Triple paratopic antibodies and methods of use thereof

Abstract

Provided herein are nanobody-based triple- and tetra-paratopic antibodies (NTAbs) and pharmaceutical compositions comprising the same. Also provided herein are methods of using NTAbs for treating infection (e.g., Clostridioides difficile infection or Clostridium botulinum infection) or intoxication by a toxin (e.g., botulinum neurotoxin, Toxin A, Toxin B).

Description

[0001] TRIPLE PARATOPIC ANTIBODIES AND METHODS OF USE THEREOF RELATED APPLICATIONS

[0002] This Application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 751022, filed on January 29, 2025, the entire contents of which are incorporated herein by reference.

[0003] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0004] The contents of the electronic sequence listing (C123370293WO00-SEQ-ACZ.xml; Size: 226,720 bytes; and Date of Creation: January 28, 2026) are herein incorporated by reference in their entirety.

[0005] FEDERALLY SPONSORED RESEARCH

[0006] This invention was made with government support under Grant Numbers AI132387 and NS080833, awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0007] BACKGROUND

[0008] Bacterial protein toxins are a cause of disease in humans and animals. Anti-toxin serum has been utilized for treatment for more than a century, with a successful track record. However, this traditional approach of using sera from large animals or human donors has many drawbacks such as batch variation, limited availability, and allergic reactions. A modern approach to address these drawbacks is the use of monoclonal antibodies. However, this modern approach has its own limitations when used against potent toxins, including lower efficacy in vivo than the traditional approach of using polyclonal anti-sera.

[0009] SUMMARY

[0010] Monoclonal antibodies are a commonly used therapeutic modality because they can be readily produced and have a long half-life in serum. However, the use of a single monoclonal antibody has been shown to be inadequate in protecting against potent toxins in vivo. Programs using a combination of monoclonal antibodies have shown to be effective in vivo in animal models. However, these programs greatly increase the complexity of drug development: the monoclonal antibodies need to be manufactured individually, co-formulations must be developed, and the quality and stability of each antibody individually and in combination needs

[0011] 12413624.1to be fully analyzed. Furthermore, antibodies can exhibit drastic differences in pharmacological kinetics, creating a major challenge for dosing. The inventors of this disclosure surprisingly found that the caveats of current antibody combination approaches can be addressed by nanobody-based triple- and tetra-epitope antibodies (NTAbs). Specifically, the inventors found that by modulating the linker length and choice of nanobodies, NTAbs could be designed such that each a single toxin molecule could be simultaneously bound by multiple NTAbs, each binding to a different epitope on the target. The inventors unexpectedly found that NTAbs promote in vivo clearance with greater than 100-fold efficacy than a cocktail of multiple individual VHH-Fcs.

[0012] Accordingly, in some aspects, this disclosure provides an antibody, comprising: a first VHH that binds to a first epitope of a botulinum toxin (BoNT); a second VHH that binds to a second epitope of the BoNT; a third VHH that binds to a third epitope of the BoNT; and a constant region (Fc) of an immunoglobulin (Ig). In some embodiments, the BoNT is BoNT type A (BoNT / A), BoNT type B (BoNT / B), BoNT type C (BoNT / C), BoNT type D (BoNT / D), BoNT type E (BoNT / E), BoNT type F (BoNT / F), or BoNT type G (BoNT / G).

[0013] In some embodiments, the first epitope, the second epitope, and the third epitope are selected from the group consisting of a BoNT light chain (LC) epitope and a BoNT heavy chain epitope. In some embodiments, the BoNT heavy chain is an amino-terminal part (HN) or a carboxy-terminal part (HC).

[0014] In some embodiments, the BoNT is BoNT / A. In some embodiments, the first epitope is a BoNT / A-LC epitope, the second epitope is a BoNT / A-HN epitope, and the third epitope is a BoNT / A-HC epitope.

[0015] In some embodiments, the first VHH comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 2 or SEQ ID NO: 5; the second VHH comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 3 or SEQ ID NO: 6; and, the third VHH comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 4 or SEQ ID NO: 7. In some embodiments, the first VHH comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 5; the second VHH comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 6; and the third VHH comprises the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 7. In some embodiments, the first VHH consists of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 5; the second VHH consists of the amino acid sequence of SEQ ID NO: 3

[0016] 12413624.1or SEQ ID NO: 6; and the third VHH consists of the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 7.

[0017] In some embodiments, the antibody comprises, from N-terminal to C-terminal, the first VHH, the second VHH, the third VHH, and the Fc. In some embodiments, the antibody comprises, from N-terminal to C-terminal, the first VHH, the third VHH, the second VHH, and the Fc. In some embodiments, the antibody comprises, from N-terminal to C-terminal, the second VHH, the first VHH, the third VHH, and the Fc. In some embodiments, the antibody comprises, from N-terminal to C-terminal, the second VHH, the third VHH, the first VHH, and the Fc. In some embodiments, the antibody comprises, from N-terminal to C-terminal, the third VHH, the first VHH, the second VHH, and the Fc. In some embodiments, the antibody comprises, from N-terminal to C-terminal, the third VHH, the second VHH, the first VHH, and the Fc.

[0018] In some embodiments, the BoNT is BoNT / B. In some embodiments, the first epitope is a BoNT / B-EC epitope, the second epitope is a BoNT / B-HN epitope, and the third epitope is a BoNT / B -HC epitope.

[0019] In some embodiments, the first VHH comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 20 or SEQ ID NO: 23; the second VHH comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 21 or SEQ ID NO: 24; and the third VHH comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 22 or SEQ ID NO: 25. In some embodiments, the first VHH comprises the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 23; the second VHH comprises the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 24; and the third VHH comprises the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 25. In some embodiments, the first VHH consists of the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 23; the second VHH consists of the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 24; and the third VHH consists of the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 25.

[0020] In some embodiments, the antibody comprises, from N-terminal to C-terminal, the first VHH, the second VHH, the third VHH, and the Fc. In some embodiments, the antibody comprises, from N-terminal to C-terminal, the first VHH, the third VHH, the second VHH, and the Fc. In some embodiments, the antibody comprises, from N-terminal to C-terminal, the second VHH, the first VHH, the third VHH, and the Fc. In some embodiments, the antibody comprises, from N-terminal to C-terminal, the second VHH , the third VHH, the first VHH, and

[0021] 12413624.1the Fc. In some embodiments, the antibody comprises, from N-terminal to C-terminal, the third VHH, the first VHH, the second VHH, and the Fc. In some embodiments, the antibody comprises, from N-terminal to C-terminal, the third VHH, the second VHH, the first VHH, and the Fc.

[0022] In some embodiments, the first VHH, the second VHH, the third VHH, and the Fc are connected by a peptide linker. In some embodiments, the peptide linker is a flexible linker. In some embodiments, the flexible linker is (GGGS)n (SEQ ID NOs: 151-155) or (GGGGS)n (SEQ ID NOs: 146-150), where n is 1, 2, 3, 4, or 5. In some embodiments, the peptide linker is a rigid linker. In some embodiments, the rigid linker is (EAAAK)n (SEQ ID NOs: 156-160), where n is 1, 2, 3, 4, or 5.

[0023] In some embodiments, the immunoglobulin (Ig) is an immunoglobulin G (IgG), an immunoglobulin M (IgM), an immunoglobulin A (IgA), an immunoglobulin D (IgD), or an immunoglobulin E (IgE). In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the Fc consists of the amino acid sequence of SEQ ID NO: 1.

[0024] In some embodiments, the antibody comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 8-19 or 26-37. In some embodiments, the antibody comprises the amino acid sequence of any one of SEQ ID NOs: 8-19 or 26-37. In some embodiments, the antibody consists of the amino acid sequence of any one of SEQ ID NOs: 8-19 or 26-37.

[0025] In some embodiments, this disclosure provides a nucleic acid which encodes, or a set of nucleic acids which collectively encode, an antibody of the present disclosure. In some embodiments, this disclosure provides a vector or vector set, comprising a nucleic acid or a set of nucleic acids described herein. In some embodiments, the vector(s) is (are) an expression vector(s).

[0026] In some embodiments, this disclosure provides a cell comprising a vector or vector set described herein. In some embodiments, the cell is a bacterial cell, a yeast cell, an insect cell, a plant cell, or a mammalian cell. In some embodiments, the cell is a Chinese Hamster Ovary (CHO) cell.

[0027] In some embodiments, this disclosure provides a method of producing an antibody, comprising: culturing a cell described herein in a culture medium; and collecting the cultured

[0028] 12413624.1cells or the culture medium for isolation of the antibody. In some embodiments, the method further comprises isolating the antibody from the cultured cells or the culture medium.

[0029] In some embodiments, this disclosure provides a pharmaceutical composition, comprising: an antibody of the present disclosure, a nucleic acid of the present disclosure, or a vector or vector set of the present disclosure; and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is for use in treating botulism.

[0030] In some aspects, this disclosure provides a method of treating a Clostridium botulinum infection in a subject, comprising administering to the subject an antibody of the present disclosure, a nucleic acid of the present disclosure, a vector or vector set of the present disclosure, or a pharmaceutical composition of the present disclosure. In some aspects, this disclosure provides a method of reducing the severity of one or more symptoms associated with a Clostridium botulinum infection or intoxication by botulinum neurotoxins in a subject, comprising administering to the subject an antibody of the present disclosure, a nucleic acid of the present disclosure, a vector or vector set of the present disclosure, or a pharmaceutical composition of the present disclosure.

[0031] In some embodiments, the Clostridium botulinum produces botulinum neurotoxin (BoNT) type A (BoNT / A), BoNT type B (BoNT / B), BoNT type C (BoNT / C), BoNT type D (BoNT / D), BoNT type E (BoNT / E), BoNT type F (BoNT / F), or BoNT type G (BoNT / G). In some embodiments, the Clostridium botulinum infection or botulinum neurotoxin intoxication is foodborne botulism, wound botulism, infant botulism, iatrogenic botulism, or adult intestinal toxemia. In some embodiments, the subject exhibits one or more symptoms of Clostridium botulinum infection or botulinum neurotoxin intoxication selected from the group consisting of fatigue, weakness, vertigo, blurred vision, dry mouth, difficulty swallowing or speaking, vomiting, diarrhea, constipation, abdominal swelling, weakness in the neck or arms, descending symmetrical paralysis, ptosis, facial palsy, ophthalmoplegia, and diplopia.

[0032] Aspects of this disclosure provide a VHH comprising an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 5-7 or 23-25.

[0033] In some aspects, this disclosure provides an antibody, comprising: a first VHH that binds to a first epitope of a Clostridioides difficile toxin (Ted); a second VHH that binds to a second epitope of the Ted; a third VHH that binds to a third epitope of the Ted; and a constant region (Fc) of an immunoglobulin (Ig). In some embodiments, the Ted is Clostridioides difficile toxin A (TcdA) or Clostridioides difficile toxin B (TcdB).

[0034] 12413624.1In some embodiments, the first epitope, the second epitope, and the third epitope are selected from the group consisting of a Ted N-terminal glucosyltransferase domain (GTD), a Ted autoprocessing domain (APD), a Ted delivery and receptor-binding domain (DRBD), and a Ted combined repetitive oligopeptides (CROPs) domain. In some embodiments, the first epitope, the second epitope, and the third epitope are TcdB epitopes. In some embodiments, the first epitope is a TcdB-DRBD epitope, the second epitope is a TcdB-GTD epitope, and the third epitope is a TcdB-GTD epitope. In some embodiments, the second epitope and the third epitope are different epitopes of TcdB-GTD.

[0035] In some embodiments, the first VHH comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 38 or SEQ ID NO: 41; the second VHH comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 39 or SEQ ID NO: 42; and, the third VHH comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 40 or SEQ ID NO: 43. In some embodiments, the first VHH comprises the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 41; the second VHH comprises the amino acid sequence of SEQ ID NO: 39 or SEQ ID NO: 42; and, the third VHH comprises the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43. In some embodiments, the first VHH consists of the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 41; the second VHH consists of the amino acid sequence of SEQ ID NO: 39 or SEQ ID NO: 42; and, the third VHH consists of the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43.

[0036] In some embodiments, the antibody comprises, from N-terminal to C-terminal, the first VHH, the second VHH, and third VHH, and the Fc. In some embodiments, the antibody comprises, from N-terminal to C-terminal, the first VHH, the third VHH, the second VHH, and the Fc. In some embodiments, the antibody comprises, from N-terminal to C-terminal, the second VHH, the first VHH, and third VHH, and the Fc. In some embodiments, the antibody comprises, from N-terminal to C-terminal, the second VHH, the third VHH, the first VHH, and the Fc. In some embodiments, the antibody comprises, from N-terminal to C-terminal, the third VHH, the first VHH, the second VHH, and the Fc. In some embodiments, the antibody comprises, from N-terminal to C-terminal, the third VHH, the second VHH, the first VHH, and the Fc.

[0037] In some embodiments, the antibody further comprises a fourth VHH that binds to a fourth epitope of the Ted, wherein the fourth epitope is a TcdA epitope. In some embodiments, the TcdA epitope is selected from the group consisting of a Ted AN-terminal glucosyltransferase

[0038] 12413624.1domain (GTD), a TcdA autoprocessing domain (APD), a TcdA delivery and receptor-binding domain (DRBD), and a TcdA combined repetitive oligopeptides (CROPs) domain.

[0039] In some embodiments, the fourth VHH comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 56-61. In some embodiments, the fourth VHH comprises the amino acid sequence of SEQ ID NO: 56-61. In some embodiments, the fourth VHH consists of the amino acid sequence of SEQ ID NO: 56-61.

[0040] In some embodiments, the antibody comprises, from N-terminal to C-terminal, the fourth VHH, the first VHH, the second VHH, the third VHH, and the Fc. In some embodiments, the antibody comprises, from N-terminal to C-terminal, the fourth VHH, the first VHH, the third VHH, the second VHH, and the Fc. In some embodiments, the antibody comprises, from N-terminal to C-terminal, the fourth VHH, the second VHH, the first VHH, the third VHH, and the Fc. In some embodiments, the antibody comprises, from N-terminal to C-terminal, the fourth VHH, the third VHH, the first VHH, the second VHH, and the Fc. In some embodiments, the antibody comprises, from N-terminal to C-terminal, the fourth VHH, the second VHH, the third VHH, the first VHH, and the Fc. In some embodiments, the antibody comprises, from N-terminal to C-terminal, the fourth VHH, the third VHH, the second VHH, the first VHH, and the Fc.

[0041] In some embodiments, the antibody comprises, from N-terminal to C-terminal, the first VHH, the second VHH, the third VHH, the Fc, and the fourth VHH. In some embodiments, the antibody comprises, from N-terminal to C-terminal, the first VHH, the third VHH, the second VHH, the Fc, and the fourth VHH. In some embodiments, the antibody comprises, from N-terminal to C-terminal, the second VHH, the first VHH, the third VHH, the Fc, and the fourth VHH. In some embodiments, the antibody comprises, from N-terminal to C-terminal, the third VHH, the first VHH, the second VHH, the Fc, and the fourth VHH. In some embodiments, the antibody comprises, from N-terminal to C-terminal, the second VHH, the third VHH, the first VHH, the Fc, and the fourth VHH. In some embodiments, the antibody comprises, from N-terminal to C-terminal, the third VHH, the second VHH, the first VHH, the Fc, and the fourth VHH.

[0042] In some embodiments, the first VHH, the second VHH, the third VHH, and the Fc are connected by a peptide linker. In some embodiments, the first VHH, the second VHH, the third VHH, the fourth VHH, and the Fc are connected by a peptide linker. In some embodiments, the peptide linker is a flexible linker. In some embodiments, the flexible linker is (GGGS)n (SEQ ID

[0043] 12413624.1NOs: 151-155) or (GGGGS)n (SEQ ID NOs: 146-150), where n is 1, 2, 3, 4, or 5. In some embodiments, the peptide linker is a rigid linker. In some embodiments, the rigid linker is (EAAAK)n (SEQ ID NOs: 156-160), where n is 1, 2, 3, 4, or 5.

[0044] In some embodiments, the immunoglobulin (Ig) is an immunoglobulin G (IgG), an immunoglobulin M (IgM), an immunoglobulin A (IgA), an immunoglobulin D (IgD), or an immunoglobulin E (IgE). In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the Fc consists of the amino acid sequence of SEQ ID NO: 1.

[0045] In some embodiments, the antibody comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 44-55, 62-72, or 74-145. In some embodiments, the antibody comprises the amino acid sequence of any one of SEQ ID NOs: 44-55, 62-72, or 74-145. In some embodiments, the antibody consists of the amino acid sequence of any one of SEQ ID NOs: 44-55, 62-72, or 74-145.

[0046] In some embodiments, this disclosure provides a nucleic acid which encodes, or a set of nucleic acids which collectively encode, an antibody of the present disclosure. In some embodiments, this disclosure provides a vector or vector set, comprising an isolated nucleic acid or a set of isolated nucleic acids described herein. In some embodiments, the vector(s) is (are) an expression vector(s).

[0047] In some embodiments, this disclosure provides a cell comprising a vector or vector set described herein. In some embodiments, the cell is a bacterial cell, a yeast cell, an insect cell, a plant cell, or a mammalian cell. In some embodiments, the cell is a Chinese Hamster Ovary (CHO) cell.

[0048] In some embodiments, this disclosure provides a method of producing an antibody, comprising: culturing cells of the present disclosure in a culture medium; and collecting the cultured cells or the culture medium for isolation of the antibody. In some embodiments, the method further comprises isolating the antibody from the cultured cells or the culture medium.

[0049] In some embodiments, this disclosure provides a pharmaceutical composition, comprising: comprising an antibody of the present disclosure, a nucleic acid of the present disclosure, or a vector or vector set of the present disclosure; and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is for use in treating Clostridioides difficile infection.

[0050] 12413624.1In some aspects, this disclosure provides a method of treating a Clostridioides difficile infection in a subject, comprising administering to the subject an antibody of the present disclosure, a nucleic acid of the present disclosure, a vector or vector set of the present disclosure, or a pharmaceutical composition of the present disclosure. In some aspects, this disclosure provides a method of reducing the severity of one or more symptoms associated with a Clostridioides difficile infection or intoxication by Clostridioides difficile toxins in a subject, comprising administering to the subject an antibody of the present disclosure, a nucleic acid of the present disclosure, a vector or vector set of the present disclosure, or a pharmaceutical composition of the present disclosure. In some embodiments, the Clostridioides difficile produces Toxin A (TcdA) and / or Toxin B (TcdB). In some embodiments, the C o idioides difficile infection of intoxication by Clostridioides difficile toxins is recurrent C. difficile infection (rCDI), antibiotic associated diarrhea, C. difficile colitis, pseudomembranous colitis, toxic megacolon, or fulminant colitis. In some embodiments, the subject exhibits one or more symptoms of Clostridioides difficile infection selected from the group consisting of watery diarrhea, abdominal cramping and pain, fast heart rate, dehydration, fever, nausea, increased white blood cell count, kidney failure, loss of appetite, abdominal swelling, weight loss, blood or pus in stool, pseudomembranous colitis, colonic ileus, toxic megacolon, and sepsis.

[0051] Other aspects of this disclosure provide a VHH comprising an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 41-43 or 59-61.

[0052] BRIEF DESCRIPTION OF DRAWINGS

[0053] The accompanying drawings are not intended to be drawn to scale. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:

[0054] FIGs. 1A-1B show schematic drawings of nanobody-based triple paratopic antibodies (NTAbs) in comparison with IgG. FIG. 1A shows a schematic of IgG and a camelid heavychain-only antibody. FIG. IB shows a schematic of an exemplary NTAb that comprises three different VHHs that bind to three distinct sites on a target.

[0055] FIG. 2A-2C show exemplary design and characterization of anti-Clostridioides difficile (C. difficile') toxin B (anti-TcdB) Fc-tagged nanobodies and NTAb. FIG. 2A is a schematic drawing of mono- VHH antibody binding to C. difficile toxin B. FIG. 2B is a schematic illustration of mono- VHH (VHHl-Fc, VHH2-Fc, VHH3-Fc) and triple-paratopic VHH (VHH123-Fc) antibodies. FIG. 2C shows SDS-PAGE gel characterization of purified mono-

[0056] 12413624.1VHH and triple-paratopic VHH antibodies. NR: non-reducing conditions; R: reducing conditions.

[0057] FIGs. 3A-3B show dxAi-Clostridioides difficile (C. difficile) toxin B (anti-TcdB) VH antibody affinity characterization data. FIG. 3A shows binding affinity between mono- VHH or triple-paratopic VHH antibodies and TcdB subtype 1 (TcdBl) measured using biolayer interferometry assay (BLI). FIG. 3B shows binding affinity between mono- VHH or tripleparatopic VHH antibodies and TcdB subtype 2 (TcdB2) measured using BLI.

[0058] FIGs. 4A-4N show nanobody-based triple paratopic antibodies (NTAbs) can neutralize all twelve Clostridioides difficile (C. difficile) toxin B (TcdB) subtypes. FIGs. 4A-4L demonstrate neutralization using mono- VHH antibodies, a triple-paratopic VHH antibody, or a control monoclonal antibody bezlotoxumab against TcdB subtypes 1-12. FIG. 4M is a summary of percentage of protection for the mono- VHH antibodies, the triple-paratopic VHH antibody, and the control monoclonal antibody bezlotoxumab against TcdB subtypes 1-12. FIG. 4N shows the EC50 (nM) of the mono-VHH antibodies, the triple-paratopic VHH antibody, and the control monoclonal antibody bezlotoxumab against TcdB subtypes 1-12.

[0059] FIGs. 5A-5B show survival curves of mice administered a triple-paratopic VHH antibody, a cocktail of three mono-VHH antibodies, or a control monoclonal antibody bezlotoxumab prior to injection of a Clostridioides difficile (C. difficile) toxin B (TcdB). FIG.

[0060] 5A shows percentage of survival over time after intraperitoneal injection of TcdB subtype 1. FIG. 5B shows percentage of survival over time after intraperitoneal injection of TcdB subtype 2.

[0061] FIG. 6 shows a schematic of antibody testing on antibiotic-induced Clostridioides difficile (C. difficile) infection in mouse models.

[0062] FIG. 7 is a schematic of the three domains of botulinum neurotoxin (BoNT).

[0063] FIGs. 8A-8C show the design, production, and in vitro characterization of an antibotulinum neurotoxin (BoNT) nanobody-based triple paratopic antibody (NTAb). FIG. 8A is an illustration of mono-VHH antibody binding to BoNT. FIG. 8B shows SDS-PAGE analysis of the purified mono-VHH antibodies and the purified NTAb. NR: non-reduction condition; R: reduction condition. FIG. 8C shows binding affinity between mono-VHH or triple-paratopic VHH antibodies and botulinum neurotoxin (BoNT) measured using biolayer interferometry assay (BLI).

[0064] 12413624.1FIGs. 9A-9C show survival curves of mice administered a triple-paratopic VHH antibody or a cocktail of three mono- VHH antibodies and botulinum neurotoxin (BoNT). FIG.

[0065] 9A shows percentage of survival over time after administration of a mixture of BoNT and a triple-paratopic VHH antibody or a mixture of BoNT and a cocktail of three mono- VHH antibodies. FIG. 9B shows percentage of survival over time after administration of a low dose triple-paratopic VHH antibody or a low dose cocktail of three mono- VHH antibodies two days prior to administration of BoNT. FIG. 9C shows percentage of survival over time after administration of a high dose triple-paratopic VHH antibody or a high dose cocktail of three mono-VHH antibodies two days prior to administration of BoNT.

[0066] DETAILED DESCRIPTION

[0067] In vivo protection against potent toxins can require a combination of multiple antibodies that bind to different sites on toxins, likely because binding of multiple antibodies is necessary to mark the toxin molecule for clearance and degradation by immune and liver cells. In contrast, toxins bound by a single antibody are not cleared and may even gain longer serum half-life due to the long half-life of the antibody. Bound toxins can eventually dissociate from the antibody and thus maintain toxicity.

[0068] Nanobodies® (also referred to as VHH) are -12-15 kDa single-domain proteins derived from the single variable domain of the heavy-chain-only antibodies in Camelidae animals such as alpacas and llamas. Fusion of two or three nanobodies with Fc (e.g., nanobody-based Fc-tagged multi- valent antibodies) have been shown to be capable of simultaneously binding to their epitopes to achieve binding of a single antibody to a single epitope (1:1), high affinity multi-valent binding, and long half-lives in vivo. In contrast, the inventors surprisingly found that by modulating the linker length and choice of nanobodies to have one nanobody-based Fc-tagged antibody (e.g., NTAb) bind to a single epitope on the target a single toxin molecule could be decorated with multiple Fc (e.g., 1:3) to promote in vivo clearance.

[0069] Accordingly, aspects of this disclosure provide antibodies (e.g., nanobody-based triple-epitopic antibodies) comprising a first VHH that binds to a first epitope of a toxin, a second VHH that binds to a second epitope of the toxin, a third VHH that binds to a third epitope of the toxin, and a constant region (Fc) of an immunoglobulin (Ig). In some embodiments, antibodies of the present disclosure (e.g., nanobody-based tetra-epitopic antibodies) further comprise a fourth VHH that binds to a fourth epitope the toxin.

[0070] 12413624.1A “nanobody-based triple-epitopic antibody” or a “nanobody-based tetra-epitope antibody” (collectively NTAbs) are antibodies that comprise three or four nanobodies respectively, fused to a constant region (Fc) of an immunoglobulin (Ig). In contrast to traditional multi-valent antibodies, which are designed to ensure simultaneous binding of multiple nanobodies of the multi- valent antibody to achieve high affinity multivalent binding to a target, Antibodies of the present disclosure (e.g., NTAbs), in some embodiments, are designed to achieve binding of multiple (e.g., 3, 4, 5, or more) NTAbs to a single toxin. Without being bound by theory, this results in decoration of the toxin with multiple (e.g., 3, 4, 5, or more) Fc, promoting clearance of the bound toxin by immune and liver cells. In some embodiments, antibodies of the present disclosure (e.g., NTAbs) bind to three epitopes of a toxin. In some embodiments, antibodies of the present disclosure (e.g., NTAbs) further bind to a fourth epitope of a toxin. In some embodiments, where an antibody (e.g., NTAb) binds to four epitopes of a toxin, all four epitopes are from the same toxin. In some embodiments, at least one (e.g., 1, 2, 3, or 4) epitope(s) is (are) from a different toxin. In some embodiments the different toxins are different toxins produced by the same microorganism (e.g., Toxin A and Toxin B), different serotypes of the same toxin (e.g., botulinum neurotoxin serotype A and botulinum neurotoxin serotype B), and / or different subtypes of the same toxin (e.g., Toxin B subtype 1 and Toxin B subtype 2).

[0071] In some embodiments, antibodies of the present disclosure (e.g., NTAbs) comprise a constant region (Fc) of an immunoglobulin (Ig). In some embodiments, the immunoglobulin (Ig) is an immunoglobulin G (IgG), an immunoglobulin M (IgM), an immunoglobulin A (IgA), an immunoglobulin D (IgD), or an immunoglobulin E (IgE). In some embodiments, antibodies of the present disclosure (e.g., NTAbs) comprise a constant region (Fc) of an immunoglobulin G (IgG). The Fc of an IgG is a portion of an IgG antibody responsible for binding to Fc receptors on immune cells, such as macrophages, natural killer cells, and neutrophils, marking the IgG bound molecule for clearance by the immune system. In some embodiments, the Fc region of an IgG comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identity to DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID

[0072] 12413624.1NO: 1). In some embodiments, the Fc region of an IgG comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the Fc region of an IgG consists of the amino acid sequence of SEQ ID NO 1.

[0073] In some aspects, antibodies of the present disclosure (e.g., NTAbs) comprise a first VHH that binds to a first epitope of a toxin, a second VHH that binds to a second epitope of the toxin, a third VHH that binds to a third epitope of the toxin, and a constant region (Fc) of an immunoglobulin. In some aspects, antibodies of the present disclosure (e.g., NTAbs) comprise a first VHH that binds to a first epitope of a first toxin, a second VHH that binds to a second epitope of the first toxin, a third VHH that binds to a third epitope of the first toxin, a fourth VHH that binds to a fourth epitope of a second toxin, and a constant region (Fc) of an immunoglobulin (IgG). In some embodiments, the first toxin and the second toxin are the same toxin. In some embodiments, the first toxin and the second toxin are different toxins.

[0074] The term “toxin” as used herein refers to proteins, peptides, or macromolecule produced by microorganisms (e.g., bacteria, fungi, protozoa, dinoflagellates) that are biologically active and can interfere with physiological processes in a host, resulting in host injury that can lead to dysfunction of cellular or organ processes, disease, and / or death. In some embodiments, the toxin is produced by a bacterium (e.g., Clostridium botulinum, Clostridioides difficile), a fungus (e.g., Asperillus flavus, Claviceps prpurea), a protozoon (e.g., Plasmodium spp., Entamoeba histolytica), or a dinoflagellate (e.g., Alexadrium spp., Karenia brevis). In some embodiments, the toxin is a bacterial toxin (e.g., botulinum neurotoxin, Toxin A, Toxin B), a fungal toxin (e.g., aflatoxins, ergot alkaloids, mycotoxins), a protozoan toxin (e.g., hemozoin), or a dinoflagellate toxin (e.g., saxitoxins, brevetoxins).

[0075] In some embodiments, the toxin is a bacterial toxin. In some embodiments, the toxin is produced by a bacterium. Bacterial toxins are typically classified into two main categories based on their origin and mechanism of action: (1) endotoxins, which are components of the outer membrane of Gram-negative bacterial released upon bacterial cell death; and (2) exotoxins, which are generated and actively secreted by bacteria into their surround environment. In some embodiments, the toxin is an endotoxin. In some embodiments, the endotoxin is produced by Escherichia coli, Salmonella spp., Neisseria meningitidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Shigella spp., Haemophilus influenzae, Vibrio cholerae, Legionella pneumophila, or Enterobacter spp. In some embodiments, the endotoxin is a lipopolysaccharide (EPS) or a lipooligosaccharide (EOS). In some embodiments, the toxin is an exotoxin. In some

[0076] 12413624.1embodiments, the exotoxin is produced by Clostridium botulinum, Clostridium tetani, Clostridioides difficile, Clostridium perfringens, Corynebacterium diphtheriae, Bacillus anthracis, Bordatella pertussis, Vibrio cholerae, Escherichia coli, Shigella dysenteriae, Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa, Listeria monocytogenes, Helicobacter pylori, or Yersinia pestis. Non-limiting examples of exotoxins are provided in Table 1.

[0077] Table 1. Exemplary bacterial exotoxins.

[0078]

[0079] In some embodiments, the toxin is produced by Clostridium botulinum. In some embodiments, the toxin produced by Clostridium botulinum is a botulinum neurotoxin (BoNT). In some embodiments, the toxin is a botulinum neurotoxin. BoNTs, which can cause botulism, are the most toxic proteins and have been characterized as Category A agents by the Center for Disease Control and Prevention (CDC). BoNTs are 150-kDa modular proteins composed of three domains: a receptor-binding domain (He), a middle membrane translocation domain (HN), and an N-terminal protease domain (light chain, LC) (FIG. 7). The LC is the cargo that is delivered into the cytosol of a host cell, where it cleaves neuronal proteins (known as SNARE proteins) required for synaptic vesicle exocytosis. This leads to blockage of neurotransmitter release (e.g., acetylcholine) from nerve terminals and thus relaxation of muscles. There are three types of human botulism: (1) wound botulism, which occurs upon the delivery of clostridial spores into an anaerobic wound where the spores germinate into the vegetative cells that produce

[0080] 12413624.1BoNT; (2) ingestion botulism (also referred to as food botulism), which results from the ingestion of preformed BONTs in improperly cooked or stored food; and (3) infant botulism, which is caused by ingestion of clostridial spores (e.g., from raw or pasteurized honey) by a child with an underdeveloped intestinal microbiota system. BoNTs are also clinically useful reagents with cosmetic use (e.g., BOTOX®). Botulism poisoning in humans is typified by slurred speech, dry mouth, blurred or double vision, peripheral muscle weakness, and paralysis. There are seven serotypes of BoNTs that cause botulism, termed A-G. In some embodiments, the toxin is a BoNT serotype A (BoNT / A), a BoNT serotype B (BoNT / B), a BoNT serotype C (BoNT / C), a BoNT serotype D (BoNT / D), a BoNT serotype E (BoNT / E), a BoNT serotype F (BoNT / F), or a BoNT serotype G (BoNT / G).

[0081] In some embodiments, the toxin is a BoNT / A. BoNT / A poses the most serious threat to humans because of its high potency and long duration of action, which works by cleaving synaptosomal-associated protein (SNAP)-25, disrupting the SNARE complex and blocking neurotransmitter release. There are 8 subtypes of BoNT / A: BoNT / Al, BoNT / A2, BoNT / A3, BoNT / A4, BoNT / A5, B0NT / A6, BoNT / A7, and B0NT / A8. In some embodiments, the toxin is a BoNT / Al, BoNT / A2, BoNT / A3, BoNT / A4, BoNT / A5, B0NT / A6, BoNT / A7, or a B0NT / A8.

[0082] In some aspects, this disclosure provides a VHH that bind to an epitope of BoNT / A (e.g., BoNT / Al, BoNT / A2, BoNT / A3, BoNT / A4, BoNT / A5, B0NT / A6, BoNT / A7, or a B0NT / A8). In some embodiments, the epitope of BoNT / A is selected from the group consisting of a BoNT / A heavy chain (HC) epitope and a BoNT / A light chain epitope (LC). In some embodiments, the BoNT / A-HC is an amino-terminal part (BONT / A-HN) epitope or a carboxyterminal part (BoNT / A-Hc). In some embodiments, the VHH comprises an amino acid sequence that is at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 2-7. In some embodiments, the VHH comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 5-7.

[0083] Table 2. BoNT / A VHH sequences.

[0084]

[0085] 12413624.1

[0086]

[0087] In some embodiments, this disclosure provides an antibody comprising a first VHH that binds to a first epitope of a BoNT / A (e.g., BoNT / Al, BoNT / A2, BoNT / A3, BoNT / A4, BoNT / A5, B0NT / A6, BoNT / A7, or a B0NT / A8), a second VHH that binds to a second epitope of the BoNT / A, a third VHH that binds to a third epitope of the BoNT / A, and a constant region (Fc) of an immunoglobulin (IgG). In some embodiments, the first epitope, the second epitope, and the third epitope are selected from the group consisting of a BoNT / A heavy chain (HC) epitope and a BoNT / A light chain (LC) epitope. In some embodiments, the BoNT / A-HC is an amino-terminal part (BONT / A-HN) epitope or a carboxy-terminal part (BoNT / A-Hc) epitope. In some embodiments, the epitope (e.g., the first epitope, the second epitope, the third epitope) is selected from the group consisting of a BoNT / A-LC epitope (e.g., BoNT / A 1-LC epitope, BoNT / A2-LC epitope, BoNT / A3-LC epitope, BoNT / A4-LC epitope, BoNT / A5-LC epitope, B0NT / A6-LC epitope, BoNT / A7-LC epitope, or B0NT / A8-LC epitope), a BONT / A-HN epitope (e.g., BONT / A1-HN epitope, BONT / A2-HN epitope, BONT / A3-HN epitope, BONT / A4-HN epitope, BONT / A5-HN epitope, BONT / A6-HN epitope, BONT / A7-HN epitope, or BONT / A8-HN epitope), and a BoNT / A-Hc epitope (e.g., BoNT / Al-Hc epitope, BoNT / A2-Hc epitope, BoNT / A3-Hc epitope, BoNT / A4-Hc epitope, BoNT / A5-Hc epitope, BoNT / A6-Hc epitope, BoNT / A7-Hc epitope, or BoNT / A8-Hc epitope). In some embodiments, at least one epitope (e.g., 1, 2, or 3) of the first epitope, the second epitope, and the third epitope are different epitopes. In some embodiments, each of the first epitope, the second epitope, and the third epitope are different epitopes.

[0088] In some embodiments, the first VHH binds to an epitope of BoNT / A-LC (e.g., BoNT / Al-LC, BoNT / A2-LC, BoNT / A3-LC, BoNT / A4-LC, BoNT / A5-LC, B0NT / A6-LC,

[0089] 12413624.1BoNT / A7-LC, or BoNT / A-LC). In some embodiments, the first VHH binds to an epitope of BoNT / Al-LC (e.g., ciA-H7 or hciA-H7). In some embodiments, the first VHH comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 2 or SEQ ID NO: 5. In some embodiments, the first VHH comprises or consists of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 5. In some embodiments, the second VHH binds to an epitope of BONT / A-HN (e.g., BONT / A1-HN, BONT / A2-HN, BONT / A3-HN, BONT / A4-HN, BONT / A5-HNepitope, BONT / A6-HNepitope, BONT / A7-HNepitope, or BONT / A8-HN epitope). In some embodiments, the second VHH binds to an epitope of BONT / A1-HN (e.g., ciA-B5 or hciA-B5). In some embodiments, the second VHH comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 3 or SEQ ID NO: 6. In some embodiments, the second VHH comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 3 or SEQ ID NO: 6. In some embodiments, the second VHH comprises or consists of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 6. In some embodiments, the third VHH binds to an epitope of BoNT / A-Hc (e.g., BoNT / Al-Hc, BoNT / A2-Hc, BoNT / A3-Hc, BoNT / A4-Hc, BoNT / A5-Hcepitope, BoNT / A6-Hc epitope, BoNT / A7-Hc epitope, or BoNT / A8-Hc epitope). In some embodiments, the third VHH binds to an epitope of BoNT / Al-Hc (e.g., ciA-C2 or hciA-C2). In some embodiments, the third VHH comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 4 or SEQ ID NO: 7. In some embodiments, the third VHH comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 4 or SEQ ID NO: 7. In some embodiments, the third VHH comprises or consists of the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 7.

[0090] In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal, the first VHH (e.g., ciA-H7 or hciA-H7), the second VHH (e.g., ciA-B5 or hciA-B5), the third VHH (e.g., ciA-C2 or hciA-C2), and the Fc. In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal, the first VHH (e.g., ciA-H7 or hciA-H7), the third VHH (e.g., ciA-C2 or hciA-C2), the second VHH (e.g., ciA-B5 or hciA-B5), and the Fc. In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal, the second VHH (e.g., ciA-B5 or hciA-B5), the first VHH (e.g., ciA-H7

[0091] 12413624.1or hciA-H7), the third VHH (e.g., ciA-C2 or hciA-C2), and the Fc. In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal, the second VHH (e.g., ciA-B5 or hciA-B5), the third VHH (e.g., ciA-C2 or hciA-C2), the first VHH (e.g., ciA-H7 or hciA-H7), and the Fc. In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal, the third VHH (e.g., ciA-C2 or hciA-C2), the first VHH (e.g., ciA-H7 or hciA-H7), the second VHH (e.g., ciA-B5 or hciA-B5), and the Fc. In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal, the third VHH (e.g., ciA-C2 or hciA-C2), the second VHH (e.g., ciA-B5 or hciA-B5), the first VHH (e.g., ciA-H7 or hciA-H7), and the Fc. In some embodiments, the first VHH is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 2, the second VHH is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 3, the third VHH is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 4, and the Fc is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 1. In some embodiments, the first VHH comprises the amino acid sequence of SEQ ID NO: 2, the second VHH comprises the amino acid sequence of SEQ ID NO: 3, the third VHH comprises the amino acid sequence of SEQ ID NO: 4, and the Fc comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the first VHH consists of the amino acid sequence of SEQ ID NO: 2, the second VHH consists of the amino acid sequence of SEQ ID NO: 3, the third VHH consists of the amino acid sequence of SEQ ID NO: 4, and the Fc consists of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the first VHH is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 5, the second VHH is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at

[0092] 12413624.1least 99%) identical to SEQ ID NO: 6, the third VHH is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 7, and the Fc is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 1. In some embodiments, the first VHH comprises the amino acid sequence of SEQ ID NO: 5, the second VHH comprises the amino acid sequence of SEQ ID NO: 6, the third VHH comprises the amino acid sequence of SEQ ID NO: 7, and the Fc comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the first VHH consists of the amino acid sequence of SEQ ID NO: 5, the second VHH consists of the amino acid sequence of SEQ ID NO: 6, the third VHH consists of the amino acid sequence of SEQ ID NO: 7, and the Fc consists of the amino acid sequence of SEQ ID NO: 1.

[0093] In some embodiments, an antibody of the present disclosure comprises an amino acid sequence having at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the amino acid sequence of any one of SEQ ID NOs: 8-19. In some embodiments, an antibody of the present disclosure comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 8-19.

[0094] Table 3. BoNT / A triple-paratopic sequences.

[0095]

[0096] 12413624.1

[0097]

[0098] 12413624.1

[0099]

[0100] 12413624.1

[0101]

[0102] In some embodiments, the toxin is a BoNT / B. BoNT / B blocks neurotransmitter release by cleavage of synaptobrevin 2 (also called VAMP2), disrupting the SNARE complex. There are 8 subtypes of BoNT / B: BoNT / B 1, BoNT / B2, BoNT / B3, BoNT / B4, BoNT / B5, B0NT / B6, BoNT / B7, and B0NT / B8. In some embodiments, the toxin is a BoNT / B 1, a BoNT / B2, a BoNT / B3, a BoNT / B4, a BoNT / B5, a B0NT / B6, a BoNT / B7, or a B0NT / B8.

[0103] In some aspects, this disclosure provides a VHH that bind to an epitope of BoNT / B (e.g., BoNT / B 1, BoNT / B2, BoNT / B3, BoNT / B4, BoNT / B5, B0NT / B6, BoNT / B7, or B0NT / B8). In some embodiments, the epitope of BoNT / B is selected from the group consisting of a BoNT / B heavy chain (HC) epitope and a BoNT / B light chain epitope (LC). In some embodiments, the BoNT / B-HC is an amino-terminal part (BONT / B-HN) epitope or a carboxy-terminal part (BoNT / A-Hc). In some embodiments, the VHH comprises an amino acid sequence that is at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 20-25. In some embodiments, the VHH comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 23-25.

[0104] Table 4. BoNT / B VHH sequences.

[0105]

[0106] 12413624.1

[0107]

[0108] In some embodiments, this disclosure provides an antibody comprising a first VHH that binds to a first epitope of a BoNT / B (e.g., BoNT / Bl, BoNT / B2, BoNT / B3, BoNT / B4, BoNT / B5, B0NT / B6, BoNT / B7, or B0NT / B8), a second VHH that binds to a second epitope of the BoNT / B, and a third VHH that binds to a third epitope of the BoNT / B, and a constant region (Fc) of an immunoglobulin (IgG). In some embodiments, the first epitope, the second epitope, and the third epitope are selected from the group consisting of a BoNT / B heavy chain (HC) epitope and a BoNT / B light chain (LC) epitope. In some embodiments, the BoNT / B-HC is an amino-terminal part (BONT / B-HN) epitope or a carboxy-terminal part (BoNT / B-Hc) epitope. In some embodiments, the epitope (e.g., the first epitope, the second epitope, the third epitope) is selected from the group consisting of a BoNT / B-LC epitope (e.g., BoNT / B 1-LC epitope, BoNT / B2-LC epitope, BoNT / B3-LC epitope, BoNT / B4-LC epitope, BoNT / B5-LC epitope, B0NT / B6-LC epitope, BoNT / B7-LC epitope, or B0NT / B8-LC epitope), a BONT / B-HN epitope (e.g., BoNT / B 1-HN epitope, BONT / B2-HN epitope, BONT / B3-HN epitope, BONT / B4-HN epitope, BONT / B5-HN epitope, BONT / B6-HN epitope, BONT / B7-HN epitope, or BONT / B8-HN epitope), and a BoNT / B-Hc epitope (e.g., BoNT / B 1-Hc epitope, BoNT / B2-Hc epitope, BoNT / B3-Hc epitope, BoNT / B4-Hc epitope, BoNT / B5-Hc epitope, BoNT / B6-Hc epitope, BoNT / B7-Hc epitope, or BoNT / B8-Hc epitope), n some embodiments, at least one epitope (e.g., 1, 2, or 3) of the first epitope, the second epitope, and the third epitope are different epitopes. In some embodiments, each of the first epitope, the second epitope, and the third epitope are different epitopes.

[0109] In some embodiments, the first VHH binds to an epitope of BoNT / B-Hc (e.g., BoNT / B 1-Hc, BoNT / B2-Hc, BoNT / B3-Hc, BoNT / B4-Hc, BoNT / B5-Hc, BoNT / B6-Hc, BoNT / B7-Hc, or BoNT / B8-Hc). In some embodiments, the first VHH binds to an epitope of BoNT / B 1-Hc (e.g., JLI-G10 or hJLI-GlO). In some embodiments, the first VHH comprises an amino acid sequence

[0110] 12413624.1having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 20 or SEQ ID NO: 23. In some embodiments, the first VHH comprises or consists of the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 23. In some embodiments, the second VHH binds to an epitope of BoNT / Bl-Hc (e.g., BoNT / Bl-Hc, BoNT / B2-Hc, BoNT / B3-Hc, BoNT / B4-Hc, BoNT / B5-Hc, BoNT / B6-Hc, BoNT / B7-Hc, or BoNT / B8-Hc). In some embodiments, the second VHH binds to an epitope of BoNT / Bl-Hc. In some embodiments, the second VHH binds to JLU-D10 or hJLU-DlO. In some embodiments, the second VHH comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 21 or SEQ ID NO: 24. In some embodiments, the second VHH comprises or consists of the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 24. In some embodiments, the third VHH binds to an epitope of BoNT / Bl-Hc (e.g., BoNT / Bl-Hc, BoNT / B2-Hc, BoNT / B3-Hc, BoNT / B4-Hc, BoNT / B5-Hc, BoNT / B6-Hc, BoNT / B7-Hc, or BoNT / B8-Hc). In some embodiments, the third VHH binds to an epitope of BoNT / Bl-Hc (e.g., JLK-G12 or hJLK-G12). In some embodiments, the third VHH comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 22 or SEQ ID NO: 25. In some embodiments, the third VHH comprises or consists of the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 25.

[0111] In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal, the first VHH (e.g., JLI-G10 or hJLI-GlO), the second VHH (e.g., JLU-D10 or hJLU-DlO), the third VHH (e.g., JLK-G12 or hJLK-G12), and the Fc. In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal, the first VHH (e.g., JLI-G10 or hJLI-GlO), the third VHH (e.g., JLK-G12 or hJLK-G12), the second VHH (e.g., JLU-D10 or hJLU-DlO), and the Fc. In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal, the second VHH (e.g., JLU-D10 or hJLU-D10), the first VHH (e.g., JLI-G10 or hJLI-GlO), the third VHH (e.g., JLK-G12 or hJLK-G12), and the Fc. In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal, the second VHH (e.g., JLU-D10 or hJLU-DlO), the third VHH (e.g., JLK-G12 or hJLK-G12), the first VHH (e.g., JLI-G10 or hJLI-GlO), and the Fc. In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal,

[0112] 12413624.1the third VHH (e.g., JLK-G12 or hJLK-G12), the first VHH (e.g., JLI-G10 or hJLI-GlO), the second VHH (e.g., JLU-D10 or hJLU-DlO), and the Fc. In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal, the third VHH (e.g., JLK-G12 or hJLK-G12), the second VHH (e.g., JLU-D10 or hJLU-DlO), the first VHH (e.g., JLI-G10 or hJLI-GlO), and the Fc. In some embodiments, the first VHH is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 20, the second VHH is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 21, the third VHH is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 22, and the Fc is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 1. In some embodiments, the first VHH comprises the amino acid sequence of SEQ ID NO: 20, the second VHH comprises the amino acid sequence of SEQ ID NO: 21, the third VHH comprises the amino acid sequence of SEQ ID NO: 22, and the Fc comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the first VHH consists of the amino acid sequence of SEQ ID NO: 20, the second VHH consists of the amino acid sequence of SEQ ID NO: 21, the third VHH consists of the amino acid sequence of SEQ ID NO: 4, and the Fc consists of the amino acid sequence of SEQ ID NO: 22. In some embodiments, the first VHH is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 23, the second VHH is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 24, the third VHH is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 25, and the Fc is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least

[0113] 12413624.188%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 1. In some embodiments, the first VHH comprises the amino acid sequence of SEQ ID NO: 23, the second VHH comprises the amino acid sequence of SEQ ID NO: 25, the third VHH comprises the amino acid sequence of SEQ ID NO: 26, and the Fc comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the first VHH consists of the amino acid sequence of SEQ ID NO: 23, the second VHH consists of the amino acid sequence of SEQ ID NO: 24, the third VHH consists of the amino acid sequence of SEQ ID NO: 25, and the Fc consists of the amino acid sequence of SEQ ID NO: 1.

[0114] In some embodiments, an antibody of the present disclosure comprises an amino acid sequence having at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the amino acid sequence of any one of SEQ ID NOs: 26-37. In some embodiments, an antibody of the present disclosure comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 26-37.

[0115] Table 5. BoNT / B triple-paratopic sequences.

[0116]

[0117] 12413624.1

[0118]

[0119] 12413624.1

[0120]

[0121] 12413624.1

[0122]

[0123] In some embodiments, the toxin is produced by Clostridioides difficile (C. difficile). C. difficile is a spore-forming opportunistic pathogen which can colonize the colon when the normal gut microbiome is disrupted. C. difficile infection (CDI) accounts for approximately 15-20% of all cases of antibiotic-associated diarrhea in developed countries, ranging from mild diarrhea to pseudomembranous colitis. CDI is a leading cause of hospital-acquired infection that can result in severe illness (e.g., toxic megacolon, fulminant colitis, pseudomembranous colitis, recurrent CDI) and death. Two homologous protein toxins produced by C. difficile , Toxin A (TcdA) and Toxin B (Ted) are major virulence factors, as strains lacking them do not cause disease. Clinical symptoms of CDI include watery diarrhea, fever, loss of appetite, nausea, and abdominal pain or tenderness.

[0124] In some embodiments, the toxin produced by C. difficile is TcdA. In some embodiments, the toxin produced by C. difficile is TcdB. In some embodiments, the toxin produced by C. difficile is TcdA and TcdB. TcdA and TcdB are 308 kDa and 270 kDa proteins, respectively, composed of four domains: a glucosyltransferase domain (GTD), a receptor binding domain (RBD) formed by combined repeated oligopeptides (CROPs), a cysteine protease domain (CPD), and a delivery and receptor-binding domain (DRBD). Glucosylation of Rho GTPases in host cells by TcdA and TcdB locks these proteins into an inactive conformation, thereby blocking all downstream signaling pathways, resulting in disruption of the actin cytoskeleton, cell rounding, and eventually apoptosis and death of the host cell.

[0125] In some embodiments, the toxin is a TcdB. TcdB is considered the more dominant and potent C. difficile toxin in humans. There are 12 subtypes of TcdB: TcdBl, TcdB2, TcdB3,

[0126] 12413624.1TcdB4, TcdB5, TcdB6, TcdB7, TcdB8, TcdB9, TcdBlO, TcdBll, and TcdB 12. TcdBl is the dominant subtype in over 70% of clinical isolates; TcdB2 is the second most prevalent subtype and accounts for around 10% of clinical isolates. While TcdB2 is less prevalent than TcdBl, TcdB2 has become a major threat presented by many hypervirulent strains.

[0127] In some aspects, this disclosure provides a VHH that bind to an epitope of TcdB (e.g., TcdBl, TcdB2, TcdB3, TcdB4, TcdB5, TcdB6, TcdB7, TcdB8, TcdB9, TcdBlO, TcdBll, or TcdB 12). In some embodiments, the epitope of TcdB is selected from the group consisting of a TcdB GTD epitope, a TcdB CROPs epitope, a TcdB CPD epitope, and a TcdB DRBD epitope. In some embodiments, the VHH comprises an amino acid sequence that is at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 38-43. In some embodiments, the VHH comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 41-43.

[0128] Table 6. TcdB VHH sequences.

[0129]

[0130] In some embodiments, this disclosure provides an antibody comprising a first VHH that binds to a first epitope of a TcdB (e.g., TcdBl, TcdB2, TcdB3, TcdB4, TcdB5, TcdB6, TcdB7, TcdB8, TcdB9, TcdBlO, TcdBll, or TcdB12), a second VHH that binds to a second epitope of the TcdB, a third VHH that binds to a third epitope of the TcdB, and a constant region (Fc) of an

[0131] 12413624.1immunoglobulin (IgG). In some embodiments, the first epitope, the second epitope, and the third epitope are selected from the group consisting of a TcdB N-terminal glucosyltransferase domain (GTD) epitope, a TcdB autoprocessing domain (APD) epitope, a TcdB delivery and receptorbinding domain (DRBD) epitope, and a TcdB combined repetitive oligopeptides (CROPs) domain epitope. In some embodiments, the epitope (e.g., the first epitope, the second epitope, the third epitope) is selected from the group consisting of a TcdB-APD epitope (e.g., TcdBl-APD epitope, TcdB2-APD epitope, TcdB3-APD epitope, TcdB4-APD epitope, TcdB5-APD epitope, TcdB6-APD epitope, TcdB7-APD epitope, TcdB8-APD epitope, TcdB9-APD epitope, TcdBlO-APD epitope, TcdBll-APD epitope, or TcdB12-APD epitope), a TcdB-GTD epitope (e.g., TcdBl-GTD epitope, TcdB2-GTD epitope, TcdB3-GTD epitope, TcdB4-GTD epitope, TcdB5-GTD epitope, TcdB6-GTD epitope, TcdB7-GTD epitope, TcdB8-GTD epitope, TcdB9-GTD epitope, TcdBlO-GTD epitope, TcdB 11 -GTD epitope, or TcdB12-GTD epitope), a TcdB-CROPs epitope (e.g., TcdBl-CROPs epitope, TcdB2-CROPs epitope, TcdB3-CROPs epitope, TcdB4-CROPs epitope, TcdB5-CROPs epitope, TcdB6-CROPs epitope, TcdB7-CROPs epitope, TcdB8-CROPs epitope, TcdB9-CROPs epitope, TcdBlO-CROPs epitope, TcdBll-CROPs epitope, or TcdB12-CROPs epitope), a TcdB-CPD epitope (e.g., TcdBl-CPD epitope, TcdB2-CPD epitope, TcdB3-CPD epitope, TcdB4-CPD epitope, TcdB5-CPD epitope, TcdB6-CPD epitope, TcdB7-CPD epitope, TcdB8-CPD epitope, TcdB9-CPD epitope, TcdBlO-CPD epitope, TcdBll-CPD epitope, or TcdB12-CPD epitope), and a TcdB-DRBD epitope (e.g., TcdBl-DRBD epitope, TcdB2-DRBD epitope, TcdB3-DRBD epitope, TcdB4-DRBD epitope, TcdB5-DRBD epitope, TcdB6-DRBD epitope, TcdB7-DRBD epitope, TcdB8-DRBD epitope, TcdB9-DRBD epitope, TcdBlO-DRBD epitope, TcdBll-DRBD epitope, or TcdB12-DRBD epitope). In some embodiments, at least one epitope (e.g., 1, 2, or 3) of the first epitope, the second epitope, and the third epitope are different epitopes. In some embodiments, each of the first epitope, the second epitope, and the third epitope are different epitopes.

[0132] In some embodiments, the first VHH binds to an epitope of TcdB-DRBD (e.g., TcdBl-DRBD, TcdB2-DRBD, TcdB3-DRBD, TcdB4-DRBD, TcdB5-DRBD, TcdB6-DRBD, TcdB7-DRBD, TcdB8-DRBD, TcdB9-DRBD, TcdBlO-DRBD, TcdBll-DRBD, or TcdB12-DRBD). In some embodiments, the first VHH binds to an epitope of TcdBl-DRBD (e.g., 5D or h5D). In some embodiments, the first VHH comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 38 or SEQ ID NO: 41. In some

[0133] 12413624.1embodiments, the first VHH comprises or consists of the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 41. In some embodiments, the second VHH binds to an epitope of TcdB-GTD (e.g., TcdBl-GTD, TcdB2-GTD, TcdB3-GTD, TcdB4-GTD, TcdB5-GTD, TcdB6-GTD, TcdB7-GTD, TcdB8-GTD, TcdB9-GTD, TcdBlO-GTD, TcdBll-GTD, or TcdB12-GTD). In some embodiments, the second VHH binds to an epitope of TcdBl-GTD (e.g., E3 or hE3). In some embodiments, the second VHH comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 39 or SEQ ID NO: 42. In some embodiments, the second VHH comprises or consists of the amino acid sequence of SEQ ID NO: 39 or SEQ ID NO: 42. In some embodiments, the third VHH binds to an epitope of TcdB-GTD (e.g., TcdBl-GTD, TcdB2-GTD, TcdB3-GTD, TcdB4-GTD, TcdB5-GTD, TcdB6-GTD, TcdB7-GTD, TcdB8-GTD, TcdB9-GTD, TcdBlO-GTD, TcdBll-GTD, or TcdB12-GTD). In some embodiments, the third VHH binds to an epitope of TcdBl-GTD (e.g., 7F or h7F). In some embodiments, the third VHH comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 40 or SEQ ID NO: 43. In some embodiments, the third VHH comprises or consists of the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43.

[0134] In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal, the first VHH (e.g., 5D or h5D), the second VHH (e.g., E3 or hE3), the third VHH (e.g., 7F or h7F), and the Fc. In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal, the first VHH (e.g., 5D or h5D), the third VHH (e.g., 7F or h7F), the second VHH (e.g., E3 or hE3), and the Fc. In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal, the second VHH (e.g., E3 or hE3), the first VHH (e.g., 5D or h5D), the third VHH (e.g., 7F or h7F), and the Fc. In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal, the second VHH (e.g., E3 or hE3), the third VHH (e.g., 7F or h7F), the first VHH (e.g., 5D or h5D), and the Fc. In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal, the third VHH (e.g., 7F or h7F), the first VHH (e.g., 5D or h5D), the second VHH (e.g., E3 or hE3), and the Fc. In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal, the third VHH (e.g., 7F or h7F), the second VHH (e.g., E3 or hE3), the first VHH (e.g., 5D or h5D), and the Fc. In some

[0135] 12413624.1embodiments, the first VHH is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 38, the second VHH is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 39, the third VHH is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 40, and the Fc is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 1. In some embodiments, the first VHH comprises the amino acid sequence of SEQ ID NO: 38, the second VHH comprises the amino acid sequence of SEQ ID NO: 39, the third VHH comprises the amino acid sequence of SEQ ID NO: 40, and the Fc comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the first VHH consists of the amino acid sequence of SEQ ID NO: 38, the second VHH consists of the amino acid sequence of SEQ ID NO: 39, the third VHH consists of the amino acid sequence of SEQ ID NO: 40, and the Fc consists of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the first VHH is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 41, the second VHH is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 42, the third VHH is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 43, and the Fc is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 1. In some embodiments, the first VHH comprises the amino acid sequence of SEQ ID NO: 41, the second VHH comprises the amino acid sequence of SEQ ID NO: 42, the third VHH comprises the amino acid sequence of SEQ ID

[0136] 12413624.1NO: 43, and the Fc comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the first VHH consists of the amino acid sequence of SEQ ID NO: 41, the second VHH consists of the amino acid sequence of SEQ ID NO: 42, the third VHH consists of the amino acid sequence of SEQ ID NO: 43, and the Fc consists of the amino acid sequence of SEQ ID NO: 1.

[0137] In some embodiments, an antibody of the present disclosure comprises an amino acid sequence having at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the amino acid sequence of any one of SEQ ID NOs: 44-55. In some embodiments, an antibody of the present disclosure comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 44-55.

[0138] Table 7. TcdB triple-paratopic sequences.

[0139]

[0140] 12413624.1

[0141]

[0142] 12413624.1

[0143]

[0144] 12413624.1In some embodiments, the toxin is a TcdA. There are 7 subtypes of TcdA: TcdAl, TcdA2, TcdA3, TcdA4, TcdA5, Tcd6, and TcdA7.

[0145] In some aspects, this disclosure provides a VHH that bind to an epitope of TcdA (e.g., TcdAl, TcdA2, TcdA3, TcdA4, TcdA5, TcdA6, or TcdA7). In some embodiments, the epitope of TcdA is selected from the group consisting of a TcdA GTD epitope, a TcdA CROPs epitope, a TcdA CPD epitope, and a TcdA DRBD epitope. In some embodiments, the VHH comprises an amino acid sequence that is at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to any one of SEQ ID NOs: 38-43. In some embodiments, the VHH comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 41-43.

[0146] Table 8. TcdA VHH sequences.

[0147]

[0148] In some embodiments, this disclosure provides an antibody comprising a first VHH that binds to a first epitope of a TcdA (e.g., TcdAl, TcdA2, TcdA3, TcdA4, TcdA5, TcdA6, or TcdA7), a second VHH that binds to a second epitope of the TcdA, a third VHH that binds to a third epitope of the TcdA, and a constant region (Fc) of an immunoglobulin (IgG). In some embodiments, the first epitope, the second epitope, and the third epitope are selected from the group consisting of a TcdA N-terminal glucosyltransferase domain (GTD) epitope, a TcdA autoprocessing domain (APD) epitope, a TcdA delivery and receptor-binding domain (DRBD)

[0149] 12413624.1epitope, and a TcdA combined repetitive oligopeptides (CROPs) domain epitope. In some embodiments, the epitope (e.g., the first epitope, the second epitope, the third epitope) is selected from the group consisting of a TcdA-APD epitope (e.g., TcdAl-APD epitope, TcdA2-APD epitope, TcdA3-APD epitope, TcdA4-APD epitope, TcdA5-APD epitope, TcdA6-APD epitope, or TcdA7-APD epitope), a TcdA-GTD epitope (e.g., TcdAl-GTD epitope, TcdA2-GTD epitope, TcdA3-GTD epitope, TcdA4-GTD epitope, TcdA5-GTD epitope, TcdA6-GTD epitope, or TcdA7-GTD epitope), a TcdA-CROPs epitope (e.g., TcdAl-CROPs epitope, TcdA2-CROPs epitope, TcdA3-CROPs epitope, TcdA4-CROPs epitope, TcdA5-CROPs epitope, TcdA6-CROPs epitope, or TcdA7-CROPs epitope), a TcdA-CPD epitope (e.g., TcdAl-CPD epitope, TcdA2-CPD epitope, TcdA3-CPD epitope, TcdA4-CPD epitope, TcdA5-CPD epitope, TcdA6-CPD epitope, or TcdA7-CPD epitope), and a TcdA-DRBD epitope (e.g., TcdAl-DRBD epitope, TcdA2-DRBD epitope, TcdA3-DRBD epitope, TcdA4-DRBD epitope, TcdA5-DRBD epitope, TcdA6-DRBD epitope, or TcdA7-DRBD epitope). In some embodiments, at least one epitope (e.g., 1, 2, or 3) of the first epitope, the second epitope, and the third epitope are different epitopes. In some embodiments, at least one epitope (e.g., 1, 2, or 3) of the first epitope, the second epitope, and the third epitope are different epitopes. In some embodiments, each of the first epitope, the second epitope, and the third epitope are different epitopes.

[0150] In some embodiments, the first VHH binds to an epitope of TcdA-CROPs (e.g., TcdAl-CROPs, TcdA2-CROPs, TcdA3-CROPs, TcdA4-CROPs, TcdA5-CROPs, TcdA6-CROPs, or TcdA7-CROPs). In some embodiments, the first VHH binds to an epitope of TcdAl-CROPs (e.g., A3H or hA3H). In some embodiments, the first VHH comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 56 or SEQ ID NO: 59. In some embodiments, the first VHH comprises or consists of the amino acid sequence of SEQ ID NO: 56 or SEQ ID NO: 59. In some embodiments, the second VHH binds to an epitope of TcdA-GTD (e.g., TcdAl-GTD, TcdA2-GTD, TcdA3-GTD, TcdA4-GTD, TcdA5-GTD, TcdA6-GTD, or TcdA7-GTD). In some embodiments, the second VHH binds to an epitope of TcdAl-GTD (e.g., AH3 or hAH3). In some embodiments, the second VHH comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 57 or SEQ ID NO: 60. In some embodiments, the second VHH comprises or consists of the amino acid sequence of SEQ ID NO: 57 or SEQ ID NO: 60. In

[0151] 12413624.1some embodiments, the third VHH binds to an epitope of TcdA-DRBD (e.g., TcdAl-DRBD, TcdA2-DRBD, TcdA3-DRBD, TcdA4-DRBD, TcdA5-DRBD, TcdA6-DRBD, or TcdA7-DRBD). In some embodiments, the third VHH binds to an epitope to TcdAl-DRBD (e.g., AA6 or hAA6). In some embodiments, the third VHH comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 58 or SEQ ID NO: 61. In some embodiments, the third VHH comprises or consists of the amino acid sequence of SEQ ID NO: 58 or SEQ ID NO: 61.

[0152] In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal, the first VHH (e.g., A3H or hA3H), the second VHH (e.g., AH3 or hAH3), the third VHH (e.g., AA6 or hAA6), and the Fc. In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal, the first VHH (e.g., A3H or hA3H), the third VHH (e.g., AA6 or hAA6), , the second VHH (e.g., AH3 or hAH3), and the Fc. In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal, the second VHH (e.g., AH3 or hAH3), the first VHH (e.g., A3H or hA3H), the third VHH (e.g., AA6 or hAA6), , and the Fc. In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal, the second VHH (e.g., AH3 or hAH3), the third VHH (e.g., AA6 or hAA6), , the first VHH (e.g., A3H or hA3H), and the Fc. In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal, the third VHH (e.g., AA6 or hAA6), , the first VHH (e.g., A3H or hA3H), the second VHH (e.g., AH3 or hAH3), and the Fc. In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal, the third VHH (e.g., AA6 or hAA6), the second VHH (e.g., AH3 or hAH3), the first VHH (e.g., A3H or hA3H), and the Fc. In some embodiments, the first VHH is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 56, the second VHH is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 57, the third VHH is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 58, and the Fc is at least 85% (e.g., at least 85%, at least 86%, at

[0153] 12413624.1least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 1. In some embodiments, the first VHH comprises the amino acid sequence of SEQ ID NO: 56, the second VHH comprises the amino acid sequence of SEQ ID NO: 57, the third VHH comprises the amino acid sequence of SEQ ID NO: 58, and the Fc comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the first VHH consists of the amino acid sequence of SEQ ID NO: 59, the second VHH consists of the amino acid sequence of SEQ ID NO: 60, the third VHH consists of the amino acid sequence of SEQ ID NO: 61, and the Fc consists of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the first VHH is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 59, the second VHH is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 60, the third VHH is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 61, and the Fc is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 1. In some embodiments, the first VHH comprises the amino acid sequence of SEQ ID NO: 59, the second VHH comprises the amino acid sequence of SEQ ID NO: 60, the third VHH comprises the amino acid sequence of SEQ ID NO: 61, and the Fc comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the first VHH consists of the amino acid sequence of SEQ ID NO: 59, the second VHH consists of the amino acid sequence of SEQ ID NO: 60, the third VHH consists of the amino acid sequence of SEQ ID NO: 61, and the Fc consists of the amino acid sequence of SEQ ID NO: 1.

[0154] In some embodiments, an antibody of the present disclosure comprises an amino acid sequence having at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the amino acid sequence of any one

[0155] 12413624.1of SEQ ID NOs: 62-72. In some embodiments, an antibody of the present disclosure comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 62-72.

[0156] Table 9. TcdA triple-paratopic antibody sequences.

[0157]

[0158] 12413624.1

[0159]

[0160] 12413624.1

[0161]

[0162] In some aspects, the present disclosure provides antibodies (e.g., nanobody-based tetraepitopic antibodies) comprising a first VHH that binds to a first epitope of a first toxin, a second

[0163] 12413624.1VHH that binds to a second epitope of the first toxin, a third epitope that binds to a third epitope of the first toxin, a fourth epitope that binds to a second toxin, and a constant region (Fc) of an immunoglobulin (IgG). In some embodiments, the first toxin and the second toxin are different toxins (e.g., the first toxin is TcdA and the second toxin is TcdB). In some embodiments, the first toxin and the second toxin are of different subtypes (e.g., the first toxin is TcdBl and the second toxin is TcdB2). In some embodiments, the first toxin and the second toxin are of different serotypes (e.g., the first toxin is BoNT / A and the second toxin is BoNT / B). In some embodiments, the first epitope, the second epitope, the third epitope, and the fourth epitope are selected from the group consisting of a BoNT / A-LC epitope (e.g., BoNT / A 1-LC epitope, BoNT / A2-LC epitope, BoNT / A3-LC epitope, BoNT / A4-LC epitope, or BoNT / A5-LC epitope), a BONT / A-HN epitope (e.g., BONT / A1-HN epitope, BONT / A2-HN epitope, BONT / A3-HN epitope, BONT / A4-HN epitope, or BONT / A5-HN epitope), a BoNT / A-Hc epitope (e.g., BoNT / Al-Hc epitope, BoNT / A2-Hc epitope, BoNT / A3-Hc epitope, BoNT / A4-Hc epitope, or BoNT / A5-Hc epitope), a BoNT / B-LC epitope (e.g., BoNT / B 1-LC epitope, BoNT / B2-LC epitope, BoNT / B3-LC epitope, BoNT / B4-LC epitope, BoNT / B5-LC epitope, B0NT / B6-LC epitope, BoNT / B7-LC epitope, or B0NT / B8-LC epitope), a BONT / B-HN epitope (e.g., BoNT / B 1-HN epitope, BONT / B2-HN epitope, BONT / B3-HN epitope, BONT / B4-HN epitope, BONT / B5-HN epitope, BONT / B6-HN epitope, BONT / B7-HN epitope, or BONT / B8-HN epitope), a BoNT / B-Hc epitope (e.g., BoNT / B 1-Hc epitope, BoNT / B2-Hc epitope, BoNT / B3-Hc epitope, BoNT / B4-Hc epitope, BoNT / B5-Hc epitope, BoNT / B6-Hc epitope, BoNT / B7-Hc epitope, or BoNT / B8-Hc epitope), a TcdB-APD epitope (e.g., TcdBl-APD epitope, TcdB2-APD epitope, TcdB3-APD epitope, TcdB4-APD epitope, TcdB5-APD epitope, TcdB6-APD epitope, TcdB7-APD epitope, TcdB8-APD epitope, TcdB9-APD epitope, TcdBlO-APD epitope, TcdBl 1-APD, or TcdB12-APD epitope), a TcdB-GTD epitope (e.g., TcdBl-GTD epitope, TcdB2-GTD epitope, TcdB3-GTD epitope, TcdB4-GTD epitope, TcdB5-GTD epitope, TcdB6-GTD epitope, TcdB7-GTD epitope, TcdB8-GTD epitope, TcdB9-GTD epitope, TcdBlO-GTD epitope, TcdBll-GTD, or TcdB12-GTD epitope), a TcdB-CROPs epitope (e.g., TcdBl-CROPs epitope, TcdB2-CROPs epitope, TcdB3-CROPs epitope, TcdB4-CROPs epitope, TcdB5-CROPs epitope, TcdB6-CROPs epitope, TcdB7-CROPs epitope, TcdB8-CROPs epitope, TcdB9-CROPs epitope, TcdBlO-CROPs epitope, TcdBll-CROPs, or TcdB12-CROPs epitope), a TcdB-CPD epitope (e.g., TcdBl-CPD epitope, TcdB2-CPD epitope, TcdB3-CPD epitope, TcdB4-CPD epitope, TcdB5-CPD epitope, TcdB6-CPD epitope, TcdB7-CPD epitope, TcdB8-CPD epitope, TcdB9-

[0164] 12413624.1CPD epitope, TcdBlO-CPD epitope, TcdBll-CPD, or TcdB12-CPD epitope), a TcdB-DRBD epitope (e.g., TcdBl-DRBD epitope, TcdB2-DRBD epitope, TcdB3-DRBD epitope, TcdB4-DRBD epitope, TcdB5-DRBD epitope, TcdB6-DRBD epitope, TcdB7-DRBD epitope, TcdB8-DRBD epitope, TcdB9-DRBD epitope, TcdBlO-DRBD epitope, TcdBll-DRBD, or TcdB 12-DRBD epitope), a TcdA-APD epitope (e.g., TcdAl-APD epitope, TcdA2-APD epitope, TcdA3-APD epitope, TcdA4-APD epitope, TcdA5-APD epitope, TcdA6-APD epitope, or TcdA7-APD epitope), a TcdA-GTD epitope (e.g., TcdAl-GTD epitope, TcdA2-GTD epitope, TcdA3-GTD epitope, TcdA4-GTD epitope, TcdA5-GTD epitope, TcdA6-GTD epitope, or TcdA7-GTD epitope), a TcdA-CROPs epitope (e.g., TcdAl-CROPs epitope, TcdA2-CROPs epitope, TcdA3-CROPs epitope, TcdA4-CROPs epitope, TcdA5-CROPs epitope, TcdA6-CROPs epitope, or TcdA7-CROPs epitope), a TcdA-CPD epitope (e.g., TcdAl-CPD epitope, TcdA2-CPD epitope, TcdA3-CPD epitope, TcdA4-CPD epitope, TcdA5-CPD epitope, TcdA6-CPD epitope, or TcdA7-CPD epitope), and a TcdA-DRBD epitope (e.g., TcdAl-DRBD epitope, TcdA2-DRBD epitope, TcdA3-DRBD epitope, TcdA4-DRBD epitope, TcdA5-DRBD epitope, TcdA6-DRBD epitope, or TcdA7-DRBD epitope). In some embodiments, at least one epitope (e.g., 1, 2, 3, or 4) of the first epitope, the second epitope, the third epitope, and the fourth epitope are different epitopes. In some embodiments, each of the first epitope, the second epitope, the third epitope, and the fourth epitope are different epitopes.

[0165] In some embodiments, the first toxin is TcdB and the second toxin is TcdA. In some embodiments, the first toxin is TcdA and the second toxin is TcdB. In some embodiments, an antibody of the present disclosure comprises a first VHH that binds to an epitope of TcdB, a second VHH that binds to an epitope of TcdB, a third VHH that binds to an epitope of TcdB, a fourth VHH that binds to an epitope of TcdA, and a constant region (Fc) of an immunoglobulin (IgG). In some embodiments, the first VHH binds to an epitope of TcdB-DRBD (e.g., TcdBl-DRBD, TcdB2-DRBD, TcdB3-DRBD, TcdB4-DRBD, TcdB5-DRBD, TcdB6-DRBD, TcdB7-DRBD, TcdB8-DRBD, TcdB9-DRBD, TcdBlO-DRBD, TcdBll-DRBD, or TcdB12-DRBD). In some embodiments, the first VHH binds to an epitope of TcdBl-DRBD (e.g., 5D or h5D). In some embodiments, the first VHH comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 38 or SEQ ID NO: 41. In some embodiments, the first VHH comprises or consists of the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 41. In some embodiments, the second VHH binds to an epitope of TcdB-

[0166] 12413624.1GTD (e.g., TcdBl-GTD, TcdB2-GTD, TcdB3-GTD, TcdB4-GTD, TcdB5-GTD, TcdB6-GTD, TcdB7-GTD, TcdB8-GTD, TcdB9-GTD, TcdBlO-GTD, TcdBll-GTD, or TcdB12-GTD). In some embodiments, the second VHH binds to an epitope of TcdBl-GTD (e.g., E3 or hE3). In some embodiments, the second VHH comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 39 or SEQ ID NO: 42. In some embodiments, the second VHH comprises or consists of the amino acid sequence of SEQ ID NO: 39 or SEQ ID NO: 42. In some embodiments, the third VHH binds to an epitope of TcdB-GTD (e.g., TcdBl-GTD, TcdB2-GTD, TcdB3-GTD, TcdB4-GTD, TcdB5-GTD, TcdB6-GTD, TcdB7-GTD, TcdB8-GTD, TcdB9-GTD, TcdBlO-GTD, TcdBll-GTD, or TcdB12-GTD). In some embodiments, the third VHH binds to an epitope of TcdBl-GTD (e.g., 7F or h7F). In some embodiments, the third VHH comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to SEQ ID NO: 40 or SEQ ID NO: 43. In some embodiments, the third VHH comprises or consists of the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43. In some embodiments, the fourth VHH binds to an epitope of TcdA-CROPs (e.g., TcdAl-CROPs, TcdA2-CROPs, TcdA3-CROPs, TcdA4-CROPs, TcdA5-CROPs, TcdA6-CROPs, or TcdA7-CROPs), TcdA-GTD (e.g., TcdAl-GTD, TcdA2-GTD, TcdA3-GTD, TcdA4-GTD, TcdA5-GTD, TcdA6-GTD, or TcdA7-GTD), or TcdA-DRBD (e.g., TcdAl-DRBD, TcdA2-DRBD, TcdA3-DRBD, TcdA4-DRBD, TcdA5-DRBD, TcdA6-DRBD, or TcdA7-DRBD). In some embodiments, the fourth VHH binds to an epitope to TcdAl-CROPs (e.g., A3H or hA3H), TcdAl-GTD (e.g., AH3 or hAH3), or TcdAl-DRBD (e.g., AA6 or hAA6). In some embodiments, the fourth VHH comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to any one of SEQ ID NOs: 56-61. In some embodiments, the fourth VHH comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 56-61.

[0167] In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal, the first VHH (e.g., 5D or h5D), the second VHH (e.g., E3 or hE3), the third VHH (e.g., 7F or h7F), the Fc, and the fourth VHH (e.g., A3H, hA3H, AH3, hAH3, AA6, or hAA6). In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal, the first VHH (e.g., 5D or h5D), the third VHH (e.g., 7F or h7F), the second VHH

[0168] 12413624.1(e.g., E3 or hE3), the Fc, and the fourth VHH (e.g., A3H, hA3H, AH3, hAH3, AA6, or hAA6). In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal, the second VHH (e.g., E3 or hE3), the first VHH (e.g., 5D or h5D), the third VHH (e.g., 7F or h7F), the Fc, and the fourth VHH (e.g., A3H, hA3H, AH3, hAH3, AA6, or hAA6). In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal, the second VHH (e.g., E3 or hE3), the third VHH (e.g., 7F or h7F), the first VHH (e.g., 5D or h5D), the Fc, and the fourth VHH (e.g., A3H, hA3H, AH3, hAH3, AA6, or hAA6). In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal, the third VHH (e.g., 7F or h7F), the first VHH (e.g., 5D or h5D), the second VHH (e.g., E3 or hE3), the Fc, and the fourth VHH (e.g., A3H, hA3H, AH3, hAH3, AA6, or hAA6). In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal, the third VHH (e.g., 7F or h7F), the second VHH (e.g., E3 or hE3), the first VHH (e.g., 5D or h5D), the Fc, and the fourth VHH (e.g., A3H, hA3H, AH3, hAH3, AA6, or hAA6). In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal, the fourth VHH (e.g., A3H, hA3H, AH3, hAH3, AA6, or hAA6), the first VHH (e.g., 5D or h5D), the second VHH (e.g., E3 or hE3), the third VHH (e.g., 7F or h7F), and the Fc. In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal, the fourth VHH (e.g., A3H, hA3H, AH3, hAH3, AA6, or hAA6), the first VHH (e.g., 5D or h5D), the third VHH (e.g., 7F or h7F), the second VHH (e.g., E3 or hE3), and the Fc. In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal, the fourth VHH (e.g., A3H, hA3H, AH3, hAH3, AA6, or hAA6), the second VHH (e.g., E3 or hE3), the first VHH (e.g., 5D or h5D), the third VHH (e.g., 7F or h7F), and the Fc. In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal, the fourth VHH (e.g., A3H, hA3H, AH3, hAH3, AA6, or hAA6), the second VHH (e.g., E3 or hE3), the third VHH (e.g., 7F or h7F), the first VHH (e.g., 5D or h5D), and the Fc. In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal, the fourth VHH (e.g., A3H, hA3H, AH3, hAH3, AA6, or hAA6), the third VHH (e.g., 7F or h7F), the first VHH (e.g., 5D or h5D), the second VHH (e.g., E3 or hE3), and the Fc. In some embodiments, an antibody of the present disclosure comprises, from N-terminal to C-terminal, the fourth VHH (e.g., A3H, hA3H, AH3, hAH3, AA6, or hAA6), the third VHH (e.g., 7F or h7F), the second VHH (e.g., E3 or hE3), the first VHH (e.g., 5D or h5D), and the Fc.

[0169] 12413624.1In some embodiments, the first VHH is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 38, the second VHH is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 39, the third VHH is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 40, the fourth VHH is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 56-58, and the Fc is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 1. In some embodiments, the first VHH comprises the amino acid sequence of SEQ ID NO: 38, the second VHH comprises the amino acid sequence of SEQ ID NO: 39, the third VHH comprises the amino acid sequence of SEQ ID NO: 40, the fourth VHH comprises the amino acid sequence of any one of SEQ ID NOs: 56-58, and the Fc comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the first VHH consists of the amino acid sequence of SEQ ID NO: 38, the second VHH consists of the amino acid sequence of SEQ ID NO: 39, the third VHH comprises the amino acid sequence of SEQ ID NO: 40, the fourth VHH consists of the amino acid sequence of any one of SEQ ID NOs: 56-58, and the Fc consists of the amino acid sequence of SEQ ID NO: 1.

[0170] In some embodiments, the first VHH is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 41, the second VHH is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 42, the third VHH is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 43, the fourth VHH is

[0171] 12413624.1at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 59-61, and the Fc is at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 1. In some embodiments, the first VHH comprises the amino acid sequence of SEQ ID NO: 41, the second VHH comprises the amino acid sequence of SEQ ID NO: 42, the third VHH comprises the amino acid sequence of SEQ ID NO: 43, the fourth VHH comprises the amino acid sequence of any one of SEQ ID NOs: 59-61, and the Fc comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the first VHH consists of the amino acid sequence of SEQ ID NO: 41, the second VHH consists of the amino acid sequence of SEQ ID NO: 42, the third VHH consists of the amino acid sequence of SEQ ID NO: 43, the fourth VHH consists of the amino acid sequence of any one of SEQ ID NOs: 59-61, and the Fc consists of the amino acid sequence of SEQ ID NO: 1.

[0172] In some embodiments, an antibody of the present disclosure comprises an amino acid sequence having at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the amino acid sequence of any one of SEQ ID NOs: 74-145. In some embodiments, an antibody of the present disclosure comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 74-145.

[0173] Table 10. Ted tetra-paratopic antibody sequences.

[0174]

[0175] 12413624.1

[0176]

[0177] 12413624.1

[0178]

[0179] 12413624.1

[0180]

[0181] 12413624.1

[0182]

[0183] 12413624.1

[0184]

[0185] 12413624.1

[0186]

[0187] 12413624.1

[0188]

[0189] 12413624.1

[0190]

[0191] 12413624.1

[0192]

[0193] 12413624.1

[0194]

[0195] 12413624.1

[0196]

[0197] 12413624.1

[0198]

[0199] 12413624.1

[0200]

[0201] 12413624.1

[0202]

[0203] 12413624.1

[0204]

[0205] 12413624.1

[0206]

[0207] 12413624.1

[0208]

[0209] 12413624.1

[0210]

[0211] 12413624.1

[0212]

[0213] In some embodiments, where an antibody of the present disclosure comprises a first VHH, a second VHH, a third VHH, and an Fc, the first VHH, the second VHH, the third VHH, and the Fc are connected by peptide linkers. For example, where an antibody comprises, from N-terminal to C-terminal, a first VHH, a second VHH, a third VHH, and an Fc, the first VHH and the second VHH are connected by a first peptide linker, the second VHH and the third VHH are connected by a second peptide linker, and the third VHH and the Fc are connected by a third peptide linker. In some embodiments, where an antibody of the present disclosure comprises a first VHH, a second VHH, a third VHH, a fourth VHH, and an Fc, the first VHH, the second VHH, the third VHH, the fourth VHH, and the Fc are connected by peptide linkers. For

[0214] 12413624.1example, where an antibody comprises, from N-terminal to C-terminal, a first VHH, a second VHH, a third VHH, an Fc, and a fourth VHH, the first VHH and the second VHH are connected by a first peptide linker, the second VHH and the third VHH are connected by a second peptide linker, the third VHH and the Fc are connected by a third peptide linker, and the Fc and the fourth VHH are connected by a fourth peptide linker. In some embodiments, the peptide linkers (e.g., the first peptide linker, the second peptide linker, the third peptide linker, the fourth peptide linker) are the same. In some embodiments, at least one (e.g., 1, 2, 3, or 4) of the peptide linkers (e.g., the first peptide linker, the second peptide linker, the third peptide linker, the fourth peptide linker) are different. In some embodiments, each of the peptide linkers (e.g., the first peptide linker, the second peptide linker, the third peptide linker, the fourth peptide linker) are different.

[0215] As used herein, the term “connected” describes a covalent linkage between a first polypeptide and a second polypeptide (e.g., between a first VHH and a second VHH). In some embodiments, the first polypeptide and the second polypeptide are attached by a peptide linker. In some embodiments, the peptide linker connects a C-terminal carboxyl group of a terminal amino acid of a first polypeptide to an N-terminal amino group of a terminal amino acid of a second polypeptide.

[0216] In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is a flexible linker. A flexible linker is a short sequence of amino acids typically composed of small, uncharged, and hydrophilic amino acids (e.g., glycine, serine) which reduces steric hindrance and promotes flexibility. In some embodiments, the flexible linker comprise a glycine- serine repeat or a glycine-alanine repeat. In some embodiments, flexible linker is (GGGGS)n or (GGGS)n, where n is 1, 2, 3, 4, or 5 (e.g., SEQ ID NOs: 146-155). In some embodiments, the peptide linker is a rigid linker. A rigid linker is a short sequence of amino acids designed to promote the formation of stable secondary structures, (e.g., a-helices or P-sheets). In some embodiments, the rigid linker is an a-helix-forming linker (e.g., comprises alanine, leucine, glutamine, and / or lysine), a proline-based linker, or a P-sheet-based linker. In some embodiments, the rigid linker is (EAAAK)n, where n is 1, 2, 34, or 5 (e.g., SEQ ID NOs: 156-160). Non-limiting examples of linker sequence are provided in Table 11.

[0217] Table 11. Linker sequences.

[0218] | Description | Sequence I SEQIDN6~|

[0219] 12413624.1

[0220]

[0221] In some aspects, the present disclosure relates to a nucleic acid comprising a polynucleotide sequence encoding an antibody of the present disclosure (e.g., an NT Ab as described above). In some embodiments, the polynucleotide sequence is operably linked to a promoter. In some embodiments, a construct comprises the nucleic acid (e.g., as described above). In some embodiments, the construct is a plasmid or vector. In some embodiments, the vector is an expression vector.

[0222] In some aspects, the present disclosure relates to a cell comprising an antibody of the present disclosure (e.g., an NTAb as described above), a nucleic acid (e.g., as described above), or the construct (e.g., as described above). In some embodiments, the cell is a bacterial cell, a yeast cell, an insect cell a plant cell, or a mammalian cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a Chinese Hamster Ovary (CHO) cell.

[0223] Aspects of this disclosure provide methods of producing an antibody of the present disclosure (e.g., an NTAb as described above). The NTAb will generally be produced by expression from recombinant nucleic acids in appropriate cells (e.g., bacterial cells or eukaryotic cells) and isolated. To produce the NTAb, nucleic acids encoding the NTAb may be introduced to a cell (e.g., a bacterial cell or a eukaryotic cell). The cells may be cultured under conditions that allow the NTAb to express from the nucleic acids encoding the NTAb (e.g., in a culture medium). The NTAb may be isolated using any methods of purifying a protein known in the art.

[0224] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an NTAb as described above, a nucleic acid encoding an NTAb as described above, or a construct comprising a nucleic acid encoding an NTAb as described above and a pharmaceutically acceptable carrier. A “pharmaceutical composition,” as used herein, refers to

[0225] 12413624.1the formulation of an antibody of the present disclosure (e.g., an NTAb as described above) in combination with a pharmaceutically acceptable carrier.

[0226] The term “pharmaceutically-acceptable carrier”, as used herein, means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the polypeptide from one site (e.g., the delivery site) of the body, to another site (e.g., organ, tissue or portion of the body).

[0227] A pharmaceutically acceptable carrier is “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissue of the subject (e.g., physiologically compatible, sterile, physiologic pH, etc.). Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethylcellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or poly anhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as “excipient”, “carrier”, “pharmaceutically acceptable carrier” or the like are used interchangeably herein.

[0228] In some embodiments, antibodies of the present disclosure (e.g., NTAbs) in a composition (e.g., a pharmaceutical composition) is administered by injection, by means of a catheter, by means of a suppository, or by means of an implant, the implant being of a porous,

[0229] 12413624.1non-porous, or gelatinous material, including a membrane, such as a sialastic membrane, or a fiber. Typically, when administering the composition (e.g., the pharmaceutical composition) materials to which the polypeptide of the disclosure does not absorb are used. In other embodiments, antibodies of the present disclosure (e.g., NTAbs) are delivered in a controlled release system. Such compositions and methods for administration are provides in U.S. Patent publication No. 2007 / 0020295, the contents of which are herein incorporated by reference. In one embodiment, a pump may be used (see, e.g., Langer, 1990, Science 249:1527-1533; Sefton, 1989, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymeric materials can be used. (See, e.g., Medical Applications of Controlled Release (Langer and Wise eds., CRC Press, Boca Raton, Fla., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., Wiley, New York, 1984); Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61. See also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105.) Other controlled release systems are discussed, for example, in Langer, supra.

[0230] Antibodies of the present disclosure (e.g., NTAbs) can be administered as pharmaceutical compositions comprising a therapeutically effective amount of a binding agent and one or more pharmaceutically compatible ingredients. In some embodiments, the pharmaceutical composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous or subcutaneous administration to a subject, e.g., a human being.

[0231] Typically, compositions for administration by injection are solutions in sterile isotonic aqueous buffer. Where necessary, the pharmaceutical can also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the pharmaceutical is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the pharmaceutical is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration. A pharmaceutical composition for systemic administration may be a liquid, e.g., sterile saline, lactated Ringer's or Hank’s solution. In addition, the

[0232] 12413624.1pharmaceutical composition can be in solid forms and re-dissolved or suspended immediately prior to use. Lyophilized forms are also contemplated. The pharmaceutical composition can be contained within a lipid particle or vesicle, such as a liposome or microcrystal, which is also suitable for parenteral administration. The particles can be of any suitable structure, such as unilamellar or plurilamellar, so long as compositions are contained therein.

[0233] Further, the pharmaceutical composition can be provided as a pharmaceutical kit comprising (a) a container containing a polypeptide of the disclosure in lyophilized form and (b) a second container containing a pharmaceutically acceptable diluent (e.g., sterile water) for injection. The pharmaceutically acceptable diluent can be used for reconstitution or dilution of the lyophilized polypeptide of the disclosure. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In another aspect, an article of manufacture containing materials useful for the treatment of the diseases described above is included. In some embodiments, the article of manufacture comprises a container and a label.

[0234] Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. In some embodiments, the container holds a composition that is effective for treating a disease described herein and may have a sterile access port. For example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle. The active agent in the composition is an isolated polypeptide of the disclosure. In some embodiments, the label on or associated with the container indicates that the composition is used for treating the disease of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0235] Antibodies of the present disclosure (e.g., NTAbs) and pharmaceutical compositions of the present disclosure may be used for the treatment of conditions associated with infection by a microorganism (e.g., bacteria, fungi, protozoa, dinoflagellates, or viruses) that produces a toxin (e.g., C. difficile or C. botulinum) or by intoxication with a toxin (e.g., botulinum neurotoxin, Toxin A, or Toxin B). Thus, further provided herein are methods of treating a condition

[0236] 12413624.1associated with infection by a microorganism (e.g., bacteria, fungi, protozoa, dinoflagellates, or viruses) that produces a toxin (e.g., C. difficile or C. botulinum) or by intoxication with a toxin (e.g., botulinum neurotoxin, Toxin A, or Toxin B), the method comprising administering to a subject a therapeutically effective amount of an antibody (e.g., an NTAb as described herein) or a pharmaceutical composition described herein to thereby treat the condition.

[0237] In some aspects, the present disclosure provides methods of treating a C. botulinum infection or intoxication by botulinum neurotoxin in a subject, the method comprising administering to the subject an effective amount of an antibody described herein (e.g., a botulinum neurotoxin binding NTAb as described herein) or a pharmaceutical composition described herein. Other aspects of the present disclosure provide methods of decreasing the severity of one or more symptoms associated with a C. botulinum infection or intoxication by botulinum neurotoxin in a subject, the method comprising administering to the subject an effective amount of an antibody (e.g., an NTAb as described herein) or a pharmaceutical composition described herein. In some embodiments, the antibody comprises an amino acid sequence having at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the amino acid sequence of any one of SEQ ID NOs: 8-19 or 26-37. In some embodiments, an antibody of the present disclosure comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 8-19 or 26-37. In some embodiments, the pharmaceutical composition comprises an antibody comprising an amino acid sequence having at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the amino acid sequence of any one of SEQ ID NOs: 8-19 or 26-37 and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises an antibody comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 8-19 or 26-37 and a pharmaceutically acceptable carrier. In some embodiments, the C. botulinum produces BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, or BoNT / G. In some embodiments, the botulinum neurotoxin is BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, or BoNT / G. In some embodiments, the C. botulinum infection or botulinum neurotoxin intoxication is foodborne botulism, wound botulism, infant botulism, iatrogenic botulism (e.g., from therapeutic or cosmetic injections, such as BOTOX®), or adult intestinal toxemia. In some

[0238] 12413624.1embodiments, the subject exhibits one or more symptoms of Clostridium botulinum infection or botulinum neurotoxin intoxication selected from the group consisting of fatigue, weakness, vertigo, blurred vision, dry mouth, difficulty swallowing or speaking, vomiting, diarrhea, constipation, abdominal swelling, weakness in the neck or arms, descending symmetrical paralysis, ptosis, facial palsy, ophthalmoplegia, and diplopia.

[0239] In some aspects, the present disclosure provides methods of treating a C. difficile infection in a subject, the method comprising administering to the subject an effective amount of an antibody (e.g., a C. difficile binding NTAb as described herein) or a pharmaceutical composition described herein. Other aspects of the present disclosure provide methods of decreasing the severity of one or more symptoms associated with a C. difficile infection in a subject, the method comprising administering to the subject an effective amount of an antibody (e.g., an NTAb as described herein) or a pharmaceutical composition described herein. In some embodiments, the antibody comprises an amino acid sequence having at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the amino acid sequence of any one of SEQ ID NOs: 44-55, 62-72, or 74-143. In some embodiments, an antibody of the present disclosure comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 44-55, 62-72, or 74-143. In some embodiments, the pharmaceutical composition comprises an antibody comprising an amino acid sequence having at least 85% (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the amino acid sequence of any one of SEQ ID NOs: 44-55, 62-72, or 74-143 and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises an antibody comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 44-55, 62-72, or 74-143 and a pharmaceutically acceptable carrier. In some embodiments, the C. difficile produces TcdA and TcdB. In some embodiments, the C. difficile produces TcdA and not TcdB. In some embodiments, the C. difficile produces TcdB and not TcdA. In some embodiments, the one or more symptoms is (are) selected from the group consisting of watery diarrhea, abdominal cramping and pain, fast heart rate, dehydration, fever, nausea, increased white blood cell count, kidney failure, loss of appetite, abdominal swelling, weight loss, blood or pus in stool, pseudomembranous colitis, colonic ileus, toxic megacolon, and sepsis.

[0240] 12413624.1A “subject” to which administration is contemplated refers to a human (i.e., male or female of any age group, e.g., pediatric subject (e.g., infant, child, or adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)), or a non-human animal. In some embodiments, the non-human animal is a mammal (e.g., primate (e.g., cynomolgus monkey or rhesus monkey), commercially relevant mammal (e.g., cattle, pig, horse, sheep, goat, cat, or dog), or bird (e.g., commercially relevant bird, such as chicken, duck, goose, or turkey)). In some embodiments, the non-human animal is a fish, reptile or amphibian. The non-human animal may be a female or male at any stage of development. The non-human animal may be a transgenic animal or a genetically engineered animal.

[0241] In some embodiments, the subject is a human subject, e.g., a human neonate, infant, child, adult, or elderly. In some embodiments, a human subject to which the contemplated antibody (e.g., an NT Ab as described herein) or a pharmaceutical composition described herein is administered is a newborn or more than 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3, years, 4 years, 5 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, or 65 years old. In some embodiments, the human subject is an adult human subject (e.g., more than 18 years old). In some embodiments, the human subject has an undeveloped (e.g., an infant or a neonate), weak (e.g., elderly, e.g.., more than 65 years of age), or compromised immune system. Immunocompromised subjects include, without limitation, subjects with primary immunodeficiency or acquired immunodeficiency, such as those suffering from sepsis, HIV infection, cancers, including those undergoing chemotherapy and / or radiotherapy, and subjects with autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, and inflammatory bowel disease. In some embodiments, the human subject has had recent (e.g., within 90 days, within 60 days, within 30 days, within 28 days, within 21 days, within 14 days, within 7 days, within 6 days, within 5 days, within 4 days, within 3 days, within 2 days, or within 1 day prior to the administration) hospitalization or exposure to healthcare services. In some embodiments, the human subject was (e.g., within 90 days, within 60 days, within 30 days, within 28 days, within 21 days, within 14 days, within 7 days, within 6 days, within 5 days, within 4 days, within 3 days, within 2 days, or within 1 day prior to the administration), is, or will be on antibiotics. In some embodiments, the antibiotic is clindamycin, a fluoroquinolone, a cephalosporin, penicillin.

[0242] 12413624.1In some embodiments, the subject is a companion animal (i.e., a pet or service animal). A “companion animal,” as used herein, refers to pets and other domestic animals. Non-limiting examples of companion animals include dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and other animals such as mice, rats, guinea pigs, and hamsters. In some embodiments, the subject is a research animal. Non-limiting examples of research animals include rodents (e.g., ferrets, pigs, rats, mice, guinea pigs, and hamsters), rabbits, or non-human primates.

[0243] The terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease described herein. In some embodiments, treatment may be administered after one or more signs or symptoms of the disease (e.g., botulism, such as foodborne botulism, infant botulism, wound botulism, and iatrogenic botulism, and C. difficile infection, such as antibiotic-associated diarrhea, C. difficile colitis, pseudomembranous colitis, toxic megacolon, recurrent CDI, and fulminant colitis) have developed or have been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease. For example, treatment may be administered to a susceptible subject prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of exposure to a pathogen). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence. Prophylactic treatment refers to the treatment of a subject who is not and was not with a disease but is at risk of developing the disease or who was with a disease, is not with the disease, but is at risk of regression of the disease. In some embodiments, the subject is at a higher risk of developing the disease or at a higher risk of regression of the disease than an average healthy member of a population.

[0244] An “effective amount” of a composition described herein refers to an amount sufficient to elicit the desired biological response. An effective amount of a composition described herein may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the mode of administration, and the age and health of the subject. In some embodiments, an effective amount is a therapeutically effective amount. In some embodiments, an effective amount is a prophylactic treatment. In some embodiments, an effective amount is the amount of a compound described herein in a single dose. In some embodiments, an effective amount is the combined amounts of a compound described herein in multiple doses. When an effective amount of a composition is referred herein, it means the amount is prophylactically and / or therapeutically effective, depending on the subject and / or the

[0245] 12413624.1disease to be treated. Determining the effective amount or dosage is within the abilities of one skilled in the art.

[0246] The terms “administer,” “administering,” or “administration” refers to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a compound described herein, or a composition thereof, in or on a subject. Antibodies (e.g., NTAbs) or pharmaceutical compositions described herein may be administered systemically (e.g., via intravenous injection) or locally (e.g., via local injection). In some embodiments, antibodies (e.g., NTAbs) or pharmaceutical compositions described herein are administered orally, intravenously, topically, intranasally, or sublingually. Parenteral administration is also contemplated. The term “parenteral” as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.

[0247] Polypeptide molecules of the present disclosure may share a certain degree of sequence similarity or identity with reference molecules (e.g., reference polypeptides), for example, wildtype molecules. The term “identity” as known in the art, refers to a relationship between the sequence of two or more polypeptides, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between them as determined by the number of matches between strings of two or more amino caid residues. Identity measures the precent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (e.g., “algorithms”). Identity of related peptides can be readily calculated by known methods. “% identity” as it applies to polypeptide sequences is defined as the percentage of residues (amino acid residues) in the candidate amino acid sequence that are identical with the residues in the amino acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for the alignment are well known in the art. Such tools for alignment include those of the BLAST suite (Stephen F. Altschul, et al (1997), "Gapped BLAST and PSLBLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402). Another popular local alignment technique is based on the Smith-Waterman algorithm (Smith, T.F. & Waterman, M.S. (1981) “Identification of common molecular subsequences.” J. Mol. Biol. 147:195-197.) A general global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, S.B. & Wunsch, C.D. (1970) “A general method applicable to the search

[0248] 12413624.1for similarities in the amino acid sequences of two proteins.” J. Mol. Biol. 48:443-453.). More recently a Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has been developed that purportedly produces global alignment of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman-Wunsch algorithm. It is understood that identity depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation.

[0249] The term “identity” refers to the overall relatedness between polymeric molecules, for example, between polypeptide molecules. Calculation of the percent identity of two polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid sequence(s) for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In some embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The amino acids at corresponding amino acid positions are then compared. When a position in the first sequence is occupied by the same amino acid as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.

[0250] EXAMPLES

[0251] Example 1.

[0252] Clostridioides difficile (C. difficile') is a spore-forming opportunistic pathogen, which can colonize the colon when the normal gut microbiome is disrupted. C. difficile infection (CDI)) accounts for approximately 15-20% of all cases of antibiotic-associated diarrhea in developed countries, ranging from mild diarrhea to pseudomembranous colitis. The incidence of multiple recurrent CDI (rCDI) has risen disproportionately, and estimated annual cost is between 4.8 and 6.3 billion for in-hospital management. In the United States, CDI is a leading cause of hospital-acquired infection that can result in severe illness and death.

[0253] 12413624.1- SO - Two homologous protein toxins, toxin A (TcdA) and toxin B (TcdB), are major virulence factors of C. difficile, as strains lacking them do not cause disease. A subset of C. difficile strains also express a third toxin (binary toxin CDT) that also contributes to pathogenesis. Among these toxins, TcdB is the dominant toxin in humans, as many clinical isolates are TcdA TcdB+, which cause full-scale disease. Therefore, TcdB is a major therapeutic target in humans.

[0254] Antibiotics (e.g., vancomycin and fidaxomicin) are approved as first-line therapy for CDI. However, antibiotic resistance has been on the rise. Furthermore, antibiotics disrupt microbiomes, leading to recurrent infection that could become difficult to treat. In 2022, the FDA approved the first fecal microbiota transplantation (FMT) for patients with rCDI, but FMT remains difficult to standardize and carries the risk of exposure to additional pathogens. Given that CDI patients are often immunocompromised and hospitalized and are especially vulnerable, FMT is not a reliable approach for treating CDI. Toxin-neutralizing antibodies could bring passive immunity to immune-compromised and elderly patients. A TcdB-neutralizing antibody, bezlotoxumab by Merck, is the only FDA-approved preventative against rCDI. However, its clinical efficacy is less than ideal, as rCDI frequency differences for bezlotoxumab and antibiotic versus placebo and antibiotic are: metronidazole: -9.7%; vancomycin: -15.4%; fidaxomicin: -11.9%. Furthermore, neither bezlotoxumab nor FMT can be used to treat severe acute CDI in hospitalized patients, which is the clinical situation most likely to cause mortality and continues to present an urgent need for new treatment options. No vaccines are currently available, and previous vaccine development by Sanofi failed Phase III trials.

[0255] Anti-sera have long been utilized for passive immunity treatment, but have many drawbacks. Attempts to replace them with monoclonal antibodies, however, have encountered their own challenges in vivo. For example, studies have shown that a single monoclonal antibody cannot provide adequate protection in vivo against a potent toxin, no matter how potent the antibody is in vitro. Protection in vivo requires combinations of multiple monoclonal antibodies that bind to different sites on toxins, with a minimum combination of three antibodies. This is likely because binding of multiple antibodies is necessary to mark the toxin molecule for clearance and degradation by immune and liver cells. In contrast, toxins bound by a single monoclonal antibody are not cleared and may even gain longer serum half-life due to the long half-life of the monoclonal antibodies. Bound toxins can eventually dissociate from the antibody and exhibit toxicity. This is why a single monoclonal antibody can be less effective in

[0256] 12413624.1vivo than polyclonal anti- sera: efficient clearance is critical for getting rid of these potent toxins in vivo. As this cannot be achieved with a single monoclonal antibody, a cocktail of monoclonal antibodies is necessary and has been shown to be highly effective in vivo for a potent toxin. However, the major downside is the greatly increased drug development complexity: three monoclonal antibodies need to be manufactured individually, co-formulations must be developed, and the quality and stability of each antibody individually and in combination need to be fully analyzed. Antibodies can also exhibit drastic differences in pharmacological kinetics (PK), creating a major challenge for dosing, CMC (chemistry, manufacturing, and controls), and clinical use. This Example demonstrates the development and characterization of a designer 3-in-1 antibody that can promote toxin clearance in vivo, thus replacing the need for complicated antibody cocktails.

[0257] Nanobodies (also known as VHH antibodies) are -12-15 kDa proteins derived from the single variable domain of the heavy-chain-only antibodies in Camelidae animals (FIG. 1A). An exemplary nanobody-based triple paratopic antibody (NTAb) was designed with three nanobodies fused with each other and then with a Fc (FIG. IB), so that it can recognize three distinct epitopes on a target. Specifically, three nanobodies were selected for binding to three different epitopes of TcdB: 5D (VHH1), which binds to the delivery and receptor-binding domain (DRBD); and E3 (VHH2) and 7F (VHH3), which bind to different sites on the glucosyltransferase domain (GTD) (FIG. 2A). An exemplary NTAb was then created by fusion of the three nanobodies to human IgGl Fc, with a flexible linker (GGGS)4 between each component (FIG. 2B). The exemplary NTAbs, as well as control antibodies containing individual VHHs (VHH1 (5D)-Fc, VHH2 (E3)-Fc, VHH3 (7F)-Fc), were produced by a transient ExpiCHO expression system. The purity and yield (>30 mg / 100 mF expression volume) of each antibody were found to be similar to those of regular monoclonal antibodies (FIG. 2C and Table 12).

[0258] Table 12. Summary of C. difficile toxin B epitope and yield of VHH-Fc antibodies.

[0259]

[0260] 12413624.1First, antibody binding to TcdB at the biochemical level was characterized using biolayer interferometry assay (BLI). Bezlotoxumab binds to TcdB 1 with an apparent KD of ~0.9 nM, but showed 10-fold lower affinity toward TcdB2. Bezlotoxumab also showed lower efficacy on the TcdB2-expression strains. All three individual VHH-Fc (VHH1 (5D)-Fc, VHH2 (E3)-Fc, VHH3 (7F)-Fc) showed low KD on both TcdBl and TcdB2. An exemplary NTAb (VHH123-Fc) showed the best binding affinity across all tested antibodies: 0.07 nM for TcdBl and < 0.001 nM for TcdB2. The exemplary NTAb (VHH123-Fc) also showed the highest level of binding, suggesting that there are more total NTAb (VHH123-Fc) bound to immobilized TcdB in comparison with other antibodies (FIGs. 3A-3B and Table 13).

[0261] Table 13. Summary of binding affinity parameters between antibodies and C. difficile toxin B subtypes 1 and 2.

[0262] < <

[0263]

[0264] Next, the capability of an exemplary NTAb to neutralize TcdB subtypes in vitro on cultured cells was analyzed by seeding HeLa cells in 96-well plates, followed by adding a TcdB toxin mixture with or without antibody into the medium. After 18 hours, the percentage of rounded cells (due to toxin action) in each well was counted and quantified. Bezlotoxumab showed good protection only against TcdBl and TcdB3, but reduced efficacy on all other subtypes, which is consistent with previous reports. VHH2 (E3)-Fc and VHH3 (7F)-Fc showed poor or no protection against TcdB subtypes 3, 4, 7, 10, 11, and 12 and TcdB subtypes 2, 3, 4, 7, 10, 11, and 12, respectively. VHH1 (5D)-Fc was found to neutralize all TcdB subtypes except for TcdB5 and TcdB8, which is consistent with residue variations (e.g., H1307 and D1358) within the DRBD epitope in TcdB 5 and TcdB 8 (FIGs. 4A-4L). A cocktail of all three individual (VHH-Fcs (VHH1 (5D)-Fc, VHH2 (E3)-Fc, VHH3 (7F)-Fc) and the exemplary NTAb protected cells from all subtypes of TcdB (FIG. 4M). However, the NTAb also surprisingly showed > 100-fold efficacy than the cocktail of three individual VHH-Fcs (FIG. 4N). The efficacy of the 12413624.1exemplary NT Ab was found to be 380-fold higher than that of bezlotoxumab on TcdBl, the main subtype found clinically, and 11,000-fold higher on TcdB2, a major subtype associated with many hypervirulent strains.

[0265] Finally, the in vivo efficacy of the exemplary NT Ab for neutralizing TcdB was assessed by injecting the exemplary NT Ab, a cocktail of three individual VHHs (VHH-Fcs (VHH1 (5D)-Fc, VHH2 (E3)-Fc, VHH3 (7F)-Fc), or bezlotoxumab into mice via the intraperitoneal route two days prior to intraperitoneal injection of TcdB. TcdB caused death in the absence of any antibody treatment (vehicle). At the concentrations utilized (0.18 pM and 0.3 pM, 100 pL / mouse), bezlotoxumab showed no protection against TcdBl (1200 ng / mouse) or TcdB2 (150 ng / mouse). The cocktail of three individual VHH-Fcs showed a protective effect and delayed death, with 12.5% of mice rescued from TcdBl and 37.5% of mice rescued from TcdB2. Only the exemplary NT Ab fully protected all mice from both TcdBl and TcdB2 (FIGs. 5A-5B).

[0266] This Example demonstrates the superior in vivo efficacy of a highly potent NTAb that can neutralize all TcdB subtypes, which would allow a much lower dose to be used via convenient intramuscular or subcutaneous injections.

[0267] Example 2.

[0268] This Example seeks to investigate the optimal design of nanobody-based triple paratopic antibodies (NTAbs). There are two main design variables to examine: (1) the order of nanobodies; and (2) the linker between nanobodies. The order of the nanobodies may influence binding affinity (e.g., there may be simultaneous binding to two binding sites), neutralizing efficacy, protein expression, and protein stability.

[0269] First, all possible nanobody order combinations (VHH1-VHH2-VHH3, VHH1-VHH3-VHH2, VHH2-VHH1-VHH3, VHH2-VHH3-VHH1, VHH3-VHH1-VHH2, VHH3-VHH2, VHH1) are screened and compared. The NTAbs are constructed and expressed using the transient ExpiCHO cell expression system and purified using a protein A column. The NTAbs are ranked based on (1) yield, purity, and stability; (2) binding to TcdBl and TcdB2 using BLI assays; (3) efficacy for neutralizing 12 TcdB subtypes on in vivo cell-rounding assays; and (4) neutralizing TcdB 1 and TcdB2 in vivo via toxin injection assay. Once the optimal order is determined, the linkers between the nanobodies are further optimized. Both shorter and longer flexible linkers (e.g., SEQ ID NOs: 146-155) as well as rigid linkers (e.g., SEQ ID NOs: 156-160) are evaluated. To assess whether the NTAbs bind a single toxin molecule, or whether the

[0270] 12413624.1NTAbs and TcdB form large oligomers, negative staining electron microscope (EM) and cryo-EM analyses are carried out to analyze the NTAb-toxin complex in vitro.

[0271] Additionally, the efficacy of the NTAbs in protecting mice from infections in vivo is evaluated using the standard antibiotic-induced infection model. Briefly, mice are given an antibiotic cocktail in water for 3 days. On day -2, antibodies (3 pM, 100 pL / mouse) are injected in mice via intraperitoneal administration. On day -1, 100 pL clindamycine (10 mg / kg) is injected into mice via intraperitoneal administration. On day 0, 200 pL C. difficile spores diluted with sterile PBS are gavaged into mice. On day 2, the ceum of mice is collected for histopathology (FIG. 6). The following strains are utilized: an engineered M7404 strain (TcdA-TcdB2+), the standard 630 strain (TcdA+TcdBl+), and the R20291 strain (TcdA+TcdB2+).

[0272] Bezlotoxumab and a cocktail of three individual VHH-Fc are analyzed in parallel for comparison.

[0273] The ability for the NTAbs to accelerate clearance of TcdB in in vivo mouse models is also evaluated by injecting into mice a high dose (e.g., 1 pg) of detoxified catalytic inactive TcdB (ciTcdB, a mutant TcdB harboring a mutation that disrupts the enzymatic activity of TcdB. The concentration of ciTcdB in mouse serum is quantified using sensitive ELISA methods. Pre-incubation of the NTAbs with ciTcdB is carried out and the mixture is injected into mice, followed by assessing ciTcdB concentrations in serum. Bezlotoxumab and a cocktail of three individual VHH-Fc are analyzed in parallel for comparison.

[0274] It is hypothesized that co-injection of individual VHH-Fc and ciTcdB may extend the half-life of ciTcdB in serum, whereas the three-antibody cocktail and the NTAbs will greatly shorten the serum half-life of TcdB. To confirm at the mechanistic level that Fc-mediated clearance is involved, NTAbs containing a mutated IgGl Fc tag with specific mutations (e.g., L234A / L235A / P329G, LALAPG) that abolish binding to human and mouse Fc gamma receptors are constructed, purified, and tested. It is hypothesized that these mutant NTAbs will show reduced efficacy in vivo for reducing ciTcdB in serum. If the involvement of Fc-mediated clearance is confirmed, further mutations (e.g., G236A, S239D, I332E) on IgGl Fc to enhance Fc-FcyR interactions are tested to evaluate whether ciTcdB clearance can be further accelerated.

[0275] Finally, the pharmacokinetics of the NTAbs are assessed in vivo by administering the NTAbs through intravenous injection to establish the mean plasma concentrations over time and the relationships among injection doses, mean plasma concentrations, and effective doses. The concentration of NT Ab in serum is quantified using ELISA (e.g., with anti-llama antibody plus

[0276] 12413624.1anti-human Fc antibody). Animals are observed for a minimum of 14 days to ensure that there are no signs of any harmful consequences.

[0277] Example 3.

[0278] Botulinum neurotoxins (BoNTs) are 150-kDa modular proteins composed of three domains: a receptor-binding domain (He), a middle membrane translocation domain (HN), and an N-terminal protease domain (light chain, LC) (FIG. 7). The LC is the cargo delivered into the cytosol, where it cleaves neuronal proteins (known as SNARE proteins) required for synaptic vesicle exocytosis. This leads to blockage of neurotransmitter release from nerve terminals, and subsequent relaxation of muscles. Local injections of small amounts of BoNTs can relax muscles around the injection site and is effective for treating many muscle spasm conditions, including spasticity caused by central nervous system disorders such as cerebral palsy and multiple sclerosis, in addition to its well-known cosmetic uses. However, BoNTs are also one of the six agents at highest risk for bioterrorism use, classified as “Category A” and “Tier 1” select agent. It is considered a major threat to both civilian and military populations due to its extreme potency, ease of production, risk for contaminating food supplies, potential for attacks through aerosolization, and lack of immunity tin the populat8ion. Thus, there is an urgent need to prepare and stockpile effective countermeasures. Besides biodefense, there is also a medical need for better therapeutics. Although rare, the occurrence of botulism persists in modem society, with -200 cases per year in the United States, and the death rate remains -3-5%. In the United States, over 70% of cases occur in infants. BoNTs cause persistent paralysis and patients need intensive care and ventilation for weeks to months. For example, a recent outbreak occurred in September 2023 in a wine tasting restaurant in France due to contaminated sardines, resulting in 12 people in critical condition in intensive care units and the death of a 32-year-old woman.

[0279] There are currently no FDA-approved BoNT vaccines. Although several recombinant vaccines are under development, vaccination of the general population would be unsuitable since the disease is rare and vaccination prevents people from future medical use. Current FDA-approved treatment includes only two anti-toxins. One of which is an equine polyclonal antitoxin (HBAT) processed from immunized horse serum. HBAT consists of the Fab and F(ab')2fragments to reduce allergic reactions, but it is still associated with severe hypersensitivity reaction in some patients and cannot be used in infants due to allergic reactions. Furthermore,

[0280] 12413624.1HBAT has a short half-life, as short as 7.5 hours in humans, and thus cannot be used prophylactically. Another FDA-approved treatment, known as BabyBIG, is a human immune globulin purified from human serum (from laboratory workers immunized with deactivated BoNT / A and BoNT / B). This is used only in infants and is in limited supply.

[0281] Monoclonal antibodies are a widely used therapeutic modality which can be readily produced and have a long half-life in serum. However, previous studies have shown that a single monoclonal antibody cannot provide adequate protection in vivo against BoNT / A, no matter how potent it is in vitro. Protection in vivo requires combinations of multiple antibodies that bind to different sites on toxins, with a minimum combination of 3 antibodies. This is likely because binding of multiple antibodies is necessary to mark the toxin molecule for clearance and degradation by immune and liver cells. In contrast, toxins bound by a single antibody are not cleared and may even gain longer serum half-life due to the long half-life of the antibody. Bound toxins can eventually dissociate from the antibody and thus maintain toxicity. This is why a single monoclonal antibody is less effective, whereas poly clonal anti-serum usually has high efficacy in neutralizing toxins in vivo.

[0282] The most advanced monoclonal antibody program under development uses three monoclonal antibodies against one BoNT. For example, NTM-1631 (formerly XOMA 3AB) contains an equal molar mixture of three monoclonal antibodies that recognize different sites on BoNT / A. This combination is highly effective in vivo in animal models and has proven safe in Phase I clinical trials. The major downside is the greatly increased drug development complexity three monoclonal antibodies need to be manufactured individually, co-formulations must be developed, and the quality and stability of each antibody individually and in combination needs to be fully analyzed. Furthermore, antibodies can exhibit drastic differences in pharmacological kinetics (PK), creating a major challenge for dosing. To address the three from the two most common BoNTs (type A and type B), a cocktail of 6 antibodies must be developed, rendering the CMC (chemistry, manufacturing, and controls) and clinical development expensive, timeconsuming, and technically challenging. This Example demonstrates the development and characterization of a designer 3-in-l antibody that can promote toxin clearance in vivo, thus replacing the need for complicated antibody cocktails.

[0283] Nanobodies (also known as VHH antibodies) are -12-15 kDa proteins derived from the single variable domain of the heavy-chain-only antibodies in Camelidae animals (FIG. 1A). An exemplary nanobody-based triple paratopic antibody (NTAb) was designed with three

[0284] 12413624.1nanobodies fused with each other and then with a Fc (FIG. IB), so that it can recognize three distinct epitopes on a target. Specifically, three previously characterized nanobodies were selected: H7 (VHH1), which binds to BoNT / A-Lc; B5 (VHH2), which binds to BONT / A-HN; and C2 (VHH3), which binds to BoNT / A-Hc (FIG. 8A). An exemplary nanobody-based triple paratopic antibody (NTAb) was designed using a flexible linker (SEQ ID NO: 148) between nanobodies, as well as individual nanobodies fused with Fc (VHH-Fcs). These antibodies were produced following the same protocol used for producing monoclonal antibodies using a transient mammalian cell protein and expression system (ExpiCHO system, ThermoFisher). The exemplary NTAb and individual VHH-Fcsshowed good yield and ease of purification using protein A columns (-80-11 mg per 30 mF culture) (FIG. 8B).

[0285] First, binding of the exemplary Ntab to BoNT / A was evaluated in vitro by producing detoxified ciBoNT / A, which was biotinylated and immobilized onto the probe in a biolayer interferometry assay. Binding affinity (KD) was estimated to be 0.7 nM for VHH1 (H7)-Fc, 0.8 nM for VHH2 (B5)-Fc, 1.8 nM for VHH3 (C2)-Fc, and 0.4 nM for the exemplary NT Ab (FIG.

[0286] 8C and Table 14). The exemplary NTAb showed higher total binding levels than the three individual nanobodies, suggesting that NTAbs likely bind to one epitope at a time and that there are more NTAbs bound to the probes than individual VHH-Fcs.

[0287] Table 14. Summary of binding affinity parameters between antibodies and botinulinum toxin (BoNT).

[0288]

[0289] Next, the in vivo efficacy of NTAbs were evaluated by utilizing a standard approach of pre-mixing the exemplary NTAb (9 nM, -0.12 pg / mouse) with BoNT / A (1000-fold of lethal dose 50, ED50, 5 ng) for 30 minutes in a test tube followed by intraperitoneal injection of the mixture into mouse. For comparison, a cocktail comprising the three individual VHH-Fcs (containing equal molar of VHH1 (H7)-Fc, VHH2 (B5)-Fc, and VHH3 (C2)-Fc) and a PBS control were used. Mice were observed for 7 days and the survival rates were plotted (FIG. 9A).

[0290] BoNT / A induced rapid death in all control mice within a few hours. The VHH-Fc cocktail

[0291] 12413624.1initially provided protection, but mice began to die after day 2 and all mice died within 3.5 days. In contrast, the exemplary NT Ab protected all mice and no rebound toxicity was observed. These data demonstrate that NTAbs are superior to three-antibody cocktails in vivo.

[0292] Finally, the potency for prophylactic treatment was evaluated by injecting mice with an exemplary NT Ab (30 nM, ~0.4 pg / mouse) or a cocktail comprising the three individual VHH-Fcs 2 days prior to injection of BoNT / A (100-fold of LD50, 0.5 ng). The VHH-Fc cocktail delayed death by only a few hours, and all mice treated with the VHH-Fc cocktail died within the first day after BoNT / A injection (FIG. 9B). In contrast, the NTAb protected all mice from BoNT / A with no rebound toxicity. Increasing the dose of the VHH-Fc cocktail by three-fold protected all mice, confirming that this cocktail can be effective in vivo but less efficacious than NTAb for prophylactic treatment (FIG. 9C).

[0293] This Example demonstrates the superior in vivo efficacy of NTAbs over a cocktail of three individual VHH-Fcs that can be used both therapeutically and prophylactically.

[0294] Example 4.

[0295] This Example seeks to investigate the optimal design of nanobody-based triple paratopic antibodies (NTAbs). There are two main design variables to examine: (1) the order of nanobodies; and (2) the linker between nanobodies. The order of the nanobodies may influence binding affinity (e.g., there may be simultaneous binding to two binding sites), neutralizing efficacy, protein expression, and protein stability.

[0296] First, all possible nanobody order combinations (VHH1-VHH2-VHH3, VHH1-VHH3-VHH2, VHH2-VHH1-VHH3, VHH2-VHH3-VHH1, VHH3-VHH1-VHH2, VHH3-VHH2, VHH1) are screened and compared. The NTAbs are constructed and expressed using the transient ExpiCHO cell expression system and purified using a protein A column. The NTAbs are ranked based on (1) binding affinity and total level of binding to BoNT / A immobilized in the biolayer interferometry assay; (2) efficacy for neutralizing BoNT / A in vivo both when pre-mixed with the toxin and in prophylactic treatment; and (3) expression levels, purity, and stability. Once the optimal order is determined, the linkers between the nanobodies are further optimized. Both shorter and longer flexible linkers (e.g., SEQ ID NOs: 146-155) as well as rigid linkers (e.g., SEQ ID NOs: 156-160) are evaluated. To confirm that the NTAbs bind a single toxin molecule, negative staining electron microscope (EM) analysis and cryo-EM analysis are used to visualize the NTAb-toxin complex. Binding site occupation is evaluated through competition

[0297] 12413624.1assays using individual nanobodies in a pull-down assay and in biolayer interferometry assays. The nanobodies are humanized via databases and software (e.g., Discovery Studio) that have proven effective in various clinical -stage antibody drugs. According to the high homology of camelid VHHs with human IGHV3, the human germline (GHV3-23) is used as a framework, with good clinical-stage developability. The mutations in the FR2 region will also improve the stability and solubility of the humanized VHH. The framework region is engineered to preserve the natural binding with BoNT / A as backmutations based on the published crystal structure. The developability is also improved by engineering the hotspots for potential post-translational modification sites (e.g., deamination sites) in all CDRs. These humanized nanobodies are characterized for binding, neutralization, and developability.

[0298] To evaluate whether NTAbs can accelerate clearance of BoNT / A in mouse models detoxified ciBoNT / A is injected into mice and the mouse serum is collected over time. The concentration of ciBoNT / A in mouse serum is quantified using sensitive mesoscale discovery (MSD)-based ELISA methods. Pre-incubation of NT Ab with ciBoNT / A is carried out and the mixture is injected into mice for analysis of ciBoNT / A changes in serum. As controls, individual VHH-Fcs as well as a three-antibody cocktail are preincubated with ciBoNT / A. Similar detoxified BoNT / B (ciBoNT / B) is utilized as a specificity control. It is hypothesized that coinjection of individual VHH-Fc and ciBoNT / A may extend the half-life of ciBoNT / A in serum, whereas the three-antibody cocktail and NTAbs will greatly shorten the serum half-life of ciBoNT / A. To further confirm that Fc-mediated clearance is involved, NTAbs containing a mutyated Fc tag with specific mutations (e.g., L234A / L235A / P329G, LALAPG) that abolish binding to human and mouse Fc gamma receptors are constructed, purified, and tested. It is hypothesized that the mutant NTAbs will show reduced in vivo efficacy.

[0299] Next, pharmacological kinetic parameters are established using rat models, which are larger than mice and permit multiple serum draws from the same animal to minimize variability. NTAbs are administered through intravenous injection. First, the safety and maximum tolerated dose (MTD) is confirmed. Next, the mean plasma concentrations over time and the relationships among injection doses, mean plasma concentrations, and effective doses are established. The concentration of NT Ab in serum is quantified using ELISA (e.g., with anti-llama antibody plus anti-human Fc antibody). Animals are observed for a minimum of 14 days.

[0300] NTAbs against BoNT / B are also developed as described above, using nanobodies against different domains of BoNT / B (e.g., JLI-G10 and JLK-G12 , which bind to He; JLU-D10, which

[0301] 12413624.1binds to HN; JNE-B10, JSG-C1, and JSG-G1, which bind to Lc). The efficacy of a combination of two NTAbs (one against BoNT / A and the other against BoNT / B) is evaluated to determine whether the same level of neutralization and clearance of BoNT / A and BoNT / B can be maintained at the same level as the individual NTAbs in in vivo mouse models.

[0302] Finally, the therapeutic efficacy of the NTAbs against BoNT / A and BoNT / B is validated in guinea pig models using the established protocol for analyzing FDA-approved HBAT.

[0303] Briefly, 4-LD50 of BoNT / A is injected into the right hind leg via the intramuscular route.

[0304] Animals are observed for clinical signs (lethargy and hind limb paralysis), which are expected to develop by 32-39 hours after injection. Additionally, survival is evaluated after intravenous injection of the NTAbs (at lx, 0.2x, 0.04x, and 0.008x scaled human dose) 12 hours after toxin injection. It is hypothesized that the NTAbs will have similar levels of protection as HBAT (-100% survival with 0.03x human dose; 55% survival with 0.008x human dose). Next, the prophylactic use of NTAbs is evaluated by pre-injection of NTAbs followed by BoNT / A (via intramuscular injection of 4-LD50) at a later time point (e.g., 48 hours). Animals are observed for clinical symptoms (e.g. degrees of lethargy and limb paralysis), body weight, and mobility for a minimum of 14 days. BoNT / B is evaluated separately following the same experimental design.

[0305] 12413624.1

Claims

CLAIMS1. An antibody, comprising:(a) a first VHH that binds to a first epitope of a botulinum toxin (BoNT);(b) a second VHH that binds to a second epitope of the BoNT;(c) a third VHH that binds to a third epitope of the BoNT; and(d) a constant region (Fc) of an immunoglobulin (Ig).

2. The antibody of claim 1, wherein the BoNT is BoNT type A (BoNT / A), BoNT type B (BoNT / B), BoNT type C (BoNT / C), BoNT type D (BoNT / D), BoNT type E (BoNT / E), BoNT type F (BoNT / F), or BoNT type G (BoNT / G).

3. The antibody of claim 1 or claim 2, wherein the first epitope, the second epitope, and the third epitope are selected from the group consisting of a BoNT light chain (LC) epitope and a BoNT heavy chain epitope.

4. The antibody of claim 3, wherein the BoNT heavy chain is an amino-terminal part (HN) or a carboxy-terminal part (HC).

5. The antibody of any one of claims 2-4, wherein the BoNT is BoNT / A.

6. The antibody of claim 5, wherein the first epitope is a BoNT / A-LC epitope, the second epitope is a BoNT / A-HN epitope, and the third epitope is a BoNT / A-HC epitope.

7. The antibody of claim 5 or claim 6, wherein the first VHH comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 2 or SEQ ID NO: 5; the second VHH comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 3 or SEQ ID NO: 6; and, the third VHH comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 4 or SEQ ID NO: 7.

8. The antibody of claim 7, wherein the first VHH comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 5; the second VHH comprises the amino acid sequence of SEQ12413624.1ID NO: 3 or SEQ ID NO: 6; and the third VHH comprises the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 7.

9. The antibody of claim 7 or claim 8, wherein the first VHH consists of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 5; the second VHH consists of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 6; and the third VHH consists of the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 7.

10. The antibody of any one of claims 7-9, wherein the antibody comprises, from N-terminal to C-terminal, the first VHH, the second VHH, the third VHH, and the Fc.

11. The antibody of any one of claims 7-9, wherein the antibody comprises, from N-terminal to C-terminal, the first VHH, the third VHH, the second VHH, and the Fc.

12. The antibody of any one of claims 7-9, wherein the antibody comprises, from N-terminal to C-terminal, the second VHH, the first VHH, the third VHH, and the Fc.

13. The antibody of any one of claims 7-9, wherein the antibody comprises, from N-terminal to C-terminal, the second VHH, the third VHH, the first VHH, and the Fc.

14. The antibody of any one of claims 7-9, wherein the antibody comprises, from N-terminal to C-terminal, the third VHH, the first VHH, the second VHH, and the Fc.

15. The antibody of any one of claims 7-9, wherein the antibody comprises, from N-terminal to C-terminal, the third VHH, the second VHH, the first VHH, and the Fc.

16. The antibody of any one of claims 2-4, wherein the BoNT is BoNT / B.

17. The antibody of claim 16, wherein the first epitope is a BoNT / B-LC epitope, the second epitope is a BoNT / B -HN epitope, and the third epitope is a BoNT / B -HC epitope.12413624.

118. The antibody of claim 16 or claim 17, wherein the first VHH comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 20 or SEQ ID NO: 23; the second VHH comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 21 or SEQ ID NO: 24; and the third VHH comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 22 or SEQ ID NO: 25.

19. The antibody of claim 18, wherein the first VHH comprises the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 23; the second VHH comprises the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 24; and the third VHH comprises the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 25.

20. The antibody of claim 18 or claim 19, wherein the first VHH consists of the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 23; the second VHH consists of the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 24; and the third VHH consists of the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 25.

21. The antibody of any one of claims 18-20, wherein the antibody comprises, from N-terminal to C-terminal, the first VHH, the second VHH, the third VHH, and the Fc.

22. The antibody of any one of claims 18-20, wherein the antibody comprises, from N-terminal to C-terminal, the first VHH, the third VHH, the second VHH, and the Fc.

23. The antibody of any one of claims 18-20, wherein the antibody comprises, from N-terminal to C-terminal, the second VHH, the first VHH, the third VHH, and the Fc.

24. The antibody of any one of claims 18-20, wherein the antibody comprises, from N-terminal to C-terminal, the second VHH , the third VHH, the first VHH, and the Fc.

25. The antibody of any one of claims 18-20, wherein the antibody comprises, from N-terminal to C-terminal, the third VHH, the first VHH, the second VHH, and the Fc.12413624.

126. The antibody of any one of claims 18-20, wherein the antibody comprises, from N-terminal to C-terminal, the third VHH, the second VHH, the first VHH, and the Fc.

27. The antibody of any one of claims 1-25, wherein the first VHH, the second VHH, the third VHH, and the Fc are connected by a peptide linker.

28. The antibody of any one of claims 27, wherein the peptide linker is a flexible linker.

29. The antibody of claim 28, wherein the flexible linker is (GGGS)n(SEQ ID NOs: 151- 155) or (GGGGS)n (SEQ ID NOs: 146-150), where n is 1, 2, 3, 4, or 5.

30. The antibody of claim 27, wherein the peptide linker is a rigid linker.

31. The antibody of claim 30, wherein the rigid linker is (EAAAK)n(SEQ ID NOs: 156- 160), where n is 1, 2, 3, 4, or 5.

32. The antibody of any one of claims 1-31, wherein the immunoglobulin (Ig) is an immunoglobulin G (IgG), an immunoglobulin M (IgM), an immunoglobulin A (IgA), an immunoglobulin D (IgD), or an immunoglobulin E (IgE).

33. The antibody of any one of claims 1-32, wherein the Fc comprises the amino acid sequence of SEQ ID NO: 1.

34. The antibody of claim 33, wherein the Fc consists of the amino acid sequence of SEQ ID NO: 1.

35. The antibody of any one of claims 1-34, wherein the antibody comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 8-19 or 26-37.

36. The antibody of claim 35, wherein the antibody comprises the amino acid sequence of any one of SEQ ID NOs: 8-19 or 26-37.12413624.

137. The antibody of claim 35 or claim 36, wherein the antibody consists of the amino acid sequence of any one of SEQ ID NOs: 8-19 or 26-37.

38. A nucleic acid which encodes, or a set of nucleic acids which collectively encode, the antibody of any one of claims 1-37.

39. A vector or vector set, comprising the nucleic acid or the set of nucleic acids of claim 38.

40. The vector or vector set of claim 39, wherein the vector(s) is (are) an expression vector(s).

41. A cell, comprising the vector or vector set of claim 39 or claim 40.

42. The cell of claim 41, wherein the cell is a bacterial cell, a yeast cell, an insect cell, a plant cell, or a mammalian cell.

43. The cell of claim 42, wherein the cell is a Chinese Hamster Ovary (CHO) cell.

44. A method of producing an antibody, comprising:(i) culturing the cell of any one of claims 41-43 in a culture medium; and(ii) collecting the cultured cells or the culture medium for isolation of the antibody.

45. The method of claim 44, wherein the method further comprises isolating the antibody from the cultured cells or the culture medium.

46. A pharmaceutical composition, comprising:(a) the antibody of any one of claims 1-37, the nucleic acid of claim 38, or the vector or vector set of claim 39 or claim 40; and(b) a pharmaceutically acceptable carrier.

47. The pharmaceutical composition of claim 46, for use in treating botulism.12413624.

148. A method of treating a Clostridium botulinum infection in a subject, comprising administering to the subject the antibody of any one of claims 1-37, the nucleic acid of claim 38, the vector or vector set of claim 39 or claim 40, or the pharmaceutical composition of claim 46 or claim 47.

49. A method of reducing the severity of one or more symptoms associated with a Clostridium botulinum infection or intoxication by botulinum neurotoxins in a subject, comprising administering to the subject the antibody of any one of claims 1-37, the nucleic acid of claim 38, the vector or vector set of claim 39 or claim 40, or the pharmaceutical composition of claim 46 or claim 47.

50. The method of claim 48 or claim 49, wherein the Clostridium botulinum produces botulinum neurotoxin (BoNT) type A (BoNT / A), BoNT type B (BoNT / B), BoNT type C (BoNT / C), BoNT type D (BoNT / D), BoNT type E (BoNT / E), BoNT type F (BoNT / F), or BoNT type G (BoNT / G).

51. The method of any one of claims 48-50, wherein the Clostridium botulinum infection or botulinum neurotoxin intoxication is foodbome botulism, wound botulism, infant botulism, iatrogenic botulism, or adult intestinal toxemia.

52. The method of any one of claims 48-51, wherein the subject exhibits one or more symptoms of Clostridium botulinum infection or botulinum neurotoxin intoxication selected from the group consisting of fatigue, weakness, vertigo, blurred vision, dry mouth, difficulty swallowing or speaking, vomiting, diarrhea, constipation, abdominal swelling, weakness in the neck or arms, descending symmetrical paralysis, ptosis, facial palsy, ophthalmoplegia, and diplopia.

53. A VHH comprising an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 5-7 or 23-25.

54. An antibody, comprising:(a) a first VHH that binds to a first epitope of a Clostridioides difficile toxin (Ted);12413624.1(b) a second VHH that binds to a second epitope of the Ted;(c) a third VHH that binds to a third epitope of the Ted; and(d) a constant region (Fc) of an immunoglobulin (Ig).

55. The antibody of claim 54, wherein the Ted is Clostridioides difficile toxin A (TcdA) or Clostridioides difficile toxin B (TcdB).

56. The antibody of claim 54 or claim 55, wherein the first epitope, the second epitope, and the third epitope are selected from the group consisting of a Ted N-terminal glucosyltransferase domain (GTD), a Ted autoprocessing domain (APD), a Ted delivery and receptor-binding domain (DRBD), and a Ted combined repetitive oligopeptides (CROPs) domain.

57. The antibody of claim 55 or claim 56, wherein the first epitope, the second epitope, and the third epitope are TcdB epitopes.

58. The antibody of claim 57, wherein the first epitope is a TcdB -DRBD epitope, the second epitope is a TcdB-GTD epitope, and the third epitope is a TcdB-GTD epitope.

59. The antibody of claim 58, wherein the second epitope and the third epitope are different epitopes of TcdB-GTD.

60. The antibody of any one of claims 57-59, wherein the first VHH comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 38 or SEQ ID NO: 41; the second VHH comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 39 or SEQ ID NO: 42; and, the third VHH comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 40 or SEQ ID NO: 43.

61. The antibody of claim 60, wherein the first VHH comprises the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 41; the second VHH comprises the amino acid sequence of SEQ ID NO: 39 or SEQ ID NO: 42; and, the third VHH comprises the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43.12413624.

162. The antibody of claim 60 or claim 61, wherein the first VHH consists of the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 41; the second VHH consists of the amino acid sequence of SEQ ID NO: 39 or SEQ ID NO: 42; and, the third VHH consists of the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43.

63. The antibody of any one of claims 60-62, wherein the antibody comprises, from N-terminal to C-terminal, the first VHH, the second VHH, and third VHH, and the Fc.

64. The antibody of any one of claims 60-62, wherein the antibody comprises, from N-terminal to C-terminal, the first VHH, the third VHH, the second VHH, and the Fc.

65. The antibody of any one of claims 60-62, wherein the antibody comprises, from N-terminal to C-terminal, the second VHH, the first VHH, and third VHH, and the Fc.

66. The antibody of any one of claims 60-62, wherein the antibody comprises, from N-terminal to C-terminal, the second VHH, the third VHH, the first VHH, and the Fc.

67. The antibody of any one of claims 60-62, wherein the antibody comprises, from N-terminal to C-terminal, the third VHH, the first VHH, the second VHH, and the Fc.

68. The antibody of any one of claims 60-62, wherein the antibody comprises, from N-terminal to C-terminal, the third VHH, the second VHH, the first VHH, and the Fc.

69. The antibody of any one of claims 54-68, wherein the antibody further comprises a fourth VHH that binds to a fourth epitope of the Ted, wherein the fourth epitope is a TcdA epitope.

70. The antibody of claim 69, wherein the TcdA epitope is selected from the group consisting of a Ted AN-terminal glucosyltransferase domain (GTD), a TcdA autoprocessing domain (APD), a TcdA delivery and receptor-binding domain (DRBD), and a TcdA combined repetitive oligopeptides (CROPs) domain.12413624.

171. The antibody of claim 69 or claim 70, wherein the fourth VHH comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 56-61.

72. The antibody of any one of claims 69-71, wherein the fourth VHH comprises the amino acid sequence of SEQ ID NO: 56-61.

73. The antibody of any one of claims 69-72, wherein the fourth VHH consists of the amino acid sequence of SEQ ID NO: 56-61.

74. The antibody of any one of claims 69-73, wherein the antibody comprises, from N-terminal to C-terminal, the fourth VHH, the first VHH, the second VHH, the third VHH, and the Fc.

75. The antibody of any one of claims 69-73, wherein the antibody comprises, from N-terminal to C-terminal, the fourth VHH, the first VHH, the third VHH, the second VHH, and the Fc.

76. The antibody of any one of claims 69-73, wherein the antibody comprises, from N-terminal to C-terminal, the fourth VHH, the second VHH, the first VHH, the third VHH, and the Fc.

77. The antibody of any one of claims 69-73, wherein the antibody comprises, from N-terminal to C-terminal, the fourth VHH, the third VHH, the first VHH, the second VHH, and the Fc.

78. The antibody of any one of claims 69-73, wherein the antibody comprises, from N-terminal to C-terminal, the fourth VHH, the second VHH, the third VHH, the first VHH, and the Fc.

79. The antibody of any one of claims 69-73, wherein the antibody comprises, from N-terminal to C-terminal, the fourth VHH, the third VHH, the second VHH, the first VHH, and the Fc.12413624.

180. The antibody of any one of claims 69-73, wherein the antibody comprises, from N-terminal to C-terminal, the first VHH, the second VHH, the third VHH, the Fc, and the fourth VHH.

81. The antibody of any one of claims 69-73, wherein the antibody comprises, from N-terminal to C-terminal, the first VHH, the third VHH, the second VHH, the Fc, and the fourth VHH.

82. The antibody of any one of claims 69-73, wherein the antibody comprises, from N-terminal to C-terminal, the second VHH, the first VHH, the third VHH, the Fc, and the fourth VHH.

83. The antibody of any one of claims 69-73, wherein the antibody comprises, from N-terminal to C-terminal, the third VHH, the first VHH, the second VHH, the Fc, and the fourth VHH.

84. The antibody of any one of claims 69-73, wherein the antibody comprises, from N-terminal to C-terminal, the second VHH, the third VHH, the first VHH, the Fc, and the fourth VHH.

85. The antibody of any one of claims 69-73, wherein the antibody comprises, from N-terminal to C-terminal, the third VHH, the second VHH, the first VHH, the Fc, and the fourth VHH.

86. The antibody of any one of claims 54-68, wherein the first VHH, the second VHH, the third VHH, and the Fc are connected by a peptide linker.

87. The antibody of any one of claims 69-85, wherein the first VHH, the second VHH, the third VHH, the fourth VHH, and the Fc are connected by a peptide linker.

88. The antibody of claim 86 or claim 87, wherein the peptide linker is a flexible linker.12413624.

189. The antibody of claim 88, wherein the flexible linker is (GGGS)n(SEQ ID NOs: 151-155) or (GGGGS)n (SEQ ID NOs: 146-150), where n is 1, 2, 3, 4, or 5.

90. The antibody of claim 86 or claim 87, wherein the peptide linker is a rigid linker.

91. The antibody of claim 90, wherein the rigid linker is (EAAAK)n(SEQ ID NOs: 156-160), where n is 1, 2, 3, 4, or 5.

92. The antibody of any one of claims 54-91, wherein the immunoglobulin (Ig) is an immunoglobulin G (IgG), an immunoglobulin M (IgM), an immunoglobulin A (IgA), an immunoglobulin D (IgD), or an immunoglobulin E (IgE).

93. The antibody of any one of claims 54-92, wherein the Fc comprises the amino acid sequence of SEQ ID NO: 1.

94. The antibody of claim 93, wherein the Fc consists of the amino acid sequence of SEQ ID NO: 1.

95. The antibody of any one of claims 54-94, wherein the antibody comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 44-55, 62-72, or 74-145.

96. The antibody of claim 95, wherein the antibody comprises the amino acid sequence of any one of SEQ ID NOs: 44-55, 62-72, or 74-145.

97. The antibody of claim 95 or claim 96, wherein the antibody consists of the amino acid sequence of any one of SEQ ID NOs: 44-55, 62-72, or 74-145.

98. A nucleic acid which encodes, or a set of nucleic acids which collectively encode, the antibody of any one of claims 54-97.

99. A vector or vector set, comprising the isolated nucleic acid or the set of isolated nucleic acids of claim 98.12413624.1100. The vector or vector set of claim 99, wherein the vector(s) is (are) an expression vector(s).

101. A cell, comprising the vector or vector set of claim 99 or claim 100.

102. The cell of claim 101, wherein the cell is a bacterial cell, a yeast cell, an insect cell, a plant cell, or a mammalian cell.

103. The cell of claim 102, wherein the cell is a Chinese Hamster Ovary (CHO) cell.

104. A method of producing an antibody, comprising:(i) culturing the cell of any one of claims 101-103 in a culture medium; and(ii) collecting the cultured cells or the culture medium for isolation of the antibody.

105. The method of claim 104, wherein the method further comprises isolating the antibody from the cultured cells or the culture medium.

106. A pharmaceutical composition, comprising:(a) the antibody of any one of claims 54-97, the nucleic acid or the set of nucleic acids of claim 98, or the vector or vector set of claim 99 or claim 100; and(b) a pharmaceutically acceptable carrier.

107. The pharmaceutical composition of claim 106, for use in treating Clostridioides difficile infection.

108. A method of treating a Clostridioides difficile infection in a subject, comprising administering to the subject the antibody of any one of claims 54-97, the nucleic acid or the set of nucleic acids of claim 98, the vector or vector set of claim 99 or claim 100, or the pharmaceutical composition of claim 106 or claim 107.12413624.1109. A method of reducing the severity of one or more symptoms associated with a Clostridioides difficile infection or intoxication by Clostridioides difficile toxins in a subject, comprising administering to the subject the antibody of any one of claims 54-97, the nucleic acid or the set of nucleic acids of claim 98, the vector or vector set of claim 99 or claim 100, or the pharmaceutical composition of claim 106 or claim 107.

110. The method of claim 108 or claim 109, wherein the Clostridioides difficile produces Toxin A (TcdA) and / or Toxin B (TcdB).

111. The method of any one of claims 108-110, wherein the Clostridioides difficile infection of intoxication by Clostridioides difficile toxins is recurrent C. difficile infection (rCDI), antibiotic associated diarrhea, C. difficile colitis, pseudomembranous colitis, toxic megacolon, or fulminant colitis.

112. The method of any one of claims 108-111, wherein the subject exhibits one or more symptoms of Clostridioides difficile infection selected from the group consisting of watery diarrhea, abdominal cramping and pain, fast heart rate, dehydration, fever, nausea, increased white blood cell count, kidney failure, loss of appetite, abdominal swelling, weight loss, blood or pus in stool, pseudomembranous colitis, colonic ileus, toxic megacolon, and sepsis.

113. A VHH comprising an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 41-43 or 59-61.12413624.1

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