 20nM or 30nM to 80nM; or 20nM or 30nM to 70nM. Specific exemplary IC 50 The values ​​are also shown in the Examples. For example, Net 14 showed an IC as low as 23 nM 50 , and Net 83 showed an IC as low as 61 nM 50 .

[0087] In certain embodiments, having an IC 50 The antibodies (or binding proteins) of the invention (e.g., for inhibiting or reducing or neutralizing NetB, e.g., inhibiting NetB hemolysis of target cells (e.g., erythrocytes, preferably chicken erythrocytes)) are at a ratio of about 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1 (antibody: NetB) when expressed as a molar ratio to the NetB toxin used in the assay. Thus, the antibodies (or binding proteins) of the invention are extremely effective. For example, Net 14 exhibits a molar ratio of about 1:1, and Net 83 exhibits a molar ratio of about 2:1 or 3:1.

[0088] Preferred IC as described above 50 The value is preferably determined in a suitable toxicity assay (e.g. a suitable hemolytic assay) performed under suitable conditions to enable the measurement or determination of the IC 50Values, for example, as described above or as described in the Examples section. These determinations can be performed using any suitable antibody (or binding protein) configuration. Thus, the exemplary values ​​provided above can be, for example, values ​​determined when evaluating a VHH or alternative single domain format of an antibody.

[0089] NetB inhibition assays as described herein can be performed at any suitable pH, such as at a pH as described elsewhere herein, typically at a neutral physiological pH (e.g., pH 7.4), or at a typical pH of the gastrointestinal tract or small intestine, such as pH 5.7 to 6.5, such as pH 6.0. Suitable amounts (doses) of NetB can also be used, and such amounts can be readily determined by one skilled in the art. For example, an amount (dosage) of NetB can be selected that causes a certain amount of lysis (e.g., as determined in a separate NetB lysis titration (e.g., NetB RBC lysis titration)), such as maximal (100%) lysis / toxicity or a different submaximal % lysis / toxicity, such as 80% lysis / toxicity (i.e., EC 80 value or dose).

[0090] The exemplary values ​​provided above may be, for example, 80 Thus, the exemplary values ​​provided above may, for example, be values ​​determined when NetB is used at a concentration of 20 nM and / or when the assay is performed at a pH of 7.4 or 6.0. Thus, the exemplary values ​​provided above may, for example, be values ​​determined when NetB is used at an EC 80 The value determined when the concentration or dose (e.g., a concentration of 20 nM) is used and / or when the measurement is performed at a pH of 7.4 or 6.0. Thus, for example, the binding protein or antibody of the present invention can reduce the toxic activity of NetB at a pH of 6.0. For example, the binding protein or antibody of the present invention can reduce the EC 80 concentration of NetB or 20 nM NetB cytotoxic activity against chicken erythrocytes, where IC 50 The value is less than 150 nM (or 150 nM or lower) at pH 6.0. Other exemplary IC 50 value.

[0091] Compared to the assay at pH 7.4, the preferred antibodies (or binding proteins) of the present invention show equivalent activity when assayed at pH 6.0, but more preferably better activity, such as a lower or decreased IC 50Such antibodies are particularly useful for being effective against NetB under conditions found in the gastrointestinal tract or small intestine, which is beneficial for treating diseases caused by or associated with NetB in that part of the body, such as diseases caused by infection with Clostridium perfringens, particularly diseases caused by NetB produced by Clostridium perfringens, such as NE. For example, Net 83 and Net 14 both showed this improved activity / lower IC 50 value.

[0092] In alternative embodiments of the invention, the binding proteins or antibodies of the invention can be used to reduce the risk of or prevent diseases caused by or associated with NetB in that part of the body, such as diseases caused by infection with Clostridium perfringens, in particular diseases caused by NetB produced by Clostridium perfringens, such as NE.

[0093] The antibodies (or binding proteins) of the present invention advantageously and preferably exhibit the further property of being resistant to proteases, in particular to various proteases found in the gastrointestinal tract and small intestine environments. Not all antibodies that bind NetB will exhibit these properties, and therefore such antibodies are particularly useful for being effective against NetB under conditions found in the gastrointestinal tract or small intestine.

[0094] For example, the antibodies (or binding proteins) of the present invention are resistant to chymotrypsin or resistant to chymotrypsin digestion. As used herein, the term "resistant to chymotrypsin" or "resistant to chymotrypsin digestion" includes that the antibody (or binding protein) does not lose or maintain significant or measurable ability to bind NetB and / or inhibit (or reduce or neutralize) the toxic activity of NetB as described elsewhere herein after exposure to or contact with chymotrypsin. For example, an antibody having such a property is able to inhibit (or reduce or neutralize) the ability of NetB to cause hemolysis of cells (e.g., suitable target cells, such as red blood cells) after contact (or exposure to) chymotrypsin. Such antibodies can also bind NetB (e.g., NetB produced by Clostridium perfringens) after contact (or exposure to) chymotrypsin. From another perspective, such antibodies retain the activity of inhibiting (or reducing or neutralizing) NetB after contact (or exposure to) chymotrypsin. These abilities of the antibodies following exposure to chymotrypsin (or in the presence of chymotrypsin) can be conveniently compared to the levels observed in the absence of chymotrypsin or in the absence of exposure to chymotrypsin.

[0095] Any suitable level or concentration of chymotrypsin can be used. Preferred and exemplary levels should be consistent with levels found in the small intestine or gastrointestinal tract. An exemplary level disclosed herein is a chymotrypsin concentration of 1 μg / ml. Other exemplary exposure conditions are a temperature of 37°C for a suitable period of time, such as for or at least for 30 minutes, such as up to 2 hours or up to 1 hour. An exemplary pH is pH 7.4. Therefore, the preferred antibodies (or binding proteins) of the present invention are resistant to chymotrypsin digestion when the chymotrypsin concentration is 1 μg / ml (e.g., at 37°C). The preferred antibodies of the present invention are resistant to chymotrypsin digestion when the chymotrypsin concentration is 1 μg / ml (e.g., at 37°C) and is exposed to the chymotrypsin for or at least for 30 minutes (e.g., 1 hour). Each of the exemplified antibodies (Net 14 and Net83) exhibits this property.

[0096] In certain embodiments, after exposure to chymotrypsin, the binding proteins or antibodies of the invention can still inhibit or reduce (or neutralize) the ability of NetB (e.g., NetB produced by Clostridium perfringens) to cause cell hemolysis, and the degree of inhibition or reduction (or neutralization) can reach at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95% or at least 98%. Preferred antibodies of the invention have the ability to almost completely inhibit NetB toxicity (e.g., NetB-induced hemolysis) after exposure to chymotrypsin, for example, at least 85% or 90% inhibition can be observed. Alternatively, at least 50%, 60%, 70%, 75% or 80% inhibition can be observed.

[0097] Typically, such percent inhibition (and other percent inhibition levels as described herein) is compared (or relatively compared) to an appropriate control assay or control level (e.g., a control assay or control level in the absence of a binding protein or antibody (anti-NetB antibody), such as a negative control or background level or assay). Thus, a 0% inhibition (control) level (or conversely, a 100% or maximum hemolysis level) is typically the level in the absence of a binding protein or antibody (anti-NetB antibody).

[0098] In addition, after exposure to chymotrypsin (in the presence of chymotrypsin), such percentage inhibition can also be conveniently compared to an equivalent percentage inhibition in the absence of chymotrypsin to assess or determine the resistance or relative resistance of the antibody (or binding protein) to chymotrypsin. Preferred antibodies (or binding proteins) of the invention retain significant or measurable ability to bind to NetB and / or inhibit (or reduce or neutralize) the toxic activity of NetB as described elsewhere herein after exposure to chymotrypsin (e.g., exposure to chymotrypsin as described herein). For example, the ability of such antibodies (or binding proteins) to bind to NetB and / or inhibit (or reduce or neutralize) the toxic activity of NetB is not significantly reduced by exposure to chymotrypsin.

[0099] In certain embodiments, following exposure to chymotrypsin, the antibodies (or binding proteins) of the invention have an IC of 300 nM or less, 280 nM or less, 260 nM or less, 250 nM or less, 240 nM or less, 220 nM or less, 210 nM or less, 200 nM or less, 190 nM or less, 180 nM or less, 170 nM or less, 160 nM or less, 150 nM or less, 140 nM or less, 130 nM or less, 120 nM or less, 110 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 35 nM or less, 30 nM or less, 25 nM or less, or 20 nM or less. 50 (e.g., for inhibiting, reducing or neutralizing NetB, such as inhibiting NetB hemolysis of target cells (e.g., erythrocytes, preferably chicken erythrocytes)). In some embodiments, IC 50 Preferably, after exposure to chymotrypsin, the antibodies (or binding proteins) of the invention have an IC of 300 nM or less, 260 nM or less, 240 nM or less, 220 nM or less, 210 nM or less, 200 nM or less, 190 nM or less, 180 nM or less, 170 nM or less, 160 nM or less, 150 nM or less, 140 nM or less, 130 nM or less, 120 nM or less. 50 Specific exemplary IC 50The values ​​and assay conditions are described elsewhere herein or shown in the Examples. For example, after exposure to 1 μg / ml chymotrypsin for 30 minutes or 1 hour at 37°C, the exemplified antibody Net 14 still exhibited an IC of 114 nM. 50 , and Net 83 still showed an IC of 195nM 50 Thus, in some embodiments, the antibodies (or binding proteins) of the invention have an IC of 220 nM or less after exposure to 1 μg / ml chymotrypsin at 37°C for 30 minutes or 1 hour. 50 In such embodiments, an exemplary pH is 7.4. In such embodiments, an exemplary concentration of NetB is EC 80 concentration or dose, or a concentration of 20 nM. Other exemplary IC 50 Values ​​are described elsewhere in this article.

[0100] Suitable assays for assessing the ability to bind NetB and / or neutralize (or inhibit or reduce) NetB toxic activity are well known in the art, and any of these assays may be used. Preferred and exemplary assays are described elsewhere herein, such as hemolytic assays, or ELISA or SPR binding assays.

[0101] Therefore, the preferred IC as described above 50 The value is preferably determined in a suitable toxicity assay (NetB inhibition assay) which is performed under suitable conditions to be able to measure or determine the IC 50 Values, as described elsewhere herein. Thus, the exemplary values ​​provided above may, for example, be values ​​determined when an antibody (preferably a VHH antibody (or binding protein)) of the invention is exposed to 1 μg / ml chymotrypsin at 37°C for 30 minutes or 1 hour, and then a suitable concentration (toxic concentration) of NetB (e.g. EC 80 The concentration or dose (or concentration) is evaluated in a toxicity assay (NetB inhibition assay), preferably a hemolytic assay, more preferably a hemolytic assay in which, for example, lysis of chicken erythrocytes is evaluated using a NetB concentration of 20 nM. An exemplary pH is pH 7.4. Thus, the exemplary values ​​provided above can be, for example, when NetB is at EC 80 The concentration (eg, a concentration of 20 nM) is the value determined when used and / or when measured at a pH of 7.4.

[0102] Preferred antibodies (or binding proteins) of the invention retain at least 50%, such as at least 55%, 60%, 65%, 70%, 75%, 80% or 85% of their ability to inhibit or neutralize NetB when exposed to chymotrypsin (or in the presence of chymotrypsin) compared to the same antibody (or binding protein) not exposed to chymotrypsin (or in the absence of chymotrypsin). Likewise, these values ​​may be values ​​determined when an antibody (preferably a VHH antibody (or binding protein)) of the invention is exposed to 1 μg / ml chymotrypsin for 1 hour at 37°C and then assessed in a toxicity assay (NetB inhibition assay) using a suitable concentration (toxic concentration) of NetB, preferably a hemolytic assay, more preferably a hemolytic assay using, for example, a NetB concentration of 20 nM to assess the lysis of chicken erythrocytes. Specific exemplary retention percentage values ​​and assay conditions are also shown in the Examples. For example, the exemplified antibody Net 14 retained 66% of its activity after exposure to chymotrypsin, and Net 83 retained 80% of its activity after exposure to chymotrypsin. Thus, in some embodiments, antibodies (binding proteins) having these or at least these retention percentage values ​​under these conditions are preferred.

[0103] In some embodiments, the antibodies (or binding proteins) of the present invention are resistant to pepsin or resistant to pepsin digestion. As used herein, the term "resistant to pepsin" or "resistant to pepsin digestion" includes that the antibody (or binding protein) does not lose or maintain significant or measurable ability to bind NetB and / or inhibit (or reduce or neutralize) the toxic activity of NetB as described elsewhere herein after exposure to or contact with pepsin. For example, an antibody having such a property is able to inhibit (or reduce or neutralize) the ability of NetB to cause hemolysis of cells (e.g., suitable target cells, such as red blood cells) after contact (or exposure to) pepsin. Such antibodies are also able to bind NetB (e.g., NetB produced by Clostridium perfringens) after contact (or exposure to) pepsin. From another perspective, such antibodies retain the activity of inhibiting (or reducing or neutralizing) NetB after contact (or exposure to) pepsin. These abilities of the antibody after exposure to pepsin (or in the presence of pepsin) can be conveniently compared to the levels observed in the absence of pepsin or when not exposed to pepsin.

[0104] Any suitable level or concentration of pepsin can be used.Preferred and exemplary levels should be consistent with the levels found in the small intestine or gastrointestinal tract.Exemplary levels disclosed herein are 30U / ml (corresponding to 30μg / ml or about 30μg / ml) pepsin concentrations.However, studies have also used at least 100U / ml (corresponding to 100μg / ml or at least about 100μg / ml) higher levels, particularly 107U / ml concentrations (corresponding to 107μg / ml or at least about 107μg / ml).Other exemplary exposure conditions are that the temperature is 37°C and continues for a suitable time period, for example, continues or at least continues for 5 minutes, 15 minutes, 30 minutes or 45 minutes, and for example, up to 2 hours, up to 1.5 hours or up to 1 hour. Since pepsin is only active at acidic pH (e.g., pepsin is inactive at neutral physiological pH (such as pH 7.4)), another exemplary exposure condition is acidic pH, particularly a pH of 1.8 or 2.5, or a pH therebetween, or a pH below 3.0 or below 2.0 is suitable.

[0105] Thus, preferred antibodies of the invention are resistant to digestion with pepsin at a pepsin concentration of 30 U / ml or 107 U / ml (e.g., at 37°C). Preferred antibodies of the invention are resistant to digestion with pepsin at a pepsin concentration of 30 U / ml (at 37°C) when exposed to the pepsin for or at least for 15 minutes, 30 minutes, 45 minutes, 60 minutes, or 90 minutes. Each of the exemplified antibodies (Net 14 and Net 83) exhibits this property. For example, Net 14 and Net 83 both exhibit resistance to pepsin when exposed to 30 U / ml pepsin for 1 hour, and Net 83 exhibits resistance to pepsin when exposed for 1.5 hours (90 minutes). Both antibodies also exhibit resistance to pepsin when exposed to 107 U / ml pepsin for 15 minutes or 30 minutes. As described above, this resistance is observed at acidic pH, for example, a pH below 3.0 or 2.0, such as a pH of 1.8 or 2.5.

[0106] Thus, such antibodies retain the ability to reduce the toxic (cytotoxic) activity of NetB after exposure to pepsin under these conditions or other conditions as described herein. Alternatively or additionally, such antibodies retain the ability to bind NetB after exposure to pepsin under these conditions or other conditions as described herein.

[0107] Thus, it is also preferred that the antibody (or binding protein) has a detectable or measurable activity, e.g., binding to NetB or inhibiting NetB, after exposure to pepsin (e.g., exposure to pepsin as described herein, e.g., exposure to 30 U / ml or at least 100 U / ml pepsin, e.g., 107 U / ml pepsin, for 15, 30, 45, 60, or 90 minutes, e.g., at 37°C). More preferably, the antibody (or binding protein) has significant NetB binding or inhibition activity after such exposure, e.g., the activity or binding is not significantly reduced compared to the activity observed when no pepsin exposure occurs.

[0108] In certain embodiments, after exposure to pepsin, the binding proteins or antibodies of the invention can still inhibit or reduce (or neutralize) the ability of NetB (e.g., NetB produced by Clostridium perfringens) to cause cell hemolysis, and the degree of inhibition or reduction (or neutralization) can reach at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95% or at least 98%. Preferred antibodies of the invention have the ability to almost completely inhibit NetB toxicity (e.g., NetB-induced hemolysis) after exposure to pepsin, for example, at least 70%, 75%, 80%, 85%, 90% or 95% inhibition can be observed. Alternatively, at least 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% inhibition can be observed.

[0109] Typically, such percent inhibition (and other percent inhibition levels as described herein) is compared (or relatively compared) to an appropriate control assay or control level (e.g., a control assay or control level in the absence of a binding protein or antibody (anti-NetB antibody), such as a negative control or background level or assay). Thus, a 0% inhibition (control) level (or conversely, a 100% or maximum hemolysis level) is typically the level in the absence of a binding protein or antibody (anti-NetB antibody).

[0110] In addition, after exposure to pepsin (in the presence of pepsin), such percentage inhibition can also be conveniently compared to an equivalent percentage inhibition in the absence of pepsin to assess or determine the resistance or relative resistance of the antibody (or binding protein) to pepsin. Preferred antibodies (or binding proteins) of the present invention retain significant or measurable ability to bind to NetB and / or inhibit (or reduce or neutralize) the toxic activity of NetB as described elsewhere herein after exposure to pepsin. For example, the ability of such antibodies (or binding proteins) to bind to NetB and / or inhibit (or reduce or neutralize) the toxic activity of NetB is not significantly reduced by exposure to pepsin.

[0111] In certain embodiments, after exposure to pepsin, the antibodies (or binding proteins) of the invention have a pepsin concentration of 400 nM or less, 350 nM or less, 300 nM or less, 280 nM or less, 260 nM or less, 250 nM or less, 240 nM or less, 220 nM or less, 210 nM or less, 200 nM or less, 190 nM or less, 180 nM or less, 170 nM or less. The IC values ​​are as follows: 1) 100 nM or less, 2) 150 nM or less, 3) 140 nM or less, 3) 130 nM or less, 3) 120 nM or less, 4) 110 nM or less, 5) 100 nM or less, 6) 150 nM or less, 7) 140 nM or less, 8) 130 nM or less, 8) 120 nM or less, 9) 110 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 35 nM or less, 30 nM or less, 25 nM or less, or 20 nM or less. 50 (e.g., for inhibiting, reducing or neutralizing NetB, such as inhibiting NetB hemolysis of target cells (e.g., erythrocytes, preferably chicken erythrocytes)). In some embodiments, IC 50 Preferably, after exposure to pepsin, the antibodies (or binding proteins) of the invention have an IC of 400 nM or less, 350 nM or less, 300 nM or less, 260 nM or less, 240 nM or less, 220 nM or less, 210 nM or less, 200 nM or less, 190 nM or less, 180 nM or less, 170 nM or less, 160 nM or less, 150 nM or less, 140 nM or less, 130 nM or less, 120 nM or less. 50 Specific exemplary IC 50 Values ​​and assay conditions are described elsewhere herein or shown in the Examples. For example, after exposure to 30 U / ml pepsin at 37°C for 30 minutes, the exemplified antibody Net 14 still exhibited an IC of 113 nM. 50 , and Net 83 still showed an IC of 208nM 50 Thus, in some embodiments, the antibodies (or binding proteins) of the invention have an IC of 220 nM or less after exposure to 30 U / ml pepsin at 37° C. for 30 minutes. 50 In such embodiments, an exemplary pH is 1.8 or 2.5. In such embodiments, an exemplary concentration of NetB is EC 80 concentration or dose, or a concentration of 20 nM. Other exemplary IC 50 Values ​​are described elsewhere in this article.

[0112] Suitable assays for assessing the ability to bind NetB and / or neutralize (or inhibit or reduce) NetB toxic activity are well known in the art, and any of these assays may be used. Preferred and exemplary assays are described elsewhere herein, such as hemolytic assays, or ELISA or SPR binding assays.

[0113] Therefore, the preferred IC as described above 50 The value is preferably determined in a suitable toxicity assay (NetB inhibition assay) which is performed under suitable conditions to be able to measure or determine the IC 50 Values, as described elsewhere herein. Thus, the exemplary values ​​provided above may, for example, be values ​​determined when an antibody (preferably a VHH antibody (or binding protein)) of the invention is exposed to 30 U / ml or 107 U / ml pepsin at 37°C for or at least for 15 minutes, 30 minutes, 45 minutes, 60 minutes or 90 minutes, and then a suitable concentration (toxic concentration) of NetB (e.g. EC 80 The concentration or dose (or concentration) is evaluated in a toxicity assay (NetB inhibition assay), preferably a hemolytic assay, more preferably a hemolytic assay in which, for example, lysis of chicken erythrocytes is evaluated using a NetB concentration of 20 nM. Exemplary pH values ​​are pH 1.8 or pH 2.5. Thus, the exemplary values ​​provided above may be, for example, when NetB is at EC 80 The value is determined when the concentration (eg, a concentration of 20 nM) is used and / or when the measurement is performed at a pH of 1.8 or 2.5.

[0114] Preferred antibodies (or binding proteins) of the invention retain at least 50%, such as at least 55%, 60%, 65%, 70%, 75%, 80% or 85% of the ability to inhibit or neutralize NetB when exposed to pepsin (or in the presence of pepsin) compared to the same antibody (or binding protein) not exposed to pepsin (or in the absence of pepsin). Likewise, such values ​​may be values ​​determined when an antibody (preferably a VHH antibody (or binding protein)) of the invention is exposed to 30 U / ml or 107 U / ml pepsin for or at least for 15 minutes, 30 minutes, 45 minutes, 60 minutes or 90 minutes at 37°C and then assessed in a toxicity assay (NetB inhibition assay) using a suitable concentration (toxic concentration) of NetB, preferably a hemolytic assay, more preferably a hemolytic assay using, for example, a NetB concentration of 20 nM to assess the lysis of chicken erythrocytes. Exemplary pH is pH 1.8 or pH 2.5. Specific exemplary retention percentage values ​​and assay conditions are also shown in the Examples. For example, the exemplified antibody Net 14 retains 84% ​​of its activity after exposure to 30 U / ml pepsin at pH 1.8 for 30 minutes, and retains 45% of its activity after exposure for 45 minutes or 60 minutes, while Net 83 retains 72% of its activity after exposure to 30 U / ml pepsin for 30 minutes, and retains 35% of its activity after exposure for 45 minutes, 60 minutes or 90 minutes. Therefore, in some embodiments, antibodies (or binding proteins) having these or at least these retention percentage values ​​under these conditions are preferred.

[0115] In some embodiments, the antibodies (or binding proteins) of the present invention are resistant to pancreatic enzymes or resistant to pancreatic digestion. As used herein, the term "resistant to pancreatic enzymes" or "resistant to pancreatic digestion" includes that the antibody (or binding protein) does not lose or maintain significant or measurable ability to bind NetB and / or inhibit (or reduce or neutralize) the toxic activity of NetB as described elsewhere herein after exposure to or contact with pancreatic enzymes. For example, an antibody having such a property is able to inhibit (or reduce or neutralize) the ability of NetB to cause hemolysis of cells (e.g., suitable target cells, such as red blood cells) after contact (or exposure to) pancreatic enzymes. Such antibodies can also bind NetB (e.g., NetB produced by Clostridium perfringens) after contact (or exposure to) pancreatic enzymes. From another perspective, such antibodies retain the activity of inhibiting (or reducing or neutralizing) NetB after contact (or exposure to) pancreatic enzymes. These abilities of the antibody after exposure to pancreatic enzymes (or in the presence of pancreatic enzymes) can be conveniently compared to the levels observed in the absence of pancreatic enzymes or when not exposed to pancreatic enzymes.

[0116] Any suitable level or concentration of pancreatic enzyme can be used.Preferred and exemplary levels should be consistent with the levels found in the small intestine or gastrointestinal tract.Exemplary levels disclosed herein are 0.5 mg / ml concentrations, because this is associated with >100 U / ml protease activity, and are common values ​​for simulated intestinal fluid / intestinal digestion.Concentrations of 1 mg / ml or 0.1 mg / ml can also be used.Other exemplary exposure conditions are that the temperature is 37 ° C and continues for a suitable time period, such as continuing or at least continuing for 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes or 120 minutes, and for example up to 2.5 hours, up to 2 hours, up to 1.5 hours or up to 1 hour.Exemplary pH is or about pH 6.8.

[0117] Thus, preferred antibodies (or binding proteins) of the invention are resistant to digestion with trypsin at a concentration of 0.5 mg / ml (e.g., at a temperature of 37°C). Preferred antibodies (or binding proteins) of the invention are resistant to digestion with trypsin at a concentration of 0.5 mg / ml or 1 mg / ml of trypsin when exposed to the trypsin for a period of or at least 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, or 120 minutes at 37°C. For example, Net 83 exhibits resistance to trypsin when exposed to 0.5 mg / ml trypsin for 15 minutes, 30 minutes, 45 minutes, 60 minutes, or 90 minutes, and still exhibits some resistance at 120 minutes. Net 83 also exhibits resistance to trypsin when exposed to 1 mg / ml trypsin for 30 minutes. This resistance is conveniently observed at a pH of or about pH 6.8.

[0118] Such antibodies preferably retain the ability to reduce the toxic (cytotoxic) activity of NetB after exposure to pancreatic enzymes under these conditions or other conditions as described herein. Alternatively or additionally, such antibodies retain the ability to bind NetB after exposure to pancreatic enzymes under these conditions or other conditions as described herein.

[0119] Suitable assays for assessing the ability to bind NetB and / or neutralize (or inhibit or reduce) NetB toxic activity are well known in the art, and any of these assays may be used. Preferred and exemplary assays are described elsewhere herein, such as hemolytic assays, or ELISA or SPR binding assays.

[0120] Although the hemolysis-based NetB inhibition assay can be conveniently used to assess the activity of the antibodies of the invention after exposure to proteases, it is also convenient to assess protease resistance on a gel (e.g., an SDS-PAGE gel) and make the following assessment: whether the antibody protein has been significantly or completely digested, or whether undigested antibody protein is still present. Such an assessment can be conveniently made by detecting a band of the appropriate molecular weight on the gel.

[0121] In some embodiments, the antibodies (or binding proteins) of the invention advantageously exhibit the further property of showing stability at low pH (eg, a pH below 3.0 or below 2.0, such as a pH of 1.8 or 2.5).

[0122] Thus, preferred antibodies of the invention are also stable, e.g., are not degraded or destroyed, or retain their functional activity, e.g., one or more of the functional properties described herein, such as the ability to bind NetB, or the ability to reduce the toxic (cytotoxic) activity of NetB, or other functional properties, after exposure to (or contact or incubation at) an acidic pH (e.g., a pH below 3.0 or below 2.0, e.g., a pH of 1.8 or 2.5).

[0123] Thus, such antibodies retain the ability to reduce the toxic (cytotoxic) activity of NetB after exposure to such low pH.Alternatively or additionally, such antibodies retain the ability to bind NetB after exposure to such low pH.

[0124] Suitable assays are described elsewhere herein for assessing the retention of these functional activities after the antibody (or binding protein) has been exposed to low pH. Exemplary exposure, contact or incubation times under the above low pH conditions may be exposure for 15, 30, 45, 60, 90 or 120 minutes, or at least 15, 30, 45, 60, 90 or 120 minutes, or up to 15, 30, 45, 60, 90 or 120 minutes, etc. An exemplary temperature may be 37°C.

[0125] In some embodiments, the antibodies (or binding proteins) of the invention advantageously exhibit the further property of being stable in water, particularly in tap water. In some areas of animal health, such as when a relatively large number of animals need to be treated, this property is particularly advantageous, as it provides the possibility of administering or delivering the active agent in drinking water or as a component of feed provided to the animals. Therefore, this property is highly desirable for the use of the antibodies of the invention in the treatment of NetB-related diseases (such as NE) in avian animals.

[0126] In some embodiments, the antibodies (or binding proteins) of the present invention have a stability of at least 4 days or 7 days in water (particularly tap water), for example at least or up to 14 days, 21 days or 28 days, or at least or up to 2 months or 3 months. These time points refer to the stability at room temperature.

[0127] Preferably, the above abilities and properties are observed at a measurable or significant level, more preferably at a statistically significant level, when compared to an appropriate control level. Suitable significant levels are discussed elsewhere herein. More preferably, one or more of the above abilities and properties are observed at a level that is measurably better or more preferably significantly better (preferably statistically significantly better) when compared to the abilities observed with prior art antibodies.

[0128] In any statistical analysis mentioned herein, preferably, a statistically significant difference relative to a relevant control or other comparative entity or measurement has a probability value of ≤0.1 or <0.1, preferably ≤0.05 or <0.05. Suitable methods for determining statistical significance are well known and documented in the art, and any of these methods may be used.

[0129] In some embodiments, the binding proteins or antibodies of the present invention have one or more, preferably two or more, or three or more, or four or more, or five or more, most preferably all, functional properties described herein, particularly the preferred functional properties. Examples of preferred functional properties and further details about said properties are described elsewhere herein, and include i) high affinity for NetB, e.g., when measured by Biacore (the ability to bind NetB can be observed at both pH 6.0 and pH 7.4), ii) the ability to reduce the toxic activity of NetB (this ability can be observed at both pH 6.0 and pH 7.4, and preferably at both 42°C and 37°C); resistance to various proteases, e.g., iii) resistance to digestion with pepsin, (e.g., at a pepsin concentration of 30 U / ml at a temperature of 37°C), iv) resistance to digestion with chymotrypsin (e.g., at a chymotrypsin concentration of 1 μg / ml at a temperature of 37°C), and optionally v) resistance to digestion with pancreatin (e.g., at a pancreatin concentration of 0.5 mg / ml at a temperature of 37°C). Preferred antibodies (or binding proteins) also exhibit stability in water.

[0130] As used throughout this application, the terms "a" and "an" mean that they refer to "at least one," "at least a first," "one or more," or "a plurality" of the referenced components or steps, unless an upper limit is specifically stated thereafter. Thus, an "antibody" as used herein refers to "at least a first antibody."

[0131] In addition, when the terms "comprising," "including," "having," or "with," or other equivalent terms are used herein, in some more specific embodiments, such as in the definition of CDR or FR sequences herein, these terms include the terms "consisting of," or "consisting essentially of," or other equivalent terms.

[0132] Nucleic acid molecules (e.g., one or more nucleic acid molecules) comprising a nucleotide sequence encoding a binding protein or antibody of the invention as defined herein, or a part or fragment thereof, or nucleic acid molecules substantially homologous to these nucleic acid molecules, constitute further aspects of the invention.

[0133] Preferred nucleic acid molecules are nucleic acid molecules encoding VHH antibodies or VH regions or domains of the invention (e.g., nucleic acid molecules encoding SEQ ID NO: 1 or SEQ ID NO: 9). Other preferred nucleic acid molecules are nucleic acid molecules encoding three CDR sequence groups defined in any one of Tables A or B. Preferred such nucleic acid molecules also encode suitable framework regions, such as FR1, FR2, FR3 and FR4 regions, preferably the FR sequence groups defined in any one of Tables A or B.

[0134] The term "substantially homologous" as used herein in relation to an amino acid or nucleic acid sequence includes sequences having at least 60%, 65%, 70% or 75%, preferably at least 80%, and even more preferably at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the disclosed amino acid or nucleic acid sequence.

[0135] Therefore, the substantially homologous sequences of the present invention include changes (additions, substitutions, insertions or deletions) to one or more bases or amino acids of the sequences of the present invention. At the amino acid level, preferred substantially homologous sequences contain up to 5, such as only 1, 2, 3, 4 or 5, preferably 1, 2, 3 or 4, preferably 1, 2 or 3, more preferably 1 or 2 changed amino acids in one or more framework regions and / or one or more CDRs in the framework regions constituting the sequences of the present invention. In addition, at the amino acid level, preferred substantially homologous sequences contain up to 5, such as only 1, 2, 3, 4 or 5, preferably 1, 2, 3 or 4, preferably 1, 2 or 3, more preferably 1 or 2 changed amino acids in the combined framework regions (e.g., four framework regions) and / or combined CDRs (e.g., three CDR regions) constituting the sequences of the present invention. Furthermore, at the amino acid level, preferred substantially homologous sequences contain up to 5, such as only 1, 2, 3, 4 or 5, preferably 1, 2, 3 or 4, preferably 1, 2 or 3, more preferably 1 or 2 altered amino acids in one or more of the VHH domains of the invention. The alterations may be conservative amino acids or non-conservative amino acids, or a mixture of both. Preferably, the alterations are substitutions, preferably conservative amino acid substitutions.

[0136] In certain embodiments, if a given starting sequence is relatively short (e.g., five amino acids in length), fewer amino acid substitutions may be present in a sequence substantially homologous to the shorter starting sequence than in a longer starting sequence, because more amino acid substitutions may optionally be made in a sequence substantially homologous to the longer starting sequence. For example, in certain embodiments, a sequence substantially homologous to a starting VH CDR1 sequence according to the present invention (e.g., a starting VH CDR1 sequence that may be five amino acid residues in length, in some embodiments) preferably has 1 or 2 (more preferably 1) altered amino acids compared to the starting sequence. Thus, in some embodiments, the number of altered amino acids in a substantially homologous sequence (e.g., a substantially homologous CDR sequence) may be adjusted according to the length of a given starting CDR sequence. For example, depending on the length of a given starting CDR sequence, there may be different numbers of altered amino acids in order to achieve a certain percentage of sequence identity in the CDR, such as at least 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0137] Routine methods in the art (such as alanine scanning mutagenesis and / or deep mutation scanning (aiming to perform all possible single substitutions on all selected residues in a given protein sequence), and / or crystal structure analysis of antigen-antibody complexes) can be used to determine which amino acid residues in the CDRs do not contribute or contribute insignificantly to antigen binding and are therefore good candidates for modification or substitution in embodiments of the present invention involving substantially homologous sequences.

[0138] Once identified, one or more amino acids can be added, deleted, substituted or inserted into the amino acid sequence of a parent antibody using routine techniques in the art to form new antibodies, wherein the parent antibody is one of the antibodies of the invention as defined elsewhere herein, and the resulting new antibodies are tested to identify antibodies that bind to NetB according to the invention. Such methods can be used to form a plurality of new antibodies, and all of these antibodies can be tested for their ability to bind NetB. Preferably, said addition, deletion, substitution or insertion of one or more amino acids occurs in one or more of the CDR domains.

[0139] For example, such manipulations may be conveniently accomplished by genetic engineering at the nucleic acid level, wherein nucleic acid molecules encoding suitable binding proteins and domains thereof are modified, thereby modifying the amino acid sequence of the resulting expressed protein in a suitable manner. The ability of one or more of the modified antibodies / binding proteins to bind NetB may be tested by any suitable method, which methods are well known and described in the art. Suitable methods are also described elsewhere herein and in the Examples section.

[0140] New antibodies produced, obtained or obtainable by these methods form a further aspect of the invention.

[0141] The term "substantially homologous" also includes modifications or chemical equivalents of the amino acid and nucleotide sequences of the invention that perform substantially the same function as the protein or nucleic acid molecules of the invention in substantially the same manner. For example, any substantially homologous antibody should retain the ability to bind NetB as described above. Preferably, any substantially homologous antibody should retain one or more (or all) functional capabilities of the starting antibody.

[0142] Substantially homologous sequences of the proteins of the present invention include, but are not limited to, conservative amino acid substitutions; or, for example, changes that do not affect the VH, VL or CDR domains of the antibody (e.g., antibodies to which tag sequences, toxins or other components are added), which do not contribute to antigen binding; or the following changes that convert one type or configuration of binding protein, antibody molecule or fragment into another type or configuration of binding protein, antibody molecule or fragment (e.g., from VHH to Fab or scFv or a complete antibody, such as an antibody with only a full-length heavy chain, and vice versa); or convert an antibody molecule into an antibody molecule of a specific class or subclass (e.g., converting an antibody molecule into IgG or its subclass, such as IgG 2 or IgG 3 , for example, into camelid antibodies, or into IgA or IgY class antibodies); or to prepare Fc fusions, such as VHH-Fc fusions.

[0143] As used herein, "conservative amino acid substitution" refers to the replacement of an amino acid residue by another amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In other examples, families of amino acid residues can be grouped based on hydrophobic side groups or hydrophilic side groups.

[0144] Homology or sequence identity can be assessed by any convenient method. However, in order to determine the homology or degree of identity between sequences, it is useful to carry out a computer program for multiple alignment of sequences, such as Clustal W (Thompson, Higgins, Gibson, Nucleic Acids Res., 22: 4673-4680, 1994). If necessary, the Clustal W algorithm can be used together with the BLOSUM 62 scoring matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 89: 10915–10919, 1992), and a gap opening penalty of 10 and a gap extension penalty of 0.1 are set to obtain the highest order match between the two sequences, wherein at least 50% of the total length of one of the two sequences is involved in the alignment. Other methods can be used to align sequences, such as the alignment method of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 48:443, 1970) as revised by Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 2:482, 1981), to obtain the highest order match between the two sequences and determine the number of identical amino acids between the two sequences. Other methods for calculating the percent identity between two amino acid sequences are generally recognized in the art and include, for example, the method described by Carillo and Lipton (Carillo and Lipton, SIAM J. Applied Math., 48:1073, 1988) and in Computational Molecular Biology, Lesk, ed., Oxford University Press, New York, 1988, "Biocomputing: Informatics and Genomics Projects".

[0145] Typically, such calculations are performed using a computer program. Programs for comparing and aligning sequence pairs, such as ALIGN (Myers and Miller, CABIOS, 4: 11-17, 1988), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85: 2444-2448, 1988; Pearson, Methods in Enzymology, 183: 63-98, 1990), and GAP BLAST (Altschul et al., Nucleic Acids Res., 25: 3389-3402, 1997), BLASTP, BLASTN or GCG (Devereux, Haeberli, Smithies, Nucleic Acids Res., 12: 387, 1984) can also be used for this purpose. In addition, the Dali server of the European Bioinformatics Institute provides structure-based protein sequence alignments (Holm, Trends in Biochemical Sciences, 20:478-480, 1995; Holm, J. Mol. Biol., 233:123-38, 1993; Holm, Nucleic Acid Res., 26:316-9, 1998).

[0146] By providing a reference point, sequences of the invention having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, sequence identity, etc. can be determined using the ALIGN program with default parameters (e.g., available on the Internet at the GENESTREAM web server, IGH, Montpellier, France).

[0147] Preferably, any substantially homologous antibody should retain the ability to specifically bind to a NetB epitope that is identical to the NetB epitope recognized by the starting antibody in question (e.g., the same epitope recognized by the CDR domains of one or more of the antibodies of the invention as described herein or the VH (VHH) domains of the invention); for example, binding to the same epitope as the epitope recognized by one or more of the various antibodies of the invention (e.g., one or more of the VHH antibodies Net 83 or Net 14, as shown in Tables A and B, respectively). Thus, preferably, any substantially homologous antibody should retain the ability to compete with one or more of the various antibodies of the invention (e.g., one or more of the VHH antibodies Net 83 or Net 14, as shown in Tables A and B, respectively) for binding to NetB in a suitable assay.

[0148] Binding to the same epitope can be easily tested by methods well known and described in the art, such as using binding assays (e.g., competitive assays), or by analyzing the crystal structure of antigen-antibody complexes. Therefore, antibodies that bind to the same epitope as one or more of the various antibodies of the present invention (e.g., assessed or determined by competitive assays, crystal structure analysis of antigen-antibody complexes, or by mutation studies of each residue (e.g., using alanine scanning and / or deep mutation scanning)) constitute further aspects of the present invention. The retention of other functional properties can also be easily tested by methods well known and described in the art or herein.

[0149] Thus, one skilled in the art will appreciate that binding assays (e.g., binding assays as described elsewhere herein, such as competition assays or ELISA assays) can be used to test whether any antibody (e.g., a "substantially homologous" antibody) has the same binding specificity (e.g., binds to the same epitope) as the antibodies and antibody fragments of the invention, or the same or equivalent affinity. SPR assays (such as BIAcore assays as described elsewhere herein) can also be readily used to determine whether an antibody (e.g., a "substantially homologous" antibody) is able to bind NetB, and optionally determine the affinity of such binding, such as KD. One skilled in the art will appreciate other suitable methods and variations.

[0150] As described below, competitive binding assays can be used to test whether an antibody (e.g., a "substantially homologous" antibody) retains the ability to specifically bind to a NetB epitope that is identical or substantially identical to a NetB epitope recognized by one or more of the antibodies of the invention as shown in the various sequence listings herein; or whether it has the ability to compete with one or more of the various antibodies of the invention as shown in the various sequence listings herein. The method described below is only one example of a suitable competition assay. Other suitable methods and variations will be apparent to those skilled in the art.

[0151] An exemplary competition assay involves evaluating various effective concentrations of an antibody of the invention for binding to NetB in the presence of varying concentrations of a test antibody (e.g., a substantially homologous antibody). The amount of binding inhibition induced by the test antibody can then be evaluated. A test antibody that shows increased competition with an antibody of the invention at increasing concentrations (i.e., increasing concentrations of the test antibody cause a corresponding decrease in the amount of the antibody of the invention that binds NetB) demonstrates that the test antibody binds to the same or substantially the same epitope as the antibody of the invention. Preferably, the test antibody significantly reduces the amount of the antibody of the invention that binds NetB. Preferably, the test antibody reduces the amount of the antibody of the invention that binds NetB by at least about 95%. ELISA or flow cytometry assays can be used to evaluate binding inhibition in such competition assays, but other suitable techniques are also well known to those of skill in the art.

[0152] Such antibodies (monoclonal antibodies) have the ability to specifically bind to a NetB epitope that is substantially identical (or identical) to or overlaps with the NetB epitope recognized by an antibody of the present invention (e.g., one or more of the VHH antibodies Net 83 or Net 14, as shown in Tables A and B, respectively); or have the ability to compete with one or more of the various antibodies of the present invention (e.g., one or more of the VHH antibodies Net 83 or Net 14, as shown in Tables A and B, respectively), and such antibodies are further embodiments of the present invention.

[0153] As used herein, the term "competing antibody" refers to an antibody that binds to an epitope that is about the same, substantially the same, essentially the same, or even identical to a "reference antibody". A "competing antibody" includes antibodies with overlapping epitope specificities. Thus, a competing antibody is able to effectively compete with a reference antibody for binding to NetB. Preferably, a competing antibody can bind to the same epitope as a reference antibody. From another perspective, a competing antibody preferably has the same epitope specificity as a reference antibody.

[0154] A "reference antibody" as used herein is an antibody that can bind to NetB according to the present invention, which preferably has a VH domain as defined herein, more preferably has a VH domain, or is a VHH antibody comprising SEQ ID NO: 1 or SEQ ID NO: 9 (or the relevant three CDR sequences of said sequences) listed in Table A or Table B.

[0155] Now that a reference antibody (such as an antibody listed in the sequence listing herein) has been provided, it is a simple technical matter to identify one or more competing antibodies or antibodies that bind to the same epitope. Because the identification of competing antibodies or antibodies that bind to the same epitope can be determined by comparison with the reference antibody, it should be understood that it is not actually necessary to determine the specific epitope to which either or both antibodies bind in order to identify competing antibodies or antibodies that bind to the same epitope. However, if desired, epitope mapping can be performed using standard techniques, some of which are summarized elsewhere herein.

[0156] As used herein, the terms "antibody" and "immunoglobulin" refer to any immunobinder comprising an antigen binding domain, including polyclonal antibodies and monoclonal antibodies. However, monoclonal antibodies are preferred. In other words, in some embodiments, the antibodies of the present invention are not polyclonal antibodies. According to the type of constant domain in the heavy chain, complete antibodies are divided into one of five major categories: IgA, IgD, IgE, IgG and IgM, and the antibodies of the present invention may belong to any of these categories, or may be chicken IgY antibodies. In some embodiments, IgY antibodies are not used. Several of these categories are further divided into subclasses or isotypes, such as IgG1, IgG2, IgG3, IgG4, etc., for example, camelid antibodies are IgG antibodies, which generally have IgG2 or IgG3 constant domains. The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ and μ, respectively. The subunit structure and three-dimensional configuration of different classes of immunoglobulins are well known.

[0157] Generally, when whole antibodies are used in the present invention rather than antigen binding regions, IgG is preferred because it is the most common antibody under physiological conditions and is the easiest to produce in a laboratory setting.

[0158] The "light chains" of mammalian antibodies are assigned to one of two clearly distinct types: kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains and a few amino acids in the framework regions of their variable domains.

[0159] As used herein, the term "heavy chain complementarity determining region" ("heavy chain CDR") refers to the variable region of the heavy chain (V H The heavy chain variable region has three CDRs from the amino terminus to the carboxyl terminus, which are called heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3. The heavy chain variable region also has four framework regions (FR1, FR2, FR3 and FR4 from the amino terminus to the carboxyl terminus). These framework regions separate the CDRs.

[0160] As used herein, the term "heavy chain variable region" (V HThe term "domain" refers to the variable region of the heavy chain of an antibody molecule.

[0161] As used herein, the term "light chain complementarity determining region" ("light chain CDR") refers to the variable region of the light chain of an antibody molecule (V L The light chain variable region has three CDRs from the amino terminus to the carboxyl terminus, called light chain CDR1, light chain CDR2, and light chain CDR3. The light chain variable region also has four framework regions (FR1, FR2, FR3, and FR4 from the amino terminus to the carboxyl terminus). These framework regions separate the CDRs.

[0162] As used herein, the term "light chain variable region" (V L The variable domain refers to the light chain variable region of an antibody molecule.

[0163] As will be understood by those skilled in the art, the immunobinders encompassed by the term "antibody" include or extend to all antibodies and antigen-binding fragments thereof, including whole antibodies, dimers, trimers and multimers; bispecific antibodies; chimeric antibodies; recombinant and engineered antibodies and fragments thereof.

[0164] Thus, the term "antibody" is used to refer to any antibody-like molecule having an antigen binding region, and the term includes antibody fragments that contain the antigen binding domain, such as Fab', Fab, F(ab'), 2 , single domain antibody (DAB), TandAb dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibody, mini antibody, diabody, bispecific antibody fragment, bibody, tribody (scFv-Fab fusion, bispecific or trispecific, respectively); sc-diabody; kappa (lambda) body (scFv-CL fusion); BiTE (bispecific T cell engager, scFv-scFv tandem to attract T cells); DVD-Ig (dual variable domain antibody, bispecific configuration); SIP (small immune protein, a mini antibody); SMIP ("small modular immunopharmaceutical" scFv-Fc dimer; DART (ds-stable diabody "dual affinity retargeting"); small antibody mimics containing one or more CDRs, etc.

[0165] The techniques for making and using various antibody-based constructs and fragments are well known in the art.

[0166] Antibodies can be fragmented using conventional techniques. For example, F(ab') can be generated by treating an antibody with pepsin. 2 The resulting F(ab') 2Fragments are then digested to reduce disulfide bonds, thereby producing Fab' fragments. Papain digestion can produce Fab fragments. Fab, Fab' and F(ab') 2 , scFv, Fv, dsFv, Fd, dAb, TandAb, ds-scFv, dimers, miniantibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant technology, or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art.

[0167] In all embodiments of the present invention, single domain antibodies (also called VHH antibodies, sdAbs, DABs, dAbs, nanobodies, camelid antibodies, vNAR (shark) antibodies, VH antibodies or VL antibodies) are preferred, especially VHH antibodies, nanobodies, camelid antibodies and vNAR (shark) antibodies. Such antibodies contain a single monomeric variable antibody domain, usually a VH domain that can bind to an antigen (although a single VL domain with the ability to bind to an antigen has been described, and the VL domain can be used). Therefore, in some such preferred embodiments, the antibody (or antigen-binding domain) of the present invention comprises one (or a single, or only a single) heavy chain variable region (VH or VHH) (or consists of it), although in some embodiments, many of these individual heavy chain variable regions with the same or different sequences may be present together in the same construct or molecule. Therefore, in some such embodiments, the antibody (or antigen-binding domain) of the present invention has only three CDRs (usually together with four FR regions of a standard or conventional order). Sometimes such antibodies may be referred to as antibodies having only a heavy chain.

[0168] Such antibodies can be obtained or prepared using standard techniques well known and described in the art. For example, such antibodies can be obtained by immunizing a suitable animal (e.g., a camelid such as an alpaca, or a shark) with the desired antigen, then cloning the VH domain of the resulting antibody into a suitable expression vector, and selecting binders. VH domain libraries (e.g., phage display libraries of human VH domains) can also be used or generated and then screened.

[0169] In certain embodiments, the antibody or antibody fragment of the present invention comprises all or part of a heavy chain constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, IgM, IgD or IgY constant region. Preferably, the heavy chain constant region is an IgG, IgA or IgY heavy chain constant region or a portion thereof. In addition, the antibody or antibody fragment may comprise all or part of a κ light chain constant region or a λ light chain constant region, or a portion thereof. All or part of such constant regions may be naturally occurring, or may be synthesized in whole or in part. Suitable sequences of such constant regions are well known in the art and documented. When a complete complement of a heavy chain constant region and / or a light chain constant region is included in an antibody of the present invention, such antibodies are generally referred to herein as "full length" antibodies or "complete" antibodies.

[0170] In other embodiments, preferably no constant region is present (eg, no heavy chain constant region or light chain constant region is present), eg, the variable domain or heavy chain variable domain (VH) is the only portion present in the antibody.

[0171] The antibodies or antibody fragments may be naturally produced, or may be produced in whole or in part synthetically.

[0172] Many antibodies or antibody fragments contain an antibody light chain variable region (V L ) and an antibody heavy chain variable region (V H ). The VL and VH usually form an antigen binding site.

[0173] However, it is well documented in the art that the presence of three CDRs of the light chain variable domain and three CDRs of the heavy chain variable domain of an antibody is not always necessary for antigen binding. Thus, smaller constructs than the classical antibody fragments described above are known to be effective.

[0174] For example, camelid antibodies have a wide range of antigen binding repertoires, but lack light chains. In addition, the results of single-domain antibodies comprising only VH domains or VL domains show that these domains can bind antigens with acceptable high affinity and have other advantages, such as small size and ease of production. Therefore, three CDRs can effectively bind antigens, and such single-domain antibodies (e.g., VHH antibodies, sdAbs, DABs, dAbs, nanobodies, camelid antibodies, vNAR (shark) antibodies, VH antibodies or VL antibodies, particularly VHH antibodies, nanobodies, camelid antibodies and vNAR (shark) antibodies) are exemplified herein, and such antibodies (e.g., VHH antibodies) are preferred.

[0175] The antibodies, binding proteins and nucleic acid molecules of the invention are generally "isolated" or "purified" molecules because they are distinguished from any such components that may be present in situ in the human or animal body (e.g., Camelids) or derived from a tissue sample from the human or animal body (e.g., Camelids). However, the sequences may correspond to or be substantially homologous to sequences found in the human or animal body (e.g., Camelids). Thus, as used herein, the terms "isolated" or "purified" in relation to nucleic acid molecules or sequences and proteins or polypeptides (e.g., antibodies) refer to such molecules being separated, purified or substantially freed from their natural environment (e.g., separated or purified from the human or animal body (if they do occur naturally)), or to such molecules being produced by a technological process, i.e., including recombinant and synthetically produced molecules.

[0176] It is noteworthy that, in embodiments, the antibodies, etc. of the present invention do not exist in nature, and in this regard, they are artificial constructs because they do not correspond to molecules that exist in nature. For example, preferred antibodies are single domain antibodies that can be engineered or recombinantly produced, and even in species that naturally produce such antibodies (e.g., camelids), such species have been experimentally induced to produce such antibodies, such as by immunization. In other words, in embodiments, the antibodies, etc. of the present invention are non-native.

[0177] As used herein, the term "fragment" refers to a fragment that has biological relevance, such as a fragment that contributes to antigen binding (e.g., forms part or all of an antigen binding site), and / or a fragment that contributes to the functional properties of a NetB antibody. Certain preferred fragments comprise or consist of a heavy chain variable region (V H domain / VHH domain or three VH CDRs).

[0178] Those skilled in the art will appreciate that the proteins and polypeptides of the present invention (such as heavy and light chain CDRs, heavy and light chain variable regions, antibodies and antibody fragments) can be prepared by any of several methods well known and described in the art, but are most preferably prepared using recombinant methods.

[0179] Nucleic acid fragments encoding the heavy chain variable region and / or light chain variable region of an antibody of the invention (as the case may be) can be obtained or generated by any suitable method (eg, by cloning or synthesis).

[0180] Once the nucleic acid fragments encoding the heavy chain variable region and / or light chain variable region of the antibody of the present invention are obtained, these fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region fragments into full-length antibody molecules with appropriate constant region domains, or into antibody fragments of a particular configuration discussed elsewhere herein, for example, single domain antibodies, such as VHH, Fab fragments, scFv fragments, etc. Typically, or as part of this further manipulation procedure, the nucleic acid fragments encoding the antibody molecules of the present invention are usually integrated into one or more suitable expression vectors to facilitate the production of the antibodies of the present invention, or, for example, to facilitate selection or screening, such as by integration into a phage display vector.

[0181] As long as the vector is compatible with the host cell used, possible expression vectors include, but are not limited to, cosmids, plasmids, or modified viruses (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses). The expression vector is "suitable for transforming a host cell," which means that the expression vector contains a nucleic acid molecule of the invention and regulatory sequences selected based on the host cell for expression, which are operably linked to the nucleic acid molecule. Operably linked means that the nucleic acid is linked to a regulatory sequence in a manner that allows the expression of the nucleic acid.

[0182] Therefore, the present invention relates to an expression vector (eg, a recombinant expression vector) containing or comprising the nucleic acid molecule of the present invention or a fragment thereof, and a regulatory sequence necessary for the transcription and translation of the protein sequence encoded by the nucleic acid molecule of the present invention.

[0183] The expression vector can be introduced into a host cell to produce a transformed host cell. The terms "transformed with," "transfected with," "transformation," and "transfection" are intended to encompass the introduction of a nucleic acid (e.g., a vector) into a cell by one of many possible techniques known in the art. Suitable methods for transforming and transfecting host cells can be found in Sambrook et al., 1989 (Sambrook, Fritsch and Maniatis, "Molecular Cloning: A Laboratory Manual," 2nd ed., Cold Spring Harbor Press, Cold Spring Harbor, New York, 1989) and other laboratory textbooks.

[0184] As is well known to those skilled in the art, suitable host cells include a variety of eukaryotic host cells and prokaryotic cells, or plant cell-based expression systems or fungal expression systems (e.g., Saccharomyces cerevisiae) may be used. For example, the protein of the present invention may be expressed in yeast cells (such as Pichia or Saccharomyces, such as Saccharomyces cerevisiae) or mammalian cells. In addition, the protein of the present invention may be expressed in prokaryotic cells, such as Escherichia coli (E. coli). In some embodiments, expression in Pichia or E. coli is preferred.

[0185] The proteins of the invention may also be prepared by chemical synthesis using techniques well known in protein chemistry, such as solid phase synthesis.

[0186] Another aspect provides an expression construct or expression vector or expression system (e.g., virus or bacteria or other expression construct, vector or system) comprising one or more of the nucleic acid fragments or sections or molecules of the present invention, such as one or more expression constructs or expression vectors. Preferably, the expression construct or vector or system is recombinant. Preferably, the construct or vector or system also comprises a regulatory sequence, which is necessary for transcription and translation of the protein sequence encoded by the nucleic acid molecule of the present invention. Preferred constructs, etc. are constructs that allow the antibody of the present invention (or binding protein) to be extended or continuously expressed in a host target species (e.g., avian species). Such expression can be transient (e.g., episomes), or more persistent (e.g., by genome integration), and as long as sufficient expression levels and duration are reached, treatment or biological effects can be observed.

[0187] Another aspect provides a host cell (e.g., a mammalian host cell, a bacterial host cell, or a yeast host cell) or a virus, e.g., one or more host cells or viruses, comprising one or more expression constructs or expression vectors of the present invention. Also provided is a host cell (e.g., a mammalian host cell, a bacterial host cell, or a yeast host cell) or a virus, e.g., one or more host cells or viruses, comprising one or more nucleic acid molecules in a nucleic acid molecule of the present invention. A host cell (e.g., a mammalian host cell, a bacterial host cell, or a yeast host cell) or a virus expressing an antibody (or conjugated protein) of the present invention constitutes another aspect.

[0188] Such expression constructs or vectors or systems, or host cells or viruses, or other nucleic acid products or fragments encoding antibodies (or binding proteins) of the present invention can be administered to subjects as therapeutic agents to allow the antibodies (or binding proteins) of the present invention to be produced in situ in the subject, thereby exerting their therapeutic effects. Such host cells can also be referred to as live delivery vectors.

[0189] Thus, as described elsewhere herein, preferred host cells are bacterial cells or strains (e.g., probiotic bacterial cells or strains) that can naturally occur or survive in the gastrointestinal tract or small intestine and are suitable for administration to animals, e.g., for therapeutic administration. Examples of suitable bacteria are known in the art and include Bacillus, e.g., Bacillus subtilis; Lactobacillus, such as Lactobacillus reuteri; Lactococcus; Salmonella; Escherichia coli and Listeria, particularly probiotic strains (e.g., Bacillus, e.g., Bacillus subtilis; Lactobacillus, such as Lactobacillus reuteri). Yeast cells, such as Pichia pastoris or yeast (Saccharomyces cerevisiae) can also be used. Such host cells are modified using suitable methods to express antibodies of the invention.

[0190] Another aspect of the present invention provides a method for producing (or manufacturing) an antibody (or binding protein) of the present invention, comprising the step of culturing a host cell of the present invention. A preferred method comprises the following steps: (i) culturing a host cell comprising one or more expression vectors in the expression vector of the present invention or one or more nucleic acid sequences in the nucleic acid sequence under conditions suitable for expressing the encoded antibody or binding protein; and optionally (ii) isolating or obtaining the antibody or binding protein from the host cell or from the growth medium / supernatant. Such a production (or manufacturing) method may also include the step of purifying the antibody or protein product and / or the step of formulating the antibody or product into a composition (e.g., a pharmaceutical composition) comprising at least one additional component, such as a pharmaceutically acceptable carrier or excipient.

[0191] In an embodiment, when an antibody or protein of the present invention consists of more than one polypeptide chain (e.g., certain fragments such as Fab fragments or intact antibodies), all polypeptides (from the same or different expression vectors) are preferably expressed in a host cell so that the complete protein (e.g., an antibody protein of the present invention) can be assembled in the host cell and isolated or purified therefrom.

[0192] In another aspect, the invention provides a method of binding NetB, the method comprising contacting a composition comprising NetB with an antibody or binding protein of the invention.

[0193] In yet another aspect, the present invention provides a method for detecting NetB, the method comprising contacting a composition suspected of containing NetB with an antibody of the present invention under conditions effective to form a NetB / antibody complex, and detecting the formed complex.

[0194] Compositions comprising at least one first antibody (or binding protein) of the present invention, or at least one first nucleic acid molecule or expression vector of the present invention, or at least one first host cell of the present invention constitute other aspects of the present invention. Preparations (compositions) comprising one or more antibodies of the present invention, etc., optionally mixed with suitable diluents, carriers or excipients, constitute preferred embodiments of the present invention. Such preparations can be used for pharmaceutical purposes, such as for animal health applications or veterinary purposes, such as for breeding, and therefore the compositions of the present invention are preferably pharmaceutically acceptable or acceptable, so as to be applied to humans or non-human animals, particularly avian species or flocks, such as poultry. Suitable diluents, excipients and carriers are known to those skilled in the art. In some embodiments, the composition may include sodium bicarbonate (or sodium bicarbonate, NaHCO3), such as at a concentration of or about 0.5 g / l.

[0195] The compositions of the present invention may be presented, for example, in a form suitable for oral, nasal, parenteral, intravenous, topical or rectal administration or mucosal delivery, and any of these modes of administration or indeed any other suitable mode of administration may be used. The preferred mode of administration is oral administration, and therefore forms suitable for such oral administration are also preferred.

[0196] Active compounds as defined herein (e.g. antibodies of the invention) can be presented in conventional pharmacological administration forms, such as tablets, coated tablets, nasal sprays, solutions, emulsions, liposomes, powders, capsules or sustained release forms. Conventional pharmaceutical excipients and common production methods can be used to prepare these forms.

[0197] Injection solutions can be produced, for example, in a conventional manner, such as by adding preservatives (such as parabens) or stabilizers, such as EDTA. The solution is then filled into injection vials or ampoules.

[0198] Oral administration is extremely convenient. However, as described elsewhere herein, the antibodies (or associated proteins) of the present invention are advantageously highly stable, including at low pH (e.g., pH less than 3.0 or less than 2.0, e.g., pH 1.8), and are resistant to various enzymes and proteases, which means that oral administration is feasible in practice. Importantly, direct oral administration (e.g., without coating or other protective means) is also feasible in practice. Therefore, the antibodies (or associated proteins) of the present invention can be, for example, administered by drinking water, or by adding the antibodies (or associated proteins) to suitable animal feed (e.g., by incorporating them into feed pellets or the like, or by using them as surface adjuvants / sprinkled on feed for feed), all of which methods can be conveniently administered regularly, e.g., daily or more frequently as needed. Such antibodies can also be administered by spraying the subject to be treated, or by administering in eggs, or via the respiratory system (e.g., by inhalation).

[0199] Therefore, another aspect of the present invention provides a food product or food additive or food supplement comprising an antibody (or binding protein) of the present invention or a host cell of the present invention, such as an animal feed product or additive or supplement. Such feed products can be in the form of feed premixes or surface adjuvant powders (e.g., for sprinkling on feed). The present invention also provides an animal feed product in which the antibody (or binding protein) of the present invention is mixed with animal feed or incorporated into animal feed.

[0200] Alternative methods for oral delivery are described in the art. Preferred methods may involve the use of live delivery systems and carriers, such as microbial carriers. Microbial carriers include bacteria, such as bacillus, for example, Bacillus subtilis; Lactobacillus, such as Lactobacillus reuteri; Lactococcus; Salmonella; Escherichia coli and Listeria. Microbial carriers can also use yeast cells, such as Pichia pastoris or yeast. These microbial carriers or live carriers are engineered to express, preferably secrete the antibodies (or binding proteins) of the present invention, and deliver (preferably directly) these antibodies to the site of action, for example, by oral delivery or mucosal delivery. Therefore, such microbial carriers are sometimes referred to as direct feeding microorganisms (DFM). Therefore, such carriers (e.g., recombinant vectors) are capable of delivering biotherapeutics in situ because the antibodies (or binding proteins) can be secreted or otherwise produced by the microbial carriers in the animal body. The advantage of using such carriers for delivery is that before the antibodies are secreted, the antibodies can be protected from the harsh gastrointestinal environment, while also maximizing the effectiveness of the antibodies and reducing off-target effects. Such vectors are also capable of continuously producing antibodies (or binding proteins) in the gastrointestinal tract, and preferably are also capable of persisting by colonizing the gastrointestinal tract, just like the parent (unmodified) strain.

[0201] Such vectors can also be relatively easily (and cost-effectively) manufactured on a large scale, and can generally be administered in high doses without causing side effects, e.g., at least 10 8 CFU dosage, and can be applied once or twice a day as needed. Therefore, preferred microbial carriers are engineered or recombinant bacterial strains such as probiotic strains (such as bacillus, such as Bacillus subtilis; and lactobacillus, such as Lactobacillus reuteri), and these microbial carriers containing antibodies / VHH antibodies (e.g., by secretion) are described as biomolecules that can be used for oral administration (see, e.g., Del Rio, B. et al., 2019, Frontiers in Microbiology, 9, 3179). In some embodiments, the preferred carrier is Bacillus subtilis.

[0202] Appropriate dosage units can be determined by one skilled in the art.

[0203] In the case of a co-administration regimen or a combination regimen, the pharmaceutical composition may additionally comprise further active ingredients / agents (eg, as described elsewhere herein).

[0204] Another aspect of the invention provides an anti-NetB antibody (or binding protein) as defined herein, or a nucleic acid molecule, expression vector or host cell or composition as defined herein, for use in treatment, in particular for the treatment or prevention of any disease or condition associated with (or characterized by) NetB or in which NetB plays a role, such as a pathogenic role (e.g., a full or partial pathogenic role) or an essential role. For example, the anti-NetB antibody (or binding protein) and the like of the invention may be used to treat or prevent any infection caused by bacteria or other pathogens, wherein the infection is associated with NetB or in which NetB plays a role, such as a pathogenic role (e.g., a full or partial pathogenic role) or an essential role. In other words, according to the invention, the anti-NetB antibody (or binding protein) and the like may target NetB and inhibit or reduce the function of NetB (e.g., the toxic function of NetB), in particular NetB produced by Clostridium perfringens or other bacteria or pathogens. Thus, the anti-NetB antibody (or binding protein) and the like as defined herein may be used to treat or prevent any disease or condition that can be treated by inhibiting NetB or blocking or reducing the function of NetB.

[0205] In other embodiments, the present invention provides antibodies (or binding proteins) that have the ability to reduce or inhibit NetB function, and are therefore useful for treating or preventing NE, such as NE caused by or associated with Clostridium perfringens.

[0206] Preferred embodiments provide an anti-NetB antibody (or binding protein) of the invention, or a nucleic acid molecule, expression vector or host cell or composition of the invention, for use in treating or preventing bacterial infections, e.g., gastrointestinal bacterial infections, preferably Clostridium perfringens infections, in avian species (such as poultry). Particularly preferred is the treatment or prevention of necrotic enteritis (NE), e.g., NE caused by or associated with the production of NetB (particularly NetB produced by Clostridium perfringens).

[0207] In the therapeutic methods and uses of the present invention, the administration of the binding protein or antibody or nucleic acid molecule, expression vector or host cell or composition is carried out in a pharmaceutically, therapeutically or physiologically effective amount on a subject (animal, such as a mammal or avian species, such as poultry) in need of treatment. Therefore, the methods and uses may involve the additional step of identifying a subject (or multiple subjects / subject groups) in need of treatment. A person skilled in the art can easily determine the appropriate and effective concentration / dose to be administered.

[0208] Treatment of a disease or condition according to the invention (e.g., treatment of an existing disease) includes curing the disease or condition, or any reduction or alleviation of the disease, such as a reduction in disease severity or disease symptoms. Exemplary parameters assessed may include a reduction in mortality or intestinal lesion (necrotic lesion) scores, or an improvement (or increase) in final weight, or a reduction (or decrease or fall) in feed conversion. Such an increase or decrease may be compared to any suitable control (examples of which are described herein), such as compared to an untreated flock (e.g., a flock not treated with a molecule of the invention).

[0209] The methods and uses of the invention are suitable for preventing diseases as well as for actively treating diseases (e.g. treating existing diseases). Thus, the invention also encompasses prophylactic and post-prophylactic (treatment at the time of an outbreak, e.g. treatment of a group of subjects after diagnosis of infection and / or clinical disease in a subset of subjects, with the goal of preventing the spread of infectious diseases to animals in close contact and / or at significant risk) treatment, in particular prophylactic treatment. For this reason, in the methods and uses of the invention, treatment also includes appropriate prevention, post-prophylaxis or prevention.

[0210] Such preventive (or protective) aspects can be conveniently performed on healthy or normal or at-risk subjects, and can include both complete prevention and significant prevention. Similarly, significant prevention can include situations in which the severity of the disease or disease symptoms are reduced (e.g., measurably or significantly reduced) compared to the severity or symptoms expected when no treatment is given.

[0211] Symptoms resulting from clinical disease caused by or associated with NetB production (e.g., disease caused by infection with Clostridium perfringens, such as NE) include sudden increases in flock mortality, dehydration, lethargy, diarrhea, wet litter, ruffled feathers, and decreased feed consumption. Symptoms of subclinical disease include poor overall production performance and increased feed conversion (feed conversion is feed consumed / weight gain) due to chronic damage to the intestinal mucosa caused by the toxin. Additionally, in the subclinical form, damage to the intestine allows the bacteria to enter the portal bloodstream and colonize the liver, causing liver disease, leading to increased rejection rates during processing at the slaughterhouse. Both clinical and subclinical forms of the disease, such as NE, can be treated.

[0212] Thus, suitable subjects for treatment according to the present invention include any type of animal susceptible to infection by a pathogen that produces NetB or a toxin similar to (or substantially homologous to) NetB. Thus, suitable subjects generally include any type of animal susceptible to infection by Clostridium perfringens, including human subjects. Preferred subjects include all types of poultry or chickens, such as any domestic, farmed or wild poultry or chicken species, provided that they are susceptible or capable of infection and suffer from a disease associated with a NetB-producing pathogen as defined herein, particularly Clostridium perfringens. Specific examples include subjects from the order Galloanserae. For example, preferred subjects are poultry animals or species, such as chickens, turkeys, ducks, geese, pigeons, squabs or quails. Particularly preferred are chickens. Avian subjects may suffer from a clinical or subclinical form of the disease, such as NE. For the avoidance of doubt, references herein to a subject, such as treatment or administration to a subject, include a plurality of subjects or a group of subjects.

[0213] In some embodiments, for example when prevention is involved, the subject (or group of subjects) is a subject at risk of being affected by the disease or condition in question, for example, at risk of infection with a NetB-producing pathogen (e.g., Clostridium perfringens) as described above and developing the disease. Such a subject may be a healthy subject or a subject that does not show any symptoms of the disease or any other suitable "at risk" subject. In another embodiment, the subject is a subject that has, or is suspected of having (or developing), or is likely to have (or develop) a disease or condition as described above.

[0214] From another perspective, the invention provides a method for treating or preventing a disease or condition associated with (or characterized by) NetB or in which NetB plays a role, such as a pathogenic effect (e.g., a full or partial pathogenic effect) or an essential effect, the method comprising administering to a subject (or subject group) in need thereof an effective amount of an anti-NetB antibody (or binding protein) of the invention, or a nucleic acid molecule, expression vector, host cell or composition of the invention as defined herein, including a combination of agents. Suitable diseases or conditions and subjects are described elsewhere herein.

[0215] Preferred is the treatment or prevention of disease in avian species, in particular poultry, such as chickens. Particularly preferred is a method of treating or preventing infection with Clostridium perfringens. Particularly preferred is a method of treating or preventing necrotic enteritis (NE), such as NE caused by or associated with the production of NetB, in particular NetB produced by Clostridium perfringens.

[0216] The anti-NetB antibody (or binding protein) and the like of the present invention are usually administered to a subject (or subject group) in need thereof in a pharmaceutically, physiologically or therapeutically effective amount.

[0217] A "pharmaceutically or physiologically or therapeutically effective amount" refers to an amount sufficient to produce a benefit to a condition in a subject (or group of subjects). One skilled in the art can easily determine whether an amount is sufficient to produce a benefit to a condition in a subject. A pharmaceutically or physiologically or therapeutically effective amount can be determined based on a clinical assessment and can be easily monitored.

[0218] The embodiments of the invention described herein for therapeutic uses apply mutatis mutandis to this aspect of the invention.

[0219] From yet another perspective, the invention provides the use of an anti-NetB antibody (or binding protein) of the invention or a nucleic acid molecule, expression vector, host cell or composition (including pharmaceutical combination) of the invention as defined herein in the manufacture of a medicament for treating a subject (or group of subjects), or for treating or preventing a disease or condition in a subject (or group of subjects). Preferred therapeutic uses are described elsewhere herein, in particular for treating or preventing any disease or condition associated with (or characterized by) NetB or in which NetB plays a role, such as a pathogenic effect (e.g., a full or partial pathogenic effect) or an essential effect. Suitable diseases or conditions and subjects are described elsewhere herein.

[0220] Preferred embodiments provide the use of the anti-NetB antibody (or binding protein) of the present invention, etc., in the manufacture of a medicament for treating or preventing infection with Clostridium perfringens. The use of the anti-NetB antibody (or binding protein) of the present invention, etc., in the manufacture of a medicament for treating or preventing necrotic enteritis (NE), such as NE caused by or associated with the production of NetB, particularly NetB produced by Clostridium perfringens, is also preferred. Preferred is the treatment or prevention of diseases of avian species, particularly poultry (e.g., chickens).

[0221] The embodiments of the invention described herein for therapeutic uses apply mutatis mutandis to this aspect of the invention.

[0222] In some embodiments, the antibodies (or binding proteins) of the invention may be used in combination.

[0223] For example, the specific VHH antibodies Net 83 and Net 14 shown in Table A and Table B, respectively, can be used in combination. Thus, two (or more) antibodies (or binding proteins) of the present invention can be used in combination. One or more antibodies (or binding proteins) of the present invention can also be used in combination with other active agents, including other active agents that target, inhibit or reduce NetB activity, such as other anti-NetB antibodies.

[0224] Alternatively, in the above combinations, antibodies having three CDRs as shown in Table A and / or Table B, or antibodies having substantially homologous CDR sequences as defined elsewhere herein, may also be used as desired.

[0225] Preferred combinations are combinations that result in an improved or enhanced, preferably significantly improved or enhanced, therapeutic effect compared to any antibody (or binding protein) (e.g., VHH) of the invention administered as the sole active agent (monotherapy), or the sole antibody, or the sole anti-NetB agent. Other preferred combinations are combinations in which each anti-NetB antibody binds to a different epitope on the NetB molecule.

[0226] For such combination therapies using two or more anti-NetB antibodies (or binding proteins), the second (or subsequent) anti-NetB antibody can be administered to the subject substantially simultaneously with the first anti-NetB antibody, such as by administering a single pharmaceutical composition (thus, administered simultaneously), or by administering two pharmaceutical compositions closely together (simultaneous or similar times). Alternatively, the second (or subsequent) anti-NetB antibody can be administered to the subject at a time before or after the administration of the first anti-NetB antibody. As used herein, "before or after" means "staggered," such that the second antibody is administered to the subject at a different time than the administration of the first anti-NetB antibody component. Typically, the two (or more) components can be administered at effective intervals or together to allow each component to exert its respective therapeutic effect, i.e., they are administered in a "biologically effective amount" at a "biologically effective time interval," and as part of the same treatment regimen.

[0227] If appropriate, combinations of anti-NetB antibodies (or binding proteins) may conveniently be administered as part of the same molecule or construct, for example, they may be conjugated or linked together, for example using an artificial linker. This mode of administration is particularly suitable for VHH antibodies (or other types of antibody molecules consisting of a single polypeptide chain), wherein the individual antibodies may conveniently be linked by suitable peptide (or other) linkers (e.g., non-native peptides or artificial linkers) in a single polypeptide chain comprising multiple VHH (or other) antibodies, which may be antibodies of the present invention or in combination with other VHH or other antibodies, for example VHH or other antibodies directed against other related entities such as alpha toxin (CPA). In such embodiments, the agents are typically linked together using suitable techniques (e.g., spacers) so that each component can perform its respective function, for example, binding to NetB. For example, in embodiments where the anti-NetB antibodies bind to different epitopes on NetB, then combinations of such antibodies are preferred, and the construct is appropriately designed so that each individual antibody can bind to NetB, for example, to its NetB epitope. Furthermore, multispecific constructs, such as multispecific VHH constructs, are also contemplated, wherein in addition to (or in addition to) the presence of NetB, further specificities (preferably in the form of antibodies, such as VHH antibodies), are included, e.g., present in a single molecule. Thus, combinations of anti-NetB antibodies (or binding proteins) of the invention may be combined with other antibodies having specificity for entities other than NetB (e.g., CPA antibodies).

[0228] In other embodiments of the invention, multiple copies of a single NetB antibody (or binding protein) of the invention may be provided on a single molecule, for example as a dimer (homodimer) or larger multimer (homomultimer), for example with three or four copies of the same antibody or binding protein. Thus, in preferred such embodiments, a NetB antibody or binding protein of the invention (e.g., a Net 83 or Net 14 antibody as described herein, or an antibody based on a Net 83 or Net 14 antibody sequence listed in Table A or Table B as described herein) may be provided as a dimer or larger multimer. In such dimers or larger multimers, the multiple copies of the antibody or binding protein may be linked in any suitable manner. Conveniently, particularly for VHH antibodies (or other types of antibody molecules consisting of a single polypeptide chain), the individual antibodies may be linked in a single polypeptide chain by a suitable peptide (or other) linker as described above. Such dimers or larger multimers constitute a further aspect of the invention.

[0229] Thus, in some embodiments, the anti-NetB antibodies (or binding proteins) of the invention can be used as the sole active agent in a treatment regimen (monotherapy), or more than one anti-NetB antibody of the invention can be used in combination, e.g., as described above. In some embodiments, the anti-NetB antibodies (or binding proteins) of the invention (or combination, as appropriate) can be used as the sole active anti-NetB agent or the sole active anti-NetB antibody in a treatment regimen, or they can be the sole active anti-Clostridium perfringens or anti-NE agent in a treatment regimen. However, in some embodiments, additional anti-NetB agents or anti-Clostridium perfringens or anti-NE agents can be used.

[0230] Thus, an anti-NetB binding protein or antibody of the invention (or combination, as the case may be) may be combined with one or more additional (additional) NetB-targeted or non-NetB-targeted active agents, e.g., with at least a second therapeutic agent or biologic, of which an anti-NetB binding protein or antibody of the invention (or combination of such binding proteins or antibodies) is the first.

[0231] The anti-NetB antibodies (or binding proteins) of the invention (or combinations as the case may be) may be combined, for example, with any other therapeutic agent for treating the disease in question, for example treating a disease caused by or associated with infection with Clostridium perfringens or the production of NetB, or treating NE. Thus, the anti-NetB antibodies (or binding proteins) of the invention may be combined with other agents (e.g., other antibodies) for treating infection with Clostridium perfringens, such as agents (e.g., antibodies) that inhibit other toxins that may be produced by Clostridium perfringens, such as alpha toxin (CPA, Clostridium perfringens alpha toxin). One or more alpha toxin antibodies (e.g., VHH antibodies or single-chain antibodies) are examples of suitable and preferred agents for such combination therapies, for example, one or more anti-NetB VHH antibodies of the invention may be combined with one or more anti-alpha toxin (CPA) antibodies (e.g., VHH antibodies). Another example is a combination with an antibiotic, such as an antibiotic that can be used to treat a disease caused by or associated with infection with Clostridium perfringens or the production of NetB or NE. A further example is a combination with probiotics or feed additives (such as enzymes, vitamins and / or minerals). Another example is a combination with an agent useful for treating coccidiosis, such as a coccidiostat or an anti-coccidiosis antibody. In all such combination therapies, for example a combination of an anti-NetB agent (such as an antibody or binding protein of the invention) and an anti-CPA agent, the observed therapeutic effect is preferably synergistic.

[0232] For such combination therapies, the second (non-anti-NetB antibody of the invention) agent can be administered to the subject together (simultaneously or substantially simultaneously, e.g., mixed together in a combined formulation, or as part of the same molecule, e.g., when two or more VHH antibodies or single chain antibodies are used) or separately, as described above.

[0233] Thus, in some preferred embodiments, an anti-NetB antibody (or binding protein) of the invention is administered in combination with an anti-CPA antibody (or binding protein). The different antibodies may be administered, for example, as a mixture, or as separate components, or may be provided as part of the same molecule, for example by linking the different antibodies together, for example an anti-NetB VHH of the invention may be linked to an anti-CPA VHH, as described elsewhere herein.

[0234] Therefore, a preferred composition of the invention comprises an anti-NetB antibody (or binding protein) of the invention in combination with an agent that inhibits CPA, preferably an anti-CPA antibody (or binding protein), such as a VHH antibody.

[0235] The present invention also includes a kit comprising one or more of the antibodies or compositions of the present invention, or one or more nucleic acid molecules of the nucleic acid molecules encoding the antibodies of the present invention, or one or more recombinant expression vectors comprising the nucleic acid sequences of the present invention, or one or more host cells or viruses comprising the recombinant expression vectors or nucleic acid sequences of the present invention. Preferably, the kit is used for the methods and uses described herein, for example, the methods of treatment as described herein. Preferably, the kit comprises instructions for using the kit components. Preferably, the kit is used to treat diseases or conditions as described elsewhere herein, and optionally comprises instructions for using the kit components to treat such diseases or conditions. Equivalent embodiments of the binding proteins of the present invention are also provided.

[0236] The antibodies (or binding proteins) of the invention as defined herein can also be used as molecular tools for in vitro or in vivo applications and assays (e.g. diagnostic assays). Since antibodies (and some binding proteins) have antigen binding sites, they can function as members of specific binding pairs, and these molecules can be used in any assay requiring a specific binding pair member.

[0237] Therefore, further aspects of the invention also provide reagents comprising an antibody (or binding protein) of the invention as defined herein, and the use of such antibodies (or binding proteins) as molecular tools, for example in in vitro or in vivo assays, for example for detecting NetB in a target sample.

[0238] In one embodiment, the present invention provides a method of diagnosing NE or other diseases as described herein in a subject, the method comprising the steps of:

[0239] (a) contacting a test sample collected from the subject with one or more of the antibodies or binding proteins of the present invention.

[0240] In another embodiment, the present invention provides a method of diagnosing NE or other diseases as described herein in a subject, the method comprising the steps of:

[0241] (a) contacting a test sample collected from the subject with one or more of the antibodies or binding proteins of the present invention;

[0242] (b) measuring or detecting the presence and / or amount and / or location of an antibody-NetB or binding protein-NetB complex in a test sample; and optionally

[0243] (c) comparing the presence and / or amount of the antibody-NetB or binding protein-NetB complex in the test sample to a control.

[0244] In the above methods, the contacting step is performed under conditions that allow the antibody-NetB or binding protein-NetB complex to form (eg, detectably form). Suitable conditions can be readily determined by one skilled in the art.

[0245] In the above methods, any suitable test sample or biological sample may be used, such as a blood or serum sample, material from a tissue or organ suspected of being affected by NE or other diseases as described herein (e.g., small intestine or other suitable gastrointestinal sample), or a histological section. The sample is taken from a suitable subject as defined elsewhere herein, such as an avian subject, e.g., a poultry subject, such as a chicken.

[0246] In certain of the above methods, the presence of any amount of antibody-NetB or binding protein-NetB complex in the test sample will indicate the presence of NE or other diseases as described herein. Preferably, to make a positive diagnosis, the amount of antibody-NetB or binding protein-NetB complex in the test sample is greater, preferably measurably or significantly greater than the amount found in a suitable control sample (control value or level). More preferably, the significantly higher level is statistically significant, preferably with a probability value of <0.05. Suitable methods for determining statistical significance are well known and documented in the art, and any of these methods can be used.

[0247] A suitable control sample can be easily selected by one skilled in the art. For example, in the case of diagnosing NE, a suitable control would be a sample from a subject not suffering from NE (e.g., a healthy subject). Suitable control "values" or "levels" can also be easily determined without having to run a control "sample" in each test, for example by reference to ranges for normal or healthy subjects known in the art. Thus, a control value or level can correspond to a level in a suitable control subject or sample, for example, can correspond to a critical level or range found in a control or reference population.

[0248] In one embodiment, the method of diagnosing NE or other diseases as described herein is an in vitro method.

[0249] In one embodiment, the method of diagnosing NE or other diseases as described herein is an in vivo method.

[0250] Viewed from another perspective, the present invention provides methods for screening a subject for NE or other diseases as described herein, or methods for detecting NE or other diseases as described herein.

[0251] As used herein, the term "reduce" or "reduction" (or equivalent terms) includes any measurable reduction or decrease compared to a suitable control. Suitable controls can be easily identified by one skilled in the art, which may include untreated or placebo-treated subjects or healthy subjects, or samples or assays in which the antibodies (or binding proteins) of the invention are not present. Preferably, the reduction or decrease will be significant, e.g., clinically or statistically significant.

[0252] The term "increase" (or equivalent terms) as described herein includes any measurable increase or elevation compared to a suitable control. Suitable controls can be easily identified by one skilled in the art, which may include untreated or placebo-treated subjects or healthy subjects, or samples or assays in which the antibody (or binding protein) of the invention is not present. Preferably, the increase will be significant, e.g., clinically or statistically significant.

[0253] Preferably, such increase (and indeed other increases, improvements or positive effects as mentioned elsewhere herein) or such decrease (and indeed other decreases, reductions or negative effects as mentioned elsewhere herein) is a measurable increase, decrease, etc. (as the case may be), more preferably, such increase and such decrease are significant increases, decreases, etc. when compared to a suitable control level or value (e.g. compared to an untreated or placebo-treated subject, or compared to a healthy or normal subject, or compared to the same subject before treatment), preferably a clinically significant or statistically significant increase, e.g. a probability value ≤0.05 or <0.05.

[0254] The method of determining the statistical significance of the difference between the test groups of subjects or the difference of the specific parameter level is well known in the art and documented. For example, in this article, if a statistical comparison using a significance test, such as a student's t test, Mann-Whitney U rank sum test, chi-square test or Fisher's exact test, one-way analysis of variance or two-way analysis of variance test (as the case may be), shows a probability value of ≤0.05 or <0.05, then it is generally considered that the reduction or increase of the specific parameter level or the difference between the test groups of subjects is statistically significant.

[0255] The amino acid sequence listing disclosed herein and its sequence identifier (SEQ ID NO)

[0256] As is customary in the art, all amino acid sequences herein are listed from N-terminus to C-terminus.

[0257]

[0258]

[0259] NetB toxin (SEQ ID NO: 17)

[0260] SELNDINKIELKNLSGEIIKENGKEAIKYTSSDTASHKGWKATLSGTFIEDPHSDKKTALLNLEGFIPSDKQIFGSKYYGKMKWPETYRINVKSADVNNNIKIANSIPKNTIDKKDVSNSIGYSIGGNISVEGKTAGAGINASYNV QNTISYEQPDFRTIQRKDDANLASWDIKFVETKDGYNIDSYHAIYGNQLFMKSRLYNNGDKNFTDDRDLSTLISGGFSPNMALALTAPKNAKESVIIVEYQRFDNDYILNWETTQWRGTNKLSSTSEYNEFMFKINWQDHKIEYYL

[0261] NetB toxoid (SEQ ID NO: 18)

[0262] SELNDINKIELKNLSGEIIKENGKEAIKYTSSDTASHKGWKATLSGTFIEDPHSDKKTALLNLEGFIPSDKQIFGSKYYGKMKWPETYRINVKSADVNNNIKIANSIPKNTIDKKDVSNSIGYSIGGNISVEGKTAGAGINASYNV QNTISYEQPDFRTIQRKDDANLASWDIKFVETKDGYNIDSYHAIYGNQLFMKSRLYNNGDKNFTDDRDLSTLISGGFSPNMALALTAPKNAKESVIIVEYQRFDNDYILNWETTQARGTNKLSSTSEYNEFMFKINWQDHKIEYYL BRIEF DESCRIPTION OF THE DRAWINGS

[0263] The invention will now be further described in the following non-limiting examples with reference to the following drawings:

[0264] Figure 1A A standard curve of NetB-induced erythrocyte lysis is shown. Figure 1B Shown is the inhibition of NetB-induced chRBC lysis assay (pH 7.4).

[0265] Figure 2A Stability after incubation with chymotrypsin is shown. After incubation with chymotrypsin, the VHHs were subjected to SDS-PAGE gel analysis. Figure 2B Shown are the activities of selected clones after incubation with chymotrypsin.

[0266] Figure 3AStability and activity after incubation with pepsin are shown. Figure 3B Activity after incubation with pepsin is shown. After incubation with pepsin, VHHs can inhibit NetB-induced RBC lysis. Figure 3C The stability of selected clones after incubation with pepsin is shown. SDS-PAGE gel analysis of selected VHHs at different time points during prolonged incubation with pepsin. Figure 3D Shown are the activities of selected clones after incubation with pepsin.

[0267] Figure 4A Stability after incubation with trypsin is shown. SDS-PAGE gel analysis of VHHs incubated with increasing amounts of trypsin. Figure 4B The stability of selected VHHs after incubation with trypsin is shown. SDS-PAGE gel analysis of the VHHs at different time points during incubation with trypsin.

[0268] Figure 5A Shown is a gel demonstrating the stability of the VHH in water. Figure 5B Shown is the stability of VHH in water and its effect on potency

[0269] Figure 6 Biacore titration results of selected VHHs are shown.

[0270] Figure 7 Shown are the concentrations of Net_83 in crop digesta samples of chickens that drank antibody-supplemented water ad libitum over a 24-hour period, T01-1 = Net_83, T02-1 = Net_83 added to water containing NaHCO3.

[0271] Figure 8 The probability of death due to necrotic enteritis from the time of C. perfringens (Cp) challenge to the end of the study (days 17 to 30) is shown. Antibody treatment lasted for 6 days. T01 = untreated control after infection (IUC), T02 = unchallenged untreated (UUC), T03 = amoxicillin added to drinking water for 5 days, T04 = CPA + Net_83 antibody, T05 = NaHCO3, T06 = CPA + Net_83 antibody added to water containing NaHCO3.

[0272] Fig. 9 The probability of death of the flocks is shown for the antibody groups (T04 and T06) combination (n=260) and the T01 (IUC) and T05 (NaHCO3) combination (untreated group; n=260). The log-rank P value of the time-to-event analysis for each group is shown in the figure. When a chicken survived to day 30, the chicken was considered as censored data.

[0273] Fig.10The probability of a dead pen for the antibody group (T04 and T06) combination (n=20) and the T01 (IUC) and T05 (NaHCO3) combination (untreated group, n=20) is shown. A pen was considered alive if at least one bird was still alive at day 30. A pen was classified as a dead pen when all the birds in the pen died due to NE. The log-rank P values ​​for the time-to-event analysis are provided in the figure.

[0274] Fig.11 Shown is an ELISA of supernatants from three Bacillus subtilis clones expressing Net_14 and one expressing an unrelated VHH clone (anti-JTT-A) binding to NetB-His.

[0275] Fig.12 Shown is an ELISA of supernatants from three Bacillus subtilis clones expressing Net_83 and one unrelated VHH expressing clone (anti-JTT-B) binding to NetB-His. DETAILED DESCRIPTION

[0276] Example:

[0277] Example 1: Immunization, library generation, screening and clone selection

[0278] Materials and methods

[0279] immunity

[0280] Single domain antibodies were obtained from camels immunized with recombinant proteins. Llamas were injected with inactive Clostridium perfringens NetB toxoid antigens formulated in Freund's incomplete adjuvant. Animals were immunized subcutaneously six times per week (two injections of 100 μg / dose followed by four injections of 50 μg / dose). One week after the last booster immunization, sera were collected to determine the antibody titer against NetB toxoid.

[0281] In this ELISA, 96-well plates (Maxisorp; Nunc) were coated with the recombinant protein. After blocking and addition of diluted serum samples, the presence of anti-NetB toxoid antibodies was demonstrated by using mouse anti-llama IgG (FJB, catalog number FJ1203MAB01B09) followed by anti-mouse immunoglobulin peroxidase conjugate (JIR, catalog number 715-035-150).

[0282] Library construction and selection

[0283] RNA was extracted from PBMC of three immunized alpacas (400 ml each). cDNA synthesis was performed with 40 μg RNA using random primers. The cDNA was amplified by primary PCR using non-labeled primers annealing in the leader sequence and hinge CH1 region, followed by secondary PCR amplification, introducing restriction endonuclease sites to clone VHH genes in pDCL1 phagemid vectors. The library was electroporated into TG1 E. coli cells, and bacterial glycerol stocks of the immune library were stored at -80°C (FL2471 and FL2472).

[0284] choose

[0285] Phage products from the Ilama VHH library pool were used in three consecutive rounds of phage display selections using NetB toxoid recombinant proteins. Multiple rounds of selections were performed on the recombinant proteins using 10 μg / ml NetB toxoid (PBS buffer), non-specific phages were first removed by washing, and specific phages were subsequently eluted using trypsin (complete elution).

[0286] Eluted phages were serially diluted and used to infect exponentially growing TG1. Infected TG1 were plated on LBCarb100Glu2% plates and enrichment values ​​were calculated after background removal (no antigen for selection).

[0287] Initial screening

[0288] Screening for binding by ELISA

[0289] Individual clones selected according to the first and second round selection conditions were picked into 96-well master plates and their periplasmic extracts (PE) were tested for binding to NetB toxoid at pH 7.4 by binding ELISA. For the PE binding ELISA, MaxiSorp TMHigh protein binding capacity 96-well ELISA plate, overnight at 4°C. The next day, the plate was washed 3 times with PBS Tween 0.05% (pH 7.4) and blocked with 250 μl / well of 4% Marvel / CPA or 4% Marvel / PBS for 1 hour at room temperature. After blocking, the plate was washed 3 times with PBS Tween 0.05% (pH 7.4), and 20 μl P.E + 80 μl 1% Marvel / PBS (pH 7.4) was added to each well and incubated with shaking at room temperature for 1 hour. The plate was washed 3 times with PBS Tween 0.05% (pH 7.4) and incubated with 100 μl anti-c-Myc antibody (Roche; catalog number 11667203001), followed by incubation with secondary antibody DAM-HRP (JIR; catalog number 715-035-150) in 1% Marvel / PBS (pH 7.4), and incubated with shaking at room temperature for 1 hour. The plate was washed three times with PBS Tween 0.05% (pH 7.4), and a substrate solution (TMB solution) was added to the plate. 2 SO 4 The reaction was stopped and the plate was read at 450 nm using a microplate reader.

[0290] Sequencing

[0291] Positive binders were sent for sequencing. Clones were classified into families based on their different HCDR3 sequences.

[0292] Results and Discussion

[0293] After immunizing alpacas with recombinant NetB toxoid, phage clones were selected through two rounds of phage screening. Phage candidates were confirmed to bind to the recombinant NetB toxoid by ELISA. As shown in Table 1, several candidates were able to bind to NetB toxoid, which shows the data of some clones that bound to NetB toxoid.

[0294] Table 1: Summary of ELISA binding data for periplasmic extracts

[0295]

[0296] The clones were further screened and selected by Biacore to determine their off-rate when bound to recombinant NetB toxoid. NetB toxoid protein was immobilized on a CM5 sensor chip by amine coupling in 10mM sodium acetate at pH 5.0 at a concentration of approximately 1100RU. The PE of the selected clones was diluted 1:5 with HBS-EP pH 7.4 buffer and injected at a rate of 30μl / min onto the immobilized NetB toxoid for 2 minutes. After this binding injection, the sample was allowed to dissociate for 5 minutes. Between runs, the chip was regenerated by injecting 1M NaCl / 1mM glycine (pH 1.5). Controls included 5μg / ml of anti-histidine antibody (BioLegend, catalog number 652502) in HBS-EP pH7.4 buffer, which showed no significant decrease in binding at the beginning and end of the run. PE of an unrelated VHH (FJ1409MP03C04) was run in the same manner and showed no binding to NetB toxoid. As shown in Table 2, several candidates bound NetB toxoid with good off-rates.

[0297] Therefore, immunization of alpacas with recombinant NetB toxoid protein successfully isolated phage antibody candidates that could bind NetB with a good dissociation rate.

[0298] Table 2: Periplasmic extract dissociation rate binding data

[0299]

[0300] Example 2: Determination of binding of purified VHHs to NetB toxin at pH 7.4 Binding was assessed at pH 6.0.

[0301] Materials and methods:

[0302] Expression and purification of VHH candidate antibodies

[0303] This experiment purchased a synthetic gene encoding the VHH variable domain with an Avi tag and a His tag, and cloned the synthetic gene into a pET15b bacterial expression vector with a periplasmic secretion leader sequence. Escherichia coli (E coli) strain BL21 DE3 was transformed and cultured in ZYP-5052 autoinduction medium at 18 ° C for 68 hours. Ni-NTA beads (Qiagen) were used to capture the produced VHH antibodies from the clarified supernatant on a gravity feed column. The buffer for eluting the antibody was replaced with 1xPBS at pH 7.4 and concentrated with a rotary concentrator (Amicon, catalog number UFC801096D) with a molecular weight cutoff of 10k. The purified VHH protein was analyzed by SDS-PAGE to determine if the correct chain existed.

[0304] Screening for binding by ELISA

[0305] Individual clones as purified VHHs were tested for binding to NetB toxin by binding ELISA at pH 6.0 and pH 7.4. For VHH binding ELISA, MaxiSorp was coated with 1 μg / ml NetB toxin diluted in phosphate buffered saline (PBS) at pH 7.4 or in citrate-phosphate buffer (CPB) at pH 6.0. TM High protein binding capacity 96-well ELISA plate, overnight at 4°C. The next day, the plate was washed 3 times with PBS Tween 0.05% (pH 7.4) and blocked with 100 μl / well of 4% Marvel / CPB or 4% Marvel / PBS at room temperature for 1 hour. After blocking, the plate was washed 3 times with PBS Tween 0.05% (pH 7.4) or CPB Tween 0.05% (pH 6.0), and 100 μl of 180 nM VHH in PBS (pH 7.4) or CPB (pH 6.0) was added to each well and incubated for 1 hour at room temperature. The plate was washed three times with PBS Tween 0.05% (pH 7.4) or CPB Tween 0.05% (pH 6.0) and incubated with 50 μl of anti-Strep II antibody (Abcam; catalog number Ab252885), followed by incubation with secondary antibody goat anti-rat IgG-HRP (Alpha diagnostic International; catalog number 50320-200) in PBS (pH 7.4) or CPB (pH 6.0) for 1 hour at room temperature. The plate was washed three times with PBS Tween 0.05% (pH 7.4) or CPB Tween 0.05% (pH 6.0), and substrate solution (TMB solution) was added to the plate. The plate was incubated with H 2 SO 4 The reaction was stopped and the plate was read at 450 nm using a microplate reader.

[0306] Results and Discussion

[0307] As shown in Table 3, many clones demonstrated binding of the purified VHH to NetB toxin both at pH 7.4 and at the more physiologically relevant pH 6.0.

[0308] Table 3: Summary of purified VHH ELISA binding data

[0309]

[0310] Example 3: Inhibition of NetB-induced chRBC lysis assay

[0311] Materials and methods:

[0312] Expression and purification of VHH candidate antibodies

[0313] Proceed as described in Example 2 above.

[0314] Standard curve of NetB-induced erythrocyte lysis

[0315] Preparation of red blood cells (RBC)

[0316] 3 mL of fully resuspended fresh chicken blood (TCS Biosciences; catalog number FB011AP) was added to 25 ml PBS for washing, gently mixed, and centrifuged at 500 x g at room temperature. The supernatant was removed and the pellet was resuspended in 25 mL PBS and then subjected to one more washing step. After the second washing step, the RBC pellet was resuspended in 20 ml PBS again. OD595 was measured and the volume was adjusted so that OD595 = 0.5.

[0317] NetB-induced erythrocyte lysis assay

[0318] Active NetB toxin was serially diluted with PBS and added to RBCs at a 1:1 ratio (OD595 = 0.5 as prepared above). NetB toxin and RBCs were incubated at 37°C with shaking for 30 minutes. After incubation, the plate was centrifuged at 1100 x g for 3 minutes. 50 μl of each sample was removed from a suitable flat bottom plate, taking care not to disturb the RBC pellet. The OD405 of the plate was recorded using a spectrophotometer.

[0319] Characterization of NetB toxin and determination of submaximal dose (EC80)

[0320] Purified NetB toxin was characterized with each batch of fresh chicken blood before being subjected to the lysis inhibition assay. The concentration response was measured based on the ability of NetB to lyse chicken erythrocytes (data not shown). The calculated EC80 (20 nM) was used to test the inhibitory effect of VHH on NetB-induced RBC lysis assay.

[0321] Preparation of VHHs to inhibit NetB-induced erythrocyte lysis

[0322] VHH was serially diluted 3-fold from 4 μM to 5.49 nM, and 17.5 μL of the diluted VHH was transferred to a V-bottom 96-well assay plate. Then 17.5 μL of NetB (diluted 4-fold with PBS, i.e., EC80) was added to the corresponding wells. For the positive control, only NetB was used. The negative control did not contain NetB. NetB and VHH clones (total volume of 35 μL) were incubated in the assay plate for 15 minutes at room temperature, and then 35 μL of washed and prepared RBCs were added. The assay plate was sealed with an adhesive plate seal and incubated at 37°C with shaking (450 rpm) for 30 minutes. After incubation, the plate was centrifuged at 1100 x g for 3 minutes, and 50 μL was removed from each sample onto a suitable flat-bottom plate, taking care not to disturb the RBC pellet. The OD405 of the plate was recorded using a spectrophotometer.

[0323] Results and Discussion

[0324] Figure 1A A standard curve for NetB-induced erythrocyte lysis is shown. The EC50 of NetB toxin in these assays was 2 nM. The submaximal concentration of NetB used in these assays was equivalent to an EC80 = 20 nM.

[0325] The final range of VHH concentrations used in this assay was from 1 μM to 1.37 nM. This corresponds to a range of molar ratios (VHH:NetB) from 50:1 to 0.0685:1.

[0326] As shown in Table 4, many VHHs showed the potential to inhibit the lytic activity of NetB.

[0327] Figure 1B Titration inhibition curves of various VHHs screened at pH 7.4 are shown. Some of these VHHs showed potent inhibition of NetB-induced lysis. IC50 and molar ratios are shown in Table 4. VHHs were also screened at pH 6.0. IC5 0 The molar ratios are shown in Table 4.

[0328] It can be seen that some VHH clones showed improved bioactivity more favorably at pH 6.0 than at pH 7.4.

[0329]

[0330] Table 4: Inhibition of NetB-induced red blood cell (RBC) lysis (pH 7.4 and pH 6.0)

[0331] In subsequent experiments, the above chRBC lysis assay was performed with Net_14 and Net_83 at 42°C instead of 37°C to more closely mimic the body temperature of chickens. The results showed that NetB showed similar potency at both 42°C and 37°C, and both VHH antibodies retained the ability to inhibit NetB activity at this high temperature.

[0332] Example 4: Stability and activity after incubation with chymotrypsin (resistance to chymotrypsin)

[0333] Various purified VHH candidates were screened for their ability to inhibit NetB-induced erythrocyte lysis to determine their ability to resist proteases found in the gastrointestinal (GI) tract.

[0334] Candidate VHHs were incubated with chymotrypsin (1 μg / ml) for 1 hour, after which the reaction was terminated and the VHHs were run on SDS-PAGE gels and assessed for their ability to consistently inhibit NetB-induced erythrocyte lysis.

[0335] Materials and methods

[0336] Incubation of VHH with chymotrypsin

[0337] VHHs were prepared at 15 μM in PBS (unless the starting concentration of the VHHs was < 15 μM, in which case these VHHs were used at the highest possible concentration). A stock solution of chymotrypsin (Sigma Aldrich; Cat. No. 11418467001) was made to a concentration of 100 μg / mL in 1 mM HCl by adding 250 μL of 1 mM HCl to the vial containing 25 μg of chymotrypsin.

[0338] Chymotrypsin was then further diluted with PBS to a sub-stock concentration of 10 μg / mL.

[0339] 18 μL of prepared VHHs were added to low protein binding PCR tubes. 2 μL of 10 μg / mL chymotrypsin (or 2 μL of PBS for controls) was added to each tube. The tubes were then rotated for 15 seconds to ensure that all tubes had chymotrypsin added at the same time.

[0340] The PCR tubes were then transferred to a heating block and placed at 37°C for 1 hour to allow digestion, after which the digestion was terminated by adding 3.3 μL (7x) of protease inhibitors and transferring the PCR tubes to ice.

[0341] After incubation with chymotrypsin, VHHs were analyzed by SDS-PAGE gel

[0342] Take 5 μL of each terminated VHH sample and add it to each tube with 2 μL of 5x SDS loading buffer added and seal the tubes with foil adhesive plate sealer. Transfer the tubes to a heating block and incubate at 80 °C for 10 minutes and then spin to collect any evaporation. Run a 4% to 20% gradient SDS-PAGE gel electrophoresis on the denatured samples. After running the gel electrophoresis, stain with Coomassie Brilliant Blue staining reagent for 24 hours, then destain with water and subsequently photographed to assess degradation after digestion with chymotrypsin.

[0343] NetB-induced erythroid assay using VHH incubated with chymotrypsin

[0344] Take 10 μL of each terminated VHH sample and add it to 65 μL PBS, then dilute it 1 to 3 times, from 2 μM to 5.49 nM. The final concentration of these dilutions is 4 times to allow for dilution after the addition of chRBC and NetB toxins. NetB will be used at a submaximal concentration (EC80) of 20 nM. The maximum final concentration of VHH is 500 nM = 25:1 (VHH: NetB) molar ratio, and the minimum concentration is 1.37 nM = 0.0685:1 molar ratio (VHH: NetB).

[0345] VHH was serially diluted 3-fold from 2 μM to 5.49 nM, and 17.5 μL of the diluted VHH was transferred to a V-bottom 96-well assay plate. Then 17.5 μL of NetB (diluted 4-fold with PBS, i.e., EC80) was added to the corresponding wells. For the positive control, only NetB was used. The negative control did not contain NetB. NetB and VHH clones (total volume of 35 μL) were incubated in the assay plate for 15 minutes at room temperature, and then 35 μL of washed and prepared RBCs were added. The assay plate was sealed with an adhesive plate seal and incubated at 37°C with shaking (450 rpm) for 30 minutes. After incubation, the plate was centrifuged at 1100 x g for 3 minutes, and 50 μL was removed from each sample onto a suitable flat-bottom plate, taking care not to disturb the RBC pellet. The OD405 of the plate was recorded using a spectrophotometer.

[0346] Results and Discussion:

[0347] Data (see Figure 2A ) clearly demonstrated that while some VHHs were readily degraded upon incubation with chymotrypsin (as judged by the presence of multiple degradation bands, see e.g. Net 11, Net 15, Net 17 and Net 74), some of these VHHs remained largely intact and were still able to inhibit NetB-induced erythrocyte lysis when tested (see Figure 2B and Table 5). In the gel shown in this example (and other examples herein), the VHH band appears at approximately 15 kDa.

[0348] Table 5: Activity of VHH after incubation with chymotrypsin

[0349]

[0350]

[0351] Example 5: Stability and activity after incubation with pepsin (resistance to pepsin)

[0352] Candidate VHHs were incubated with pepsin for a period of time up to 45 minutes. The reaction was terminated after incubation with pepsin and the VHHs were run on SDS-PAGE gel electrophoresis and assessed for their ability to consistently inhibit NetB-induced erythrocyte lysis.

[0353] Materials and methods:

[0354] Pepsin (Sigma Aldrich; catalog number P7125) was prepared at a concentration of 5 mg / mL (5360 U / mL) in PBS at pH 1.5 (pH was lowered to 1.5 using concentrated HCl). Pepsin was further diluted to 300 U / mL in PBS at pH 1.5. To maximize the protease activity of pepsin, the pH in this assay must be below 2.

[0355] All VHHs were prepared to a concentration of 30 μM VHH with 10 μL PBS at pH 7.4, and then 8 μL PBS at pH 1.5 was added to obtain 18 μL VHH at a concentration of 16.67 μM. To this was added 2 μL pepsin at a concentration of 300 μ / mL in PBS at pH 1.5, resulting in a final reaction solution of 15 μM VHH and 30 μ / ml pepsin in PBS at pH 1.8. The tubes were then rotated for 15 seconds to ensure that all tubes were added with pepsin at the same time. The PCR tubes were then transferred to a heating block at 37°C and 1 tube for each VHH was removed at 5 minutes, 15 minutes, 30 minutes, and 45 minutes. After removing the tubes from the heating block, they were immediately transferred to ice to stop the digestion, and then 1.2 μL 0.1M NaOH was added to return the pH to 7.4 to inactivate the pepsin. Store samples on ice until running SDS-PAGE gel electrophoresis or using in chRBC lysis screening assay.

[0356] As a further test of selected clones, preparation of VHHs and incubation with pepsin were performed as described above, but the incubation time was extended to 120 min and samples were removed at 5, 30, 45, 60, 90 and 120 min.

[0357] After incubation with pepsin, VHHs were analyzed by SDS-PAGE gel

[0358] Take 5 μL of each terminated VHH sample and add it to each tube with 2 μL of 5x SDS loading buffer added and seal the tubes with foil adhesive plate sealer. Transfer the tubes to a heating block and incubate at 80 °C for 10 minutes and then spin to collect any evaporation. Run a 4% to 20% gradient SDS-PAGE gel electrophoresis on the denatured samples. After running the gel electrophoresis, stain with Coomassie Brilliant Blue staining reagent for 24 hours, then destain with water and subsequently photograph to assess degradation after digestion with pepsin.

[0359] NetB-induced erythroid assay using VHHs incubated with pepsin

[0360] Take 10 μL of each terminated VHH sample and add it to 65 μL PBS, then dilute it 1 to 3 times, from 2 μM to 5.49 nM. The final concentration of these dilutions is 4 times to allow for dilution after the addition of chRBC and NetB toxins. NetB will be used at a submaximal concentration (EC80) of 20 nM. The maximum final concentration of VHH is 500 nM = 25:1 (VHH: NetB) molar ratio, and the minimum concentration is 1.37 nM = 0.0685:1 molar ratio (VHH: NetB).

[0361] VHH was serially diluted 3-fold from 2 μM to 5.49 nM, and 17.5 μL of the diluted VHH was transferred to a V-bottom 96-well assay plate. Then 17.5 μL of NetB (diluted 4-fold with PBS, i.e., EC80) was added to the corresponding wells. For the positive control, only NetB was used. The negative control did not contain NetB. NetB and VHH clones (total volume of 35 μL) were incubated in the assay plate for 15 minutes at room temperature, and then 35 μL of washed and prepared RBCs were added. The assay plate was sealed with an adhesive plate seal and incubated at 37°C with shaking (450 rpm) for 30 minutes. After incubation, the plate was centrifuged at 1100 x g for 3 minutes, and 50 μL was removed from each sample onto a suitable flat-bottom plate, taking care not to disturb the RBC pellet. The OD405 of the plate was recorded using a spectrophotometer.

[0362] Results and Discussion:

[0363] The data clearly show that although some VHHs are easily degraded after incubation with pepsin (judged by the presence of multiple degradation bands and the loss of the 15 kDa VHH band, see e.g. Figure 3A and Figure 3B However, some of these VHHs remained largely intact and were still able to inhibit NetB-induced erythrocyte lysis even after incubation with 30 U / ml pepsin for 120 min. For example, Net 83 still had a 15 kDa band at 120 min (see Figure 3C ), and 250 nM Net 83 still showed 35% inhibition of NetB at 60 and 90 min (see Figure 3D ). Net 14 still showed 45% inhibition of NetB at 45 and 60 minutes (see Figure 3D ). Net 7 performed much worse in terms of pepsin resistance. Table 6 shows the IC50 of each clone in the NetB cleavage assay.

[0364] Table 6: Activity of VHH after incubation with pepsin

[0365]

[0366] Example 6: Stability and activity after incubation with pancreatin (resistance to pancreatin)

[0367] Candidate VHHs were incubated with increasing concentrations of trypsin for 30 minutes, and then the samples were run on SDS-PAGE gel electrophoresis to assess resistance to degradation. Selected clones (Net 83) were incubated with trypsin for an additional period of time. After trypsin incubation, the reactions were run on SDS-PAGE gel electrophoresis and the extent of degradation was assessed.

[0368] Materials and methods:

[0369] Incubation of VHH with different concentrations of trypsin

[0370] VHH was prepared as a solution with a final concentration of 30 μM and incubated with the following concentrations of pancreatin (specification 8×USP) (Sigma Aldrich; catalog number P7545-25G): 50 mM Tris-HCl + 10 mM CaCl2 at pH 6.8 to give a final concentration of 0 mg / ml, 0.1 mg / ml, 0.5 mg / ml, 1 mg / ml, 5 mg / ml. The samples were incubated at 37°C for 30 minutes and then the reaction was terminated by heating the samples in SDS loading buffer (95°C for 3 minutes). Gel electrophoresis was run on the samples in the control group without VHH to check the background of pancreatin.

[0371] Incubate selected clones with trypsin for a period of time

[0372] Pancreatin was prepared at a concentration of 25 mg / mL in 1 mM HCl (pH 3.0) and placed on a roller for 90 minutes.

[0373] After 60 minutes, trypsin was diluted to 1 mg / mL with assay buffer (50 mM Tris-HCl + 10 mM CaCl2 at pH 6.8) and kept on ice. VHH was diluted to 15 μM with assay buffer and kept on ice.

[0374] Trypsin and VHH were added together in a 1:1 ratio to give a final concentration of 0.5 mg / mL trypsin and 7.5 μM Net_83, and the tubes were placed on a heating block at 37 °C.

[0375] After 15, 30, 45, 60, 90, and 120 minutes, 20 μL was removed from these tubes, 4 μL of 5x SDS loading buffer was added, and the samples were denatured at 99°C for 3 minutes and then frozen.

[0376] After digestion, denaturation and freezing of all samples, SDS-PAGE electrophoresis was run on these samples.

[0377] Results and Discussion:

[0378] Data (see Figure 4A and Figure 4B ) clearly showed that the selected VHH (Net 83) remained largely intact after incubation with different concentrations of trypsin and remained intact even after incubation with 0.5 mg / ml trypsin for 90 minutes.

[0379] Example 7: Stability of VHH in water

[0380] Materials and methods

[0381] Incubate VHH in water

[0382] The buffer of VHH was replaced with water and VHH was diluted with water to 50 μg / ml (Net_14) or 100 μg / ml (Net_07 and Net_83) and incubated at room temperature. Samples were removed at different time points and frozen at -80°C until the study was completed, and then the samples were run on gel electrophoresis or tested for inhibition of NetB-induced erythrocyte lysis.

[0383] Preparation of VHHs to inhibit NetB-induced erythrocyte lysis

[0384] VHH was serially diluted 3-fold from 4 μM to 5.49 nM, and 17.5 μL of the diluted VHH was transferred to a V-bottom 96-well assay plate. Then 17.5 μL of NetB (diluted 4-fold with PBS, i.e., EC80) was added to the corresponding wells. For the positive control, only NetB was used. The negative control did not contain NetB. NetB and VHH clones (total volume of 35 μL) were incubated in the assay plate for 15 minutes at room temperature, and then 35 μL of washed and prepared RBCs were added. The assay plate was sealed with an adhesive plate seal and incubated at 37°C with shaking (450 rpm) for 30 minutes. After incubation, the plate was centrifuged at 1100 x g for 3 minutes, and 50 μL was removed from each sample onto a suitable flat-bottom plate, taking care not to disturb the RBC pellet. The OD405 of the plate was recorded using a spectrophotometer.

[0385] Results and Discussion

[0386] like Figure 5A As shown, the VHH remained unchanged during this time course: the 15 kDa band was still present after 4 days with no significant signs of degradation.

[0387] Figure 5B Both Table 7 and Table 7 show that the VHHs showed no loss of potency over the entire time course.

[0388] This time course was extended to 28 days and the stability of the VHH was still observed, including in the NetB functional assay.

[0389] Table 7: Stability of VHH in water

[0390]

[0391] Example 8: Affinity determination of VHH for NetB toxin (KD determined by Biacore)

[0392] Materials and methods:

[0393] The binding affinity of each identified lead VHH antibody to the NetB toxin of C. perfringens was determined by surface plasmon resonance.

[0394] KD determination by Biacore

[0395] To evaluate the affinity of the selected purified clones for NetB toxin, NetB toxin proteins were coated on CM5 sensor plates (Cytiva catalog number 29149603) by amine coupling. The binding kinetics of the selected VHHs at pH 7.4 were determined using single cycle kinetics (SCK) using surface plasmon resonance (SPR) (Biacore 8K+, Cytiva). Approximately 350 RU of NetB toxin in acetate buffer at pH 5.0 or pH 5.5 were immobilized on a CM5 chip using a standard amine coupling procedure. QC of immobilization was performed using a commercial antibody anti-histidine (BioLegend; catalog number 652505) diluted in 1xHBS-EP+ pH 7.4 buffer at 5 nM and 30 nM.

[0396] During the binding kinetics measurement, 1x HBS-EP at pH 7.4 was used as the running buffer. The purified VHHs were serially diluted two-fold with 1xHBS-EP at pH 7.4 from an initial concentration of 20 nM to 1.3 nM, and the diluted purified VHHs were injected at 30 μl / min for 2 min, followed by a 1 min off-rate wash during the VHH injection. In each cycle, the last off-rate wash was performed 5 min after the last VHH injection.

[0397] Affinity and maximum RU were calculated using the single cycle kinetics predefined evaluation method of the Biacore Insight evaluation software.

[0398] Results and Discussion:

[0399] As shown in Table 8 and Figure 6 As shown, the KD values ​​of the selected VHHs were 0.095 nM for Net14 and 0.213 nM for Net83.

[0400] Table 8: Summary of Biacore affinity data

[0401] VHH <![CDATA[Chi 2 (RU 2 )]]> Ka(1 / Ms) Kd(1 / s) Rmax(RU) KD(nM) Net_14 0.03 6.68E+05 6.32E-05 57 0.095 Net_83 0.05 1.17E+06 2.48E-04 62 0.213

[0402] Example 9: Study on the effectiveness of chicken necrotic enteritis model

[0403] The aim of this study was to investigate the safety and efficacy of Net_83 in combination with an antibody against Clostridium perfringens necrotic enteritis alpha (CPA) toxin in broiler chickens after experimentally induced necrotic enteritis (NE). The antibody against CPA was a VHH antibody provided by ECO Animal Health Ltd.

[0404] 1. Group Description

[0405] On the first day of the study, a total of 810 Ross 308 day-old chicks were distributed into pens with 15 birds per pen. The pens were then randomly assigned to one of the following treatment groups: T01 = untreated control after infection (IUC), T02 = unchallenged untreated (UUC), T03 = amoxicillin in drinking water for 5 days, T04 = CPA + Net-83 antibody in drinking water for 6 days, T05 = NaHCO in drinking water 3 6 days, T06 = CPA + Net_83 antibody added to water containing NaHCO3 for 6 days. Each group had 10 pens, except T02 group which had 4 pens. At the time of challenge, the number of birds in each pen was reduced to 13, and a total of 702 birds were included in the evaluation. All groups except T02 were challenged as further described below. Water treatment was started one day before the first C. perfringens challenge. Table 9 below lists the summary of each group.

[0406] Table 9: Description of the different treatment groups.

[0407]

[0408]

[0409] *Flocks were subjected to the appropriate test program and challenged with coccidia and C. perfringens (see challenges described further below).

[0410] 2. Chicken Necrotic Enteritis Model

[0411] Necrotic enteritis is caused by a variety of predisposing factors. On days 14 and 16, a 10-fold overdose of a commercial Eimeria vaccine was given by gavage (to provide a coccidia challenge). On day 17, the feed was switched to a grower feed containing 30% fish meal. From day 18, an alpha toxin-producing, NetB-positive strain of Clostridium perfringens was given by oral gavage for four consecutive days. The flocks were followed until 30 days of age.

[0412] Administration of therapeutic substances

[0413] 2.1. Feasibility of Net_83 application in water

[0414] Prior to the efficacy study, the feasibility of administering Net_83 antibodies in water was evaluated in a pilot study. The aim of the study was to evaluate whether Net_83 reaches the gastrointestinal tract of chickens when administered in drinking water alone (T01-1) or in combination with NaHCO3 (T02-1). Each treatment was administered ad libitum over a 24-hour period and 10 birds were housed in each pen. Each pen had a water pipe that simulated a commercial farm. Digestive samples were collected 24 hours after the start of administration and the crop was analyzed for the presence of Net_83 by ELISA. Comparable amounts of antibodies were extracted from crop samples in both treatment groups ( Figure 7 ).

[0415] This study demonstrated that the antibodies were stable under these conditions of administration and that this was a suitable method for administering antibodies to chickens, as the presence of antibodies in the crop indicated that the antibodies reached the digestive tract of the chickens using this method.

[0416] 2.2 Administration of therapeutic agents in efficacy studies

[0417] Treatments were administered via drinking water provided ad libitum.

[0418] The chickens in T03 were given a commercially available water-soluble amoxicillin drinking water. According to the label recommendations, a target dose of 13.1 mg amoxicillin / kg body weight was continued for 5 days. Amoxicillin treatment was administered prophylactically starting 1 day before the C. perfringens challenge and continued for 5 days (study days 17, 18, 19, 20 and 21).

[0419] The birds in T04 and T06 drank water supplemented with the same concentration of Net_14 and CPA antibody combination. From day 17 to day 22, water treatments were provided at a dose of 5 mg / bird / day of each antibody. The antibodies were mixed in the water that was consumed in one pen for one day as follows:

[0420] 1. Gently homogenize the vials containing CPA and Net-83 antibodies by inversion.

[0421] 2. Mix the contents of each vial into the amount of water added to the corresponding pen that day.

[0422] Net_83 and CPA antibodies are provided as a ready-to-use solution that can be mixed into drinking water.

[0423] Water was prepared daily for each pen using tap water. For T06, a stock solution of 0.5 g / L sodium bicarbonate was prepared in water prior to adding the antibodies. The amount of water that needed to be added depended on the expected daily water intake (DWI). The amount for each pen was calculated based on the number of birds in each pen and the daily feed intake, per Ross 308 2018. It was assumed that birds consumed twice as much water as they consumed feed. Based on the estimated water intake, the target concentration (mg / mL) of each antibody in the water was calculated to achieve a dose of 5 mg / bird.

[0424] 3. Effectiveness evaluation

[0425] The primary variable was NE-related mortality.

[0426] The high NE-related mortality rate in T01 (IUC) (79%) confirms that the necrotic enterocolitis challenge was successful. This mortality rate was higher than expected, resulting in lower statistical power. Preliminary survival analysis showed that the two groups treated with the antibody (T04 and T06) performed very similarly ( Figure 8 ). The analysis also showed that NaHCO 3 There was no effect on mortality and it can be considered a placebo ( Figure 8 ). Since no NaHCO 3 To improve the statistical power, we also analyzed the effect of T01 (IUC) and T05 (NaHCO 3 The pooled data of the 24-week group (untreated flocks) were additionally analyzed with the pooled data of the antibody-treated groups (T04 and T06).

[0427] Survival analysis of the pooled data showed that the mortality rate of the treated flocks was significantly higher than that of the untreated flocks (77%; P = 0.038; Fig. 9 ), the mortality rate of chickens treated with antibodies was significantly reduced (71%).

[0428] Several pens died completely due to the severity of the challenge. Additional survival analyses were performed to compare the probability of a pen dying completely in the treated versus untreated groups. Pooled data analysis showed that the IUC group and NaHCO 3 Compared with the pooled data from the 2 groups, fewer pens had total mortality due to NE in the antibody-treated flocks (45% vs 75%; P = 0.048; Fig.10 ). This suggests that in the wild, cells treated with these VHHs are less affected by NE.

[0429] No adverse events related to the administered product were reported.

[0430] In conclusion, this study resulted in higher than expected NE mortality and had low statistical power. In addition, severe challenges resulted in reduced water intake and failure to reach target antibody doses on some treatment days. Nevertheless, additional analyses of the pooled data showed that even under these severe challenge conditions (which are expected to be much more severe than what would be seen in flocks naturally exposed to these NE-inducing pathogens in the field), the Net_83+CPA antibody combination still achieved a statistically significant reduction in mortality compared to untreated flocks.

[0431] Example 10: Expression of Net_83 and Net_14 in Bacillus subtilis as a carrier for oral delivery

[0432] Bacillus subtilis strain (PY79, obtained from the Bacillus Genetic Resource Center, BGSID number: 1A747) was genetically engineered to express Net_14 or Net_83. For each construct, the antibody was cloned into the amyE locus of PY79 and a chloramphenicol (Cm) resistance selection marker was used as described elsewhere (Yang et al., 2020). Bacterial culture supernatants were collected and subjected to ELISA to confirm the expression of binding antibodies as described below.

[0433] Briefly, Maxisorp plates were coated with NetB-His toxin at a concentration of 2 μg / ml in 1x PBS overnight at 4°C. The coated plates were blocked with 4% skim milk in 1x PBS for 1 hour at room temperature. Supernatants from three Bacillus subtilis clones expressing Net_14, three Bacillus subtilis clones expressing Net_83, and two Bacillus subtilis clones expressing an unrelated VHH clone (anti-JTT) were titrated by performing a 7-step serial dilution (starting with 1% skim milk in 1x PBS at a ratio of 1:2).

[0434] The antibodies were detected using goat anti-VHH antibody (Jackson ImmunoResearch, Cat. No. 128-005-230) diluted 1:500 with 1% skim milk in 1x PBS.

[0435] Donkey anti-goat-HRP antibody (Jackson ImmunoResearch, catalog number 705-035-147) was diluted 1:5000 with 1% skim milk in 1xPBS and used as secondary detection.

[0436] Coating control was performed using anti-Histidine-HRP (Miltenyi Biotec, catalog number 130-092-783) diluted 1:500 with 1% skim milk in 1x PBS.

[0437] Binding to NetB-His was assessed by measuring OD at 450 nm.

[0438] Results and Conclusions:

[0439] Supernatants from all three B. subtilis clones expressing Net_14 showed positive binding to NetB toxin at dilutions as low as 1:100,000 ( Fig.11 ), and each supernatant should contain 15 μg / mL to 19 μg / mL of Net_14 VHH.

[0440] Supernatants from all three B. subtilis clones expressing Net_83 showed positive binding to NetB toxin at dilutions ranging from 1:2 to 1:1000 ( Fig.12 ), and each supernatant should contain approximately 4 μg / mL of Net_83 antibody.

[0441] It can be concluded that Bacillus subtilis is a suitable carrier for Net_14 and Net_83 antibodies, as Bacillus subtilis can be used to express functional antibodies that bind to the target NetB toxin in the supernatant. In other experiments, it was also demonstrated that these Bacillus subtilis secreted antibodies retained the function of inhibiting NetB-induced chRBC lysis.

[0442] References:

[0443] Yang M, Zhu G, Korza G, Sun X, Setlow P, Li J. 2020. Engineering Bacillus subtilis as a versatile and stable platform for production of nanobodies. ApplEnviron Microbiol 86:e02938-19.

Claims

1. A binding protein comprising at least one antigen binding domain for necrotic enteritis B-like toxin (NetB), wherein the antigen binding domain comprises a heavy chain variable region comprising three complementarity determining regions (CDRs), wherein the binding protein binds to NetB with a KD of 250 pM or less at pH 7.

4.

2. The binding protein according to claim 1, wherein The binding protein can reduce the toxic activity of NetB at pH 6.

0.

3. The binding protein according to claim 1 or 2, wherein The binding protein is capable of binding to 50 Value reduces EC 80 concentration of NetB or 20 nM NetB cytotoxic activity against chicken erythrocytes.

4. The binding protein according to any one of claims 1 to 3, wherein The binding protein showed resistance to pepsin digestion at a pepsin concentration of 30 U / ml at a temperature of 37°C.

5. The binding protein according to any one of claims 1 to 4, wherein The binding protein showed resistance to chymotrypsin digestion at a chymotrypsin concentration of 1 μg / ml at a temperature of 37°C.

6. The binding protein according to any one of claims 1 to 5, wherein The binding protein showed resistance to trypsin digestion at a trypsin concentration of 0.5 μg / ml at a temperature of 37°C.

7. The binding protein according to any one of claims 1 to 6, wherein The heavy chain variable region comprises: (i) a variable heavy chain (VH) CDR1 comprising the amino acid sequence of GSIRSISFMN (SEQ ID NO: 2) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing 1, 2, 3 or 4 amino acid substitutions compared to the given CDR sequence, (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence INQIGNT (SEQ ID NO: 3) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing 1, 2 or 3 amino acid substitutions compared to the given CDR sequence, and (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence of IVRGTVSPFQF (SEQ ID NO: 4) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing 1, 2, 3 or 4 amino acid substitutions compared to the given CDR sequence.

8. The binding protein according to any one of claims 1 to 6, wherein The heavy chain variable region comprises: (i) a variable heavy chain (VH) CDR1 comprising the amino acid sequence of GLTFSGYSLG (SEQ ID NO: 10) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing 1, 2, 3 or 4 amino acid substitutions compared to the given CDR sequence, (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence of ISSSGMIT (SEQ ID NO: 11) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing 1, 2 or 3 amino acid substitutions compared to the given CDR sequence, and (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence of RFPRPRSWLSTDSYNY (SEQ ID NO: 12) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing 1, 2, 3 or 4 amino acid substitutions compared to the given CDR sequence.

9. The binding protein according to any one of claims 1 to 8, wherein The binding protein is or comprises an antibody.

10. The binding protein according to claim 9, wherein The antibody is a single domain antibody.

11. An antibody that binds to the same epitope of NetB as an antibody having a heavy chain variable region as defined in claim 7 or 8.

12. The binding protein or antibody according to any one of claims 1 to 11, wherein The binding proteins or antibodies are provided as dimers or larger multimers.

13. One or more nucleic acid molecules comprising a nucleotide sequence encoding a binding protein or antibody according to any one of claims 1 to 12.

14. One or more expression vectors comprising one or more nucleic acid molecules according to claim 13.

15. One or more host cells comprising the expression vector of claim 14, or the nucleic acid molecule of claim 13, or expressing the antibody or binding protein of any one of claims 1-12.

16. The host cell according to claim 15, wherein The cells are bacterial cells, preferably probiotic cells, or yeast cells.

17. The host cell according to claim 16, wherein The bacterial cell is Bacillus, preferably Bacillus subtilis; Lactobacillus, preferably Lactobacillus reuteri; Lactococcus; Salmonella; Escherichia coli or Listeria; or, wherein the yeast cell is Pichia or Saccharomyces.

18. A method for producing a binding protein or antibody as described in any one of claims 1 to 12, the method comprising (i) culturing a host cell as described in any one of claims 15 to 17 under conditions suitable for expressing the encoded binding protein or antibody; and optionally (ii) isolating or obtaining the binding protein or antibody from the host cell or from the growth medium / supernatant.

19. A composition, preferably a pharmaceutically acceptable composition, comprising the binding protein or antibody according to any one of claims 1 to 12, one or more nucleic acid molecules according to claim 13, one or more expression vectors according to claim 14, or one or more host cells according to claims 15 to 17.

20. The composition of claim 19 comprising an additional active agent.

21. The composition of claim 20, wherein The additional active agent may be an agent that inhibits Clostridium perfringens alpha toxin (CPA), preferably an anti-CPA antibody.

22. A food product or food additive, preferably an animal feed product or additive, comprising the binding protein or antibody according to any one of claims 1 to 12, one or more host cells according to any one of claims 15 to 17, or the composition according to any one of claims 19 to 21.

23. The food product or food additive according to claim 22, wherein: The product or additive is a feed premix or surface auxiliary powder.

24. The binding protein or antibody of any one of claims 1-12, the one or more nucleic acid molecules of claim 13, the one or more expression vectors of claim 14, the one or more host cells of claims 15-17, or the composition of any one of claims 19-21, for use in the treatment of a subject; preferably for the treatment or prevention of Clostridium perfringens infection in a subject.

25. The binding protein or antibody of any one of claims 1-12, the one or more nucleic acid molecules of claim 13, the one or more expression vectors of claim 14, the one or more host cells of claims 15-17, or the composition of any one of claims 19-21, for treating or preventing necrotic enteritis (NE) in a subject.

26. Use of the binding protein or antibody according to any one of claims 1 to 12, one or more nucleic acid molecules according to claim 13, one or more expression vectors according to claim 14, one or more host cells according to claims 15 to 17, or the composition according to any one of claims 19 to 21 in the preparation of a medicament or composition for treating or preventing Clostridium perfringens infection in a subject.

27. Use of a binding protein or antibody as described in any one of claims 1 to 12, one or more nucleic acid molecules as described in claim 13, one or more expression vectors as described in claim 14, one or more host cells as described in claims 15 to 17, or a composition as described in any one of claims 19 to 21 in the preparation of a medicament or composition for treating or preventing necrotic enteritis (NE) in a subject.

28. A method for treating or preventing Clostridium perfringens infection in a subject, wherein: The method comprises administering to a subject in need thereof a therapeutically effective amount of a binding protein or antibody as described in any one of claims 1 to 12, one or more nucleic acid molecules as described in claim 13, one or more expression vectors as described in claim 14, one or more host cells as described in claims 15 to 17, or a composition as described in any one of claims 19 to 21.

29. A method for treating or preventing necrotic enterocolitis (NE) in a subject, wherein: The method comprises administering to a subject in need thereof a therapeutically effective amount of a binding protein or antibody as described in any one of claims 1 to 12, one or more nucleic acid molecules as described in claim 13, one or more expression vectors as described in claim 14, one or more host cells as described in claims 15 to 17, or a composition as described in any one of claims 19 to 21.

30. The use of the binding protein, antibody, nucleic acid molecule, expression vector, host cell or composition of claim 24 or claim 25, the use of claim 26 or claim 27, or the method of claim 28 or claim 29, wherein: The subject is an avian species, preferably poultry, more preferably chicken.

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