VHH POLYPEPTIDES THAT BIND TO CLOSTRIDIUM DIFFICILE TOXIN B AND METHODS OF USE THEREOF
20230192825 · 2023-06-22
Assignee
Inventors
Cpc classification
C07K2317/76
CHEMISTRY; METALLURGY
C07K2317/569
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
C07K2317/22
CHEMISTRY; METALLURGY
International classification
Abstract
Polypeptide products, methods, pharmaceutical compositions, and kits are provided for treating a subject exposed to, or at risk for exposure to, C. difficile microbial pathogens and toxin B produced by C. difficile (TcdB) pathogens. The methods, compositions and kits include a single domain, anti-TcdB VHH polypeptide (antibody), or toxin B binding portion thereof, that specifically binds to and/or neutralizes TcdB and treats or prevents illness and disease associated with C. difficile infection and TcdB intoxication. The anti-TcdB VHHs, or toxin B binding portion thereof, may be recombinantly produced.
Claims
1. A polypeptide that specifically binds to C. difficile toxin B (TcdB), or a TcdB-binding portion thereof, wherein the polypeptide comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, and four VHH framework regions (FRs), FR1, FR2, FR3 and FR4, with the general structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein CDR1 comprises amino acid sequence GSVYTF; CDR2 comprises amino acid sequence SGGTITK; and CDR3 comprises amino acid sequence NAGDTIAQAMGTRRFPFDR; wherein CDR1 comprises amino acid sequence GTSFPRNY; CDR2 comprises amino acid sequence SHDGNVE; and CDR3 comprises amino acid sequence KLVTLRRDEY; wherein CDR1 comprises amino acid sequence RFSLINYA; CDR2 comprises amino acid sequence TSGGATY; and CDR3 comprises amino acid sequence AAGPYSRTLVSRWKVGDGMEY; wherein CDR1 comprises amino acid sequence GFTSNSYY; CDR2 comprises amino acid sequence SSSGGSPN; and CDR3 comprises amino acid sequence AASKFPLTTMASNRYHY; wherein CDR1 comprises amino acid sequence GRGPGINV; CDR2 comprises amino acid sequence QTGGTTN; and CDR3 comprises amino acid sequence YLKKWRDEY; wherein CDR1 comprises amino acid sequence GSSFSMNV; CDR2 comprises amino acid sequence RSDGITN; and CDR3 comprises amino acid sequence FHGRARTGNNADLGS; wherein CDR1 comprises amino acid sequence GRLSERIFMIST; CDR2 comprises amino acid sequence SRLGRAN; and CDR3 comprises amino acid sequence NLKPFVDNYR; wherein CDR1 comprises amino acid sequence GITFSNVA; CDR2 comprises amino acid sequence STGGSSTS; and CDR3 comprises amino acid sequence VKGPKYSATIRRPE; wherein CDR1 comprises amino acid sequence GFNFSVQI; CDR2 comprises amino acid sequence STGGASKS; and CDR3 comprises amino acid sequence SKGPRTWINSSPR; wherein CDR1 comprises amino acid sequence GTAFSLDT; CDR2 comprises amino acid sequence SSSGASN; and CDR3 comprises amino acid sequence YRGRVRGVWPLDSGMMY; wherein CDR1 comprises amino acid sequence GSILSS; CDR2 comprises amino acid sequence SRTGATD; and CDR3 comprises amino acid sequence NAGLGMGDPRRPGPW; or wherein CDR1 comprises amino acid sequence ERNPGINA; CDR2 comprises amino acid sequence WQTGGSLS; and CDR3 comprises amino acid sequence YLKKWRDQY.
2. The polypeptide of claim 1, wherein the polypeptide neutralizes C. difficile toxin B (TcdB) activity.
3. The polypeptide of claim 1, which is a camelid-derived single domain anti-TcdB VHH antibody.
4.-5. (canceled)
6. A polypeptide that specifically binds to C. difficile toxin B (TcdB) wherein the polypeptide or a TcdB-binding portion thereof has at least 85%, at least 90%, at least 95%, or at least 98% amino acid sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23.
7.-9. (canceled)
10. The polypeptide of claim 6, wherein conservative amino acid substitutions in the polypeptide comprise the at least 85%, at least 90%, at least 95%, or at least 98% amino acid sequence identity.
11. A polypeptide that specifically binds to C. difficile toxin B (TcdB) wherein the polypeptide or a TcdB-binding portion thereof comprises a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23.
12. A polypeptide that specifically binds to C. difficile toxin B (TcdB) wherein the polypeptide or a TcdB-binding portion thereof consists of a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23.
13.-17. (canceled)
18. A dimeric or multimeric polypeptide comprising two or more anti-TcdB VHH polypeptides comprising a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23, or TcdB binding regions thereof, wherein the two or more anti-TcdB VHH polypeptides, or TcdB binding regions, are joined with one or more linker peptides.
19. The dimeric or multimeric polypeptide of claim 18, wherein the one or more linker peptides is selected from GGGGS; GGGGSGGGGSGGGGS, or a functional portion thereof; EPKTPKPQGGGGSGGGGSGGGGSQGVQSQVQLVE; EPKTPKPQ; or a combination thereof.
20. The dimeric or multimeric polypeptide of claim 18, comprising one or more epitope tag sequences specifically bindable by an anti-epitope tag antibody or binding portion thereof.
21. The dimeric or multimeric polypeptide of claim 20, wherein the one or more epitope tag sequences comprises at least one of DELGPRLMGK or GAPVPYPDPLEPR.
22.-37. (canceled)
38. An isolated polynucleotide comprising a nucleic acid sequence encoding an anti-TcdB VHH of any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23.
39. An isolated polynucleotide having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to a nucleic acid sequence encoding an anti-TcdB VHH of any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23.
40.-96. (canceled)
97. The polypeptide of claim 1, wherein the four VHH FRs are camelid VHH FRs.
98.-104. (canceled)
105. The polypeptide of claim 2, which is a camelid-derived single domain anti-TcdB VHH antibody.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE DISCLOSURE
[0153] Described and featured herein are single domain antibody (sAb) binding molecules, which are comprised of the heavy chain variable (V.sub.H) region of heavy-chain-only and bodies (Abs), that specifically bind to the toxin B (TcdB) exotoxin virulence factor produced by pathogenic Clostridium difficile (C. diff.) microorganisms. Single-domain antibodies (camelid single-domain antibodies) are called VHHs as they derive from the V.sub.H region of a class of heavy-chain-only antibodies. The anti-toxin B (TcdB) VHHs were produced from immunized camelids (alpacas) and were selected for their ability to specifically bind to TcdB and, in most cases, to neutralize TcdB and the critical pathogenic functions caused by this toxin. The anti-TcdB VHHs as described herein are C. diff. toxin B binding polypeptides comprising hypervariable variable regions (CDR) within framework (FR) regions. In general, the FRs of the anti-TcdB VHHs are typically highly similar in amino acid sequence, or differ by conservative amino acid substitutions at certain positions of the FR sequences, among different anti-TcdB VHHs or families of anti-TcdB VHHs. The anti-TcdB VHHs as described herein are stable over time and provide therapeutic efficacy in animal models. The anti-TcdB VHHs are employed as therapeutic antitoxin agents for the prevention and treatment of toxin B-mediated diseases, such as CDI and its symptoms, caused by Clostridium difficile. It will be understood that the terms “anti-TcdB VHH antibody,” “anti-TcdB VHH polypeptide,” “anti-TcdB VHH antibody polypeptide,” and “anti-TcdB VHH” are used interchangeably herein.
[0154] The presence of toxins produced by C. difficile, in particular, toxin B (TcdB), in the general circulation and locally in the gastrointestinal (GI) tract, causes serious illness in infected humans and other animals. C. difficile TcdB is considered to be the key exotoxin that causes CDI pathology. Antitoxins are therapeutic agents that prevent toxin infection or reduce further development of negative and adverse symptoms in patients who have been exposed to the toxin (a process referred to as “intoxication”). Antitoxins have historically included antisera obtained from large animals (e.g., sheep, horse, and pig) that were immunized with inactivated or non-functional toxin. More recently, antitoxin therapies have been developed using combinations of antitoxin monoclonal antibodies, including yeast-displayed single-chain variable fragment antibodies generated from vaccinated humans or mice. See, e.g., Nowakowski et al. 2002. Proc Natl Acad Sci USA, 99: 11346-11350; Mukherjee et al. 2002. Infect Immun, 70: 612-619; Mohamed et al. 2005 Infect Immun, 73: 795-802; Walker, K. 2010 Interscience Conference on Antimicrobial Agents and Chemotherapy—50th Annual Meeting—Research on Promising New Agents: Part 1. IDrugs 13: 743-745. Drawbacks to the production and use of antisera and monoclonal antibodies as antitoxins include their difficulty to produce economically at scale. The production of such products typically requires long development times and frequently results in problematic quality control, shelf-life and safety issues.
[0155] In general, antitoxins function through two key mechanisms, namely, neutralization of toxin function and clearance of the toxin from the body. Toxin neutralization occurs through biochemical processes including inhibition of enzymatic activity and prevention of binding to cellular receptors. Antibody mediated serum clearance occurs subsequent to the binding of multiple antibodies to the target antigen (Daeron M. 1997 Annu Rev Immunol, 15: 203-234; Davies et al. 2002 Arthritis Rheum, 46: 1028-1038; Johansson et al. 1996 Hepatology, 24: 169-175; and Lovdal et al. 2000 J Cell Sci, 113 (Pt 18): 3255-3266).
[0156] The anti-TcdB VHHs or multimeric forms thereof as described herein are provided as beneficial therapeutic agents and/or antitoxins that bind to C. difficile toxin B. In some cases, the anti-TcdB VHHs or multimeric forms thereof both promote toxin B neutralization by rapidly and effectively blocking further toxin B activity and also accelerate clearance of toxin B from the system to eliminate future pathology. In addition, increased stability and longevity of the anti-TcdB VHHs or multimeric forms thereof in the GI tract where they can bind to and neutralize the effects of toxin B contribute to the advantages of these molecules as antitoxins that provide greater therapeutic efficacy as a treatment for CDI and other diseases and symptoms caused by C. diff. infection and intoxication.
[0157] In some embodiments, the binding activity and/or neutralizing activity of the anti-TcdB VHHs described herein, or multimeric forms thereof, in the absence of any epitope tag sequences are significantly effective such that the antitoxin function of these molecules obviates the need for an anti-tag antibody or clearing antibody.
[0158] VHHs, such as the anti-TcdB VHHs described herein, have a number of advantages over conventional antibodies and recombinant antibody domains, including (i) they are small monomeric proteins (14 kDa) that express and fold efficiently in recombinant hosts; (ii) they are more stable to extremes of pH and temperature compared with conventional antibodies; (iii) they typically bind conformational epitopes, and thus are more likely to neutralize target functions; and (iv) they are amenable to designed multimerization which often leads to higher potencies and a reduction in the risk that microorganisms (e.g., C. diff) will develop resistance; and (v) they offer more therapeutic versatility, such as multispecificity, thus supporting their beneficial utility in treating enteric diseases.
[0159] The amino acid sequences of representative anti-TcdB VHH antibodies described herein are set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23, and the corresponding polynucleotide sequences encoding each of the representative anti-TcdB VHH antibodies are set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 (Example 1). The binding regions of the anti-TcdB VHHs include CDRs (CDR1, CDR2 and CDR3) as set forth in the sequences in Example 1 and in Table 1 below. The CDR binding regions are positioned within framework (FR) regions of the VHH polypeptide, which do not vary substantially in sequence between discrete anti-TcdB VHHs and which provide a “structural scaffold” for the CDRs, which bind to TcdB. By way of non-limiting example, the binding of CDRs within FRs to a target protein (antigen) may be via conformational binding or interaction, electrostatic binding interaction, hydrogen bonding, Van der Waals forces, or hydrophobic bonding, or combinations thereof, as would be appreciated by those having skill in the art.
[0160] The CDRs of the anti-TcdB VHH polypeptides described herein may vary in amino acid sequence length. By way of nonlimiting example, CDR1 of the anti-TcdB VHH polypeptides as described herein may comprise from about 6 to about 12 amino acid residues; CDR2 may comprise from about 7 to about 12 amino acid residues; and CDR3 may comprise from about 8 to about 21 amino acid residues. It will be appreciated by one skilled in the art that number of amino acids that constitute a CDR is not necessarily precise. In some cases, an amino acid residue, or 2 or 3 amino acid residues, at one end or both ends of a given CDR may be considered as part of the CDR or as part of the neighboring FR region. The CDR regions of representative anti-TcdB VHH antibody polypeptides generated from camelid alpacas as described herein (Example 1) are presented in Table 1 below. In addition,
TABLE-US-00001 TABLE 1 VHH (anti-TcdB VHH) CDR1 CDR2 CDR3 JZS-A2a GSVYTF SGGTITK NAGDTIAQAMGTRRFPFDR (XAF-1) JZS-B2 GTSFPRNY SHDGNVE KLVTLRRDEY (XAF-4) JZS-B9 RFSLINYA TSGGATY AAGPYSRTLVSRWKVGDGM (XAF-5) EY JZS-C2 GFTSNSYY SSSGGSPN AASKFPLTTMASNRYHY (XAF-6) JZS-C10 GRGPGINV QTGGTTN YLKKWRDEY (XAF-9) JZS-E4 GSSFSMNV RSDGITN FHGRARTGNNADLGS (XAF-10) JZS-E6 GRLSERIF SRLGRAN NLKPFVDNYR (XAF-11) MIST JZS-F6 GITFSNVA STGGSSTS VKGPKYSATIRRPE (XAF-12) JZS-H4 GFNFSVQI STGGASKS SKGPRTWINSSPR (XAF-13) JZS-H9 GTAFSLDT SSSGASN YRGRVRGVWPLDSGMMY (XAF-14) JZT-F7 GSILSS SRTGATD NAGLGMGDPRRPGPW (XAG-1) JZT-G9 ERNPGINA QTGGSLS YLKKWRDQY (XAG-7)
[0161] In view of the representative anti-TcdB VHH amino acid sequences shown in
[0162] The three CDRs of the anti-TcdB VHH polypeptides are arranged or positioned in the context of four FR regions as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1 to FR4 refer to the framework regions 1-4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1-3, respectively. An alignment of anti-TcdB VHHs, all of which specifically bind to toxin B, demonstrates the extensive similarities among the sequences of each of the FRs (FR1, FR2, FR3 and FR4) found in the different toxin B binding VHH polypeptides (
[0163] Table 2 presents the amino acid sequences of the four framework regions, i.e., FR1, FR2, FR3 and FR4, respectively, of ten representative anti-TcdB VHH polypeptides described herein (i.e., JZS-C10, JZS-F6, JZS-H4, JZS-B9, JZS-C2, JZS-H9, JZS-E6, JZS-E4, JZS-B2 and JZS-A2a), relative to the FR1-FR4 of a VHH reference as reported in L. S. Mitchell and L. J. Colwell, 2018, Proteins, 86(7): 697-706, termed “Reference sequence” herein), thus demonstrating the substantial similarities among the structural FRs of camelid VHHs, independent of antigen binding specificity. In Table 2, the amino acid (AA) position numbering of the camelid VHH sequences containing the FRs begins at residue “Q” as #1, as shown in the amino acid sequences of the anti-TcdB VHH polypeptides presented in Example 1. It will be appreciated that in the sequence alignments presented in
TABLE-US-00002 TABLE 2 FR1 Position # 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Reference AA Q V Q L Q/V E S G G G L/S V Q A/P G Sequence Anti- AA E S/T G G G L V Q A/P G TcdBVHHs FR1 Position # 16 17 18 19 20 21 22 23 24 25 Reference AA G S L R L S C A A S Sequence Anti- AA G S L R/T L/I S/N C A/V/T A/G/V/S S/A TcdBVHHs FR2 Position # 36 37 38 39 40 41 42 43 44 45 46 47 48 49 Reference AA W F/Y/V R Q A P G K E/C/G R/L E F/G/L/W V A/S/T Sequence Anti- AA W F/Y/V R Q A/P P G K/S/Q Q/G/E/K/T R/L E L/W/G V/D A/S TcdBVHHs FR3 Position # 60 61 62 63 64 65 66 67 68 69 Reference AA Y A/Q/T/V D/E S V/A K G R F T/A Sequence Anti- AA Y A/L/S D/N/A/E S/F/A V/A K/R/T G/S/D R F T/I TcdBVHHs FR3 Position # 70 71 72 73 74 75 76 77 78 Reference AA I/V S R/Q D N/K A K/A N T Sequence Anti- AA I S/T R D/G N/S A/L/T/V/F/P K/N/V N/K//S T/A TcdBVHHs FR3 Position # 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 Reference AA V/L/M Y L Q M N/D S/N L K/R P E/D D T A/G V/I/T/M Y Y C Sequence Anti- AA V/L Y/H/D/S L Q/E M N/D S/N/R/E L K/Q P/L/S/V E D T A/G V/T Y Y/F/S C TcdBVHHs FR4 Position # 117 118 119 120 121 122 123 124 125 126 127 Reference AA W G Q G T Q V T V S S Sequence Anti- AA W/R G Q/K/P G T Q/L V T V S/A S TcdBVHHVHHs
[0164] In embodiments, in cases in which a FR (or CDR) amino acid residue in a VHH polypeptide may be one of several alternative amino acid residues, the alternative amino acid residues will frequently share similar characteristics or properties, e.g., hydrophobicity, polarity, and/or charge. A conservative replacement (also called a conservative substitution) is an amino acid replacement or substitution in a polypeptide or region thereof that changes a given amino acid residue to a different amino acid residue with similar biochemical properties, such as charge, hydrophobicity, and/or size. By way of non-limiting example, the below Table 3 presents amino acids and their 1-letter codes categorized into six main classes based on their structure and the general chemical characteristics of their side chains (R groups).
TABLE-US-00003 TABLE 3 Amino Acids Class Glycine (G), Alanine (A), Valine (V), Leucine Aliphatic (L), Isoleucine (I) Serine (S), Cysteine (C), Selenocysteine (U), Hydroxyl Threonine (T), Methionine (M) or sulfur/ selenium containing Proline (P) Cyclic Phenylalanine (F), Tyrosine (Y), Tryptophan Aromatic (W) Histidine (H), Lysine (K), Arginine (R) Basic Aspartate (D), Glutamate (E), Asparagine (N), Acidic and Glutamine (Q) amides thereof
[0165] In an embodiment, amino acid sequence substitutions or changes in an anti-TcdB VHH polypeptide relative to another anti-TcdB VHH polypeptide comprise conservative amino acid substitutions or changes such that a given amino acid residue is substituted with or replaced by a different amino acid residue with similar biochemical properties, such as charge, hydrophobicity, and/or size. In an embodiment, sequence variation between or among anti-TcdB VHH polypeptides results from one or more conservative amino acid changes and account for the percent sequence variation, e.g., 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence variation.
[0166] In some embodiments, the VHHs as described herein are humanized using methods and techniques practiced by those having skill in the art. (See, e.g., U.S. Pat. Nos. 8,975,382 and 10,550,174, the contents of which are incorporated by reference herein).
[0167] The anti-TcdB VHH antibodies described herein have widespread application as therapeutics in the treatment of disease and pathologies resulting from infection by C. difficile pathogenic bacteria and the toxins, particularly, toxin B produced by these pathogens. The described anti-TcdB VHHs described herein are particularly useful for binding to and eradicating the disease-causing the TcdB target protein that intoxicates a subject after C. difficile infection and causes debilitating disease, and even death. In embodiments, various embodiments encompass polynucleotides (nucleic acid sequences) that encode the operably linked modular components that constitute the described anti-TcdB VHHs. In embodiments, the anti-TcdB VHHs are recombinantly produced. In embodiments, the anti-TcdB VHHs encompass the proteins (polypeptides) encoded by the polynucleotides. In embodiments, the polynucleotide is DNA, cDNA, RNA, mRNA, and the like. In an embodiment, the anti-TcdB VHHs may be humanized or codon-optimized using methods practiced by those having skill in the art.
Polynucleotides Encoding VHHs that Bind to C. difficile Toxin B (TcdB)
[0168] In some cases, more than one anti-TcdB-binding VHH antibody (i.e., anti-TcdB VHH) is coupled or linked (e.g., covalently linked) to other sequences, e.g., a leader amino acid sequence, one or more spacer (flexible spacer) amino acid sequences, or one or more epitope tag amino acid sequences, to produce a multimeric VHH binding molecule containing two or more, e.g., three, four, five, or six, VHHs linked together. In an embodiment, a polynucleotide molecule, such as a recombinant or isolated polynucleotide molecule, encodes a single anti-TcdB VHH or more than one anti-TcdB VHH linked together to form a multimer (i.e., a multimeric anti-TcdB VHH binding molecule). In an embodiment, the polynucleotide encodes a fragment or portion of the anti-TcdB VHH or multimeric anti-TcdB VHH binding molecule, in particular, a fragment or portion that maintains TcdB binding function or TcdB binding and neutralizing function. The polynucleotide sequences encoding representative anti-TcdB VHH antibodies as described herein are set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 (Example 1).
[0169] In an embodiment, an anti-TcdB VHH can be humanized, i.e., modified to increase its similarity to antibodies or antibody variants produced naturally in humans, using techniques known and practiced in the art. Briefly and by way of nonlimiting example, a humanized antibody can be generated by inserting the appropriate CDR coding sequences (e.g., ‘donor’ sequences that are responsible for the desired binding properties) into a human antibody “scaffold” (e.g., ‘acceptor’ sequences) comprising essentially invariant framework region (FR) sequences (FRs). In embodiments, the CDRs of the anti-TcdB VHH antibodies described herein may be inserted into FRs, which provide the structural scaffold that allows the CDRs to bind to, and in certain cases, to neutralize, toxin B. Recombinant DNA methods using an appropriate vector and expression in mammalian cells are employed and routinely practiced in the art to achieve the production of recombinant humanized antibodies.
[0170] In an embodiment, the polynucleotide encodes a TcdB binding and neutralizing function, or a functional binding portion thereof, that includes an epitope tag. In embodiments, antibody fragments, microproteins, darpins, anticalins, peptide mimetic molecules, aptamers, synthetic molecules, etc. can be linked to the multimeric anti-TcdB VHH binding molecule. In embodiments, a multimeric anti-TcdB VHH binding molecule may contain two of the same anti-TcdB VHHs, e.g., a dimeric form, or two different anti-TcdB VHHs described herein. In other embodiments, a multimeric anti-TcdB VHH binding molecule may contain more than two anti-TcdB VHHs in combination, e.g., a combination of three, four, or five, etc. anti-TcdB VHHs linked together. In an embodiment, the anti-TcdB VHH components of a multimeric anti-TcdB VHH binding molecule may be linked covalently.
[0171] In an embodiment, an anti-TcdB VHH can be modified, for example, by attachment (e.g., directly or indirectly via a linker or spacer) to another anti-TcdB VHH. In some embodiments, anti-TcdB VHH is attached or genetically (recombinantly) fused to another anti-TcdB VHH. Accordingly, a polynucleotide (e.g., DNA) that encodes one anti-TcdB VHH is joined (in reading frame) with the polynucleotide encoding a second anti-TcdB VHH, and so on. In certain embodiments, additional amino acids are encoded within the polynucleotide between the anti-TcdB VHHs so as to produce an unstructured region (e.g., a flexible spacer) that separates the anti-TcdB VHHs, e.g., to better promote independent folding of each anti-TcdB VHH antibody into its active or functional conformation or shape. Commercially available techniques for fusing proteins (or their encoding polynucleotides) may be employed to recombinantly join or couple the anti-TcdB VHHs into multimeric anti-TcdB VHHs containing two or more of the same or different anti-TcdB VHHs as described herein.
[0172] Polynucleotide sequences encoding the anti-TcdB VHHs or multimeric forms thereof as described herein can be recombinantly expressed and the resulting encoded anti-TcdB VHH antibody molecules can be produced at high levels and isolated and/or purified. In an embodiment, the recombinant anti-TcdB VHHs or multimeric forms thereof are produced in soluble form. In an embodiment, a recombinantly produced anti-TcdB VHH is dimeric, such that two anti-TcdB VHHs, same or different, are joined or linked together. In an embodiment, a recombinantly produced anti-TcdB VHH is multimeric, e.g., a tetramer, which contains four anti-TcdB VHH antibodies, the same or a combination of different anti-TcdB VHHs, joined together. By way of example, a tetramer may contain four of the same anti-TcdB VHHs joined together, or a combination of four different anti-TcdB VHHs, or two pairs of the same anti-TcdB VHHs, joined together. In an embodiment, the anti-TcdB VHH or multimeric forms thereof are contained in pharmaceutically acceptable compositions for use in treating CDI and/or neutralizing toxin B produced by C. diff.
[0173] The compositions and methods described herein in various embodiments include an isolated polynucleotide sequence or an isolated polynucleotide molecule that encodes an anti-TcdB VHH or multimeric form thereof. Accordingly, in some embodiments, the isolated polynucleotide sequence or isolated polynucleotide molecule comprises or consists of a polynucleotide sequence that encodes a polypeptide molecule (anti-TcdB VHH) having an amino acid sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, or a functional portion thereof, as described herein. In some embodiments, the isolated polynucleotide sequence or isolated polynucleotide molecule comprises or consists of a polynucleotide sequence of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24. In an embodiment, a composition comprises a combination of the isolated polynucleotide sequences or isolated polynucleotide molecules as described herein.
[0174] Also encompassed by the aspects and embodiments described herein are polynucleotide sequences, DNA or RNA, which are substantially complementary to the DNA sequences encoding the polypeptides described herein, and which specifically hybridize with these DNA sequences under conditions of stringency known to those of skill in the art. As referred to herein, substantially complementary means that the nucleotide sequence of the polynucleotide need not reflect the exact sequence of the original encoding sequences, but must be sufficiently similar in sequence to permit hybridization with a nucleic acid sequence under high stringency conditions. For example, non-complementary bases can be interspersed in a nucleotide sequence, or the sequences can be longer or shorter than the polynucleotide sequence, provided that the sequence has a sufficient number of bases complementary to the sequence to allow hybridization thereto. Conditions for stringency are described, e.g., in Ausubel, F. M., et al., Current Protocols in Molecular Biology, (Current Protocol, 1994), and Brown, et al., Nature, 366:575 (1993); and further defined in conjunction with certain assays.
[0175] Vectors, plasmids, bacteria, viruses, or genetically modified Spirulina algal organisms containing one or more of the polynucleotide molecules encoding the anti-TcdB VHH amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, or a functional portion thereof, are provided. Vectors, plasmids, bacteria, viruses, or genetically modified Spirulina algal organisms containing one or more of the polynucleotide molecules of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 are also provided. Suitable vectors for use in eukaryotic and prokaryotic cells are known in the art and are commercially available or readily prepared by the skilled practitioner in the art. Additional vectors can also be found, for example, in Ausubel, F. M., et al., Ibid. and in Sambrook et al., “Molecular Cloning: A Laboratory Manual,” 2nd ED. (1989), and other editions.
[0176] Any of a variety of expression vectors (prokaryotic or eukaryotic) known to and used by those of ordinary skill in the art may be employed to express recombinant polypeptides described herein. Expression can be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a polynucleotide (DNA) molecule that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast and higher eukaryotic cells. By way of example, the host cells employed include, without limitation, E. coli, yeast, insect cells, or a mammalian cell line such as COS or CHO. The DNA sequences expressed in this manner can encode any of the polypeptides described herein, including variants thereof.
[0177] Uses of plasmids, vectors or viruses containing polynucleotides encoding the anti-TcdB VHHs or multimeric forms thereof as described herein include generation of mRNA or protein in vitro or in vivo. In related embodiments, host cells transformed with the plasmids, vectors, or viruses are provided, as described above. Nucleic acid molecules can be inserted into a construct (such as a prokaryotic expression plasmid, a eukaryotic expression vector, or a viral vector construct, which can, optionally, replicate and/or integrate into a recombinant host cell by known methods. The host cell can be a eukaryote or prokaryote and can include, for example and without limitation, yeast (such as Pichia pastoris or Saccharomyces cerevisiae), bacteria (such as E. coli, or Bacillus subtilis), animal cells or tissue (CHO or COS cells), insect Sf9 cells (such as baculoviruses infected SF9 cells), or mammalian cells (somatic or embryonic cells, Human Embryonic Kidney (HEK) cells, Chinese hamster ovary (CHO) cells, HeLa cells, human 293 cells and monkey COS-7 cells). Suitable host cells also include a mammalian cell, a bacterial cell, a yeast cell, an insect cell, a plant cell, or a Spirulina algal cell.
[0178] An anti-TcdB VHH-encoding polynucleotide molecule can be incorporated or inserted into the host cell by known methods. Examples of suitable methods for transfecting or transforming host cells include, without limitation, calcium phosphate precipitation, electroporation, microinjection, infection, lipofection and direct uptake. “Transformation” or “transfection” as appreciated by the skilled practitioner refers to the acquisition of new or altered genetic features by the incorporation of additional nucleic acids, e.g., DNA, into cellular DNA. “Expression” of the genetic information of a host cell is a term of art which refers to the directed transcription of DNA to generate RNA that is, in turn, translated into a polypeptide (anti-TcdB VHH antibody). Procedures for preparing recombinant host cells and incorporating nucleic acids are described in more detail in Sambrook et al., “Molecular Cloning: A Laboratory Manual,” Second Edition (1989) and Ausubel, et al. “Current Protocols in Molecular Biology,” (1992), and later editions, for example.
[0179] A transfected or transformed host cell is maintained under suitable conditions for expression and recovery of the polypeptides described herein. In certain embodiments, the cells are maintained in a suitable buffer and/or growth medium or nutrient source for growth of the cells and expression (and secretion) of the gene product(s) into the growth medium. The type of growth medium is not critical to the aspects and embodiments herein and is generally known to those skilled in the art, such as, for example, growth medium and nutrient sources that include sources of carbon, nitrogen and sulfur. Examples include Luria-Bertani (LB) broth, Superbroth, Dulbecco's Modified Eagles Media (DMEM), RPMI-1640, M199 and Grace's insect media. The growth medium can contain a buffering agent, as commonly used in the art. The pH of the buffered growth medium may be selected and is generally a pH that is tolerated by, or optimal for, growth of the host cell, which is maintained under a suitable temperature and atmosphere.
[0180] In another aspect, an RNA polynucleotide, in particular, mRNA, encodes the anti-TcdB VHHs or multimeric forms thereof as described herein. mRNA encoding the anti-TcdB VHHs or multimeric forms thereof may contain a 5′ cap structure, a 5′ UTR, an open reading frame, a 3′ UTR and poly-A sequence followed by a C30 stretch and a histone stem loop sequence (Thess, A. et al., 2015, Mol Ther, 23(9):1456-1464; Thran, M. et al., 2017, EMBO Molecular Medicine, DOI: 10.15252/emmm.201707678). Sequences may be codon-optimized for human use using techniques and protocols known and used by those skilled in the art. In an embodiment, the mRNA sequences do not include chemically modified bases. mRNAs encoding the anti-TcdB VHHs or multimeric forms thereof as described herein may be capped enzymatically or further polyadenylated for in vivo studies/use.
[0181] Expression of proteins, which normally have a shortened serum half-life, by encoding mRNA, particularly sequence optimized, unmodified mRNA, advantageously prolongs the bioavailability of these proteins for in vivo activity. (see, e.g., K. Kariko et al, 2012, Mol. Ther., 20:948-953; Thess, A. et al., 2015, Mol Ther, 23(9):1456-1464). Accordingly, anti-TcdB VHHs or multimeric forms thereof with an estimated serum half-life of 1-2 days are likely to benefit from being encoded by mRNA. Of note, the half-lives of neutralizing VHH protein serum titers at one to three days after treatment were estimated to be, on average, 1.5-fold higher than from day three onward, even without target-specific mRNA optimization. (Mukherjee et al., 2014, PLoS ONE, 9e106422). In general, one to three days after treatment, both mRNA and protein half-lives contribute to the kinetics of serum titers, while after day three forward, the kinetics is almost exclusively determined by the properties of the expressed protein.
Multimeric Forms of the Anti-TcdB VHHs
[0182] Multimeric forms of the anti-TcdB VHH antibodies described herein are encompassed by the present disclosure. Such multimeric anti-TcdB VHHs contain more than one anti-TcdB VHH antibody that binds to TcdB. In an embodiment, a multimer of anti-TcdB VHH antibodies contains two anti-TcdB VHHs, same or different, that bind to TcdB. Such a multimeric form of the anti-TcdB VHH molecules constitutes a dimeric multimer. In an embodiment, the dimeric multimer comprises two of the same anti-TcdB VHH antibodies coupled using a flexible linker. In an embodiment, the dimeric multimer comprises two, different anti-TcdB VHH antibodies coupled using a flexible linker. In an embodiment, the two, different anti-TcdB VHH antibodies bind to different, nonoverlapping epitopes of TcdB.
[0183] In another embodiment, a multimer of anti-TcdB VHH antibodies contains more than two (e.g., three, four, five, six, etc.) anti-TcdB VHHs, same or different, that bind to TcdB. Such a multimeric form of the anti-TcdB VHH molecules may comprise three or more of the same anti-TcdB VHH antibodies coupled using flexible linkers. In an embodiment, the anti-TcdB VHH multimer comprises a combination or mixture of the anti-TcdB VHH antibodies described herein coupled using a flexible linker. In some cases, the multimeric form of the anti-TcdB VHH antibodies may contain more than one of the same anti-TcdB VHH antibody and/or different, or different combinations of, anti-TcdB VHH antibodies coupled using flexible linker or spacer peptides. Nonlimiting examples of flexible linking amino acid sequences include amino acid sequence GGGGS; GGGGSGGGGSGGGGS, or a functional portion thereof; EPKTPKPQGGGGSGGGGSGGGGSQGVQSQVQLVE; or EPKTPKPQ. In certain embodiments, the anti-TcdB VHH amino acid sequences described herein are coupled to epitope tag amino acid sequences as described infra, or to other sequences. In another embodiment, a dimerization agent that complexes peptide fragments each containing at least about 5 to 25 amino acids, 25 to 50 amino acids, 50 to 100 amino acids, 100 to 150 amino acids, and 150 amino acids to about 200 amino acids may be used. Multimerization agents and methods of using the agents for forming multimeric binding proteins can be found, for example, in U.S. Pat. Nos. 9,023,352, 8,349,326 and 7,763,445, each of which is incorporated by reference herein in its entirety.
[0184] In embodiments, the multimeric forms of the anti-TcdB VHH antibodies described herein both bind to TcdB and neutralize its activity.
Epitope Tags and Antibodies Thereto
[0185] In certain embodiments, an anti-TcdB VHH antibody, or a dimeric or multimeric form thereof, includes a single epitope tag (single tag sequence) or multiple tags (multiple tag sequences), to which anti-tag antibodies specifically bind. For example, a multimeric VHH may include at least one, or two or more, epitope tags in the molecule. Such epitope tags, which are specifically bindable by the anti-epitope tag antibodies, are useful in detecting VHHs bound to toxin antigens. In addition, such tags may facilitate clearance of VHHs bound to toxin antigens following binding of the tags by anti-tag antibody. By way of nonlimiting example, a tag may constitute an O-tag epitope of amino acid sequence DELGPRLMGK or an E-tag epitope of amino acid sequence GAPVPYPDPLEPR. The epitope tags may be placed at the amino terminus, carboxy terminus, or internally within a multimeric VHH molecule. Such tags and/or anti-tag antibodies are described for example, in (U.S. Pat. Nos. 8,349,326; 9,023,352, WO 2019/094095A1) and U.S. Pat. Nos. 7,943,345; 8,114,634 and 8,865,871), the contents of which are incorporated herein by reference in their entireties. An example of an anti-0 tag monoclonal antibody (IgG1) suitable for binding the DELGPRLMGK tag sequence is described in WO 2019/094095A1, the contents of which are fully incorporated by reference. By way of illustrative example, peroxidase labeled antibodies that bind the anti-O-tag antibody may be used to detect these anti-tag antibodies in assays in which samples are incubated with goat anti-O-tag-HRP conjugated antibody (Bethyl labs) diluted 1:5000 in blocking solution for 1 hour at RT with rocking and were washed as above before adding TMB microwell peroxidase substrate (KPL) to develop (incubated for 10-40 min). Development was stopped with 1M H.sub.2SO.sub.4 and the plates were read at 450 nm on an ELx808 Ultra Microplate Reader (Bio-Tek instruments), (Mukherjee, J. et al., 2012, PloS ONE 7:e29941).
[0186] In some cases, an albumin binding peptide (DICLPRWGCLWED), may be included at the 3′ end of an anti-TcdB VHH antibody or multimeric form thereof.
[0187] In certain embodiments, the presence of an epitope tag operably linked, coupled, or fused to a VHH antibody or multimeric form thereof, wherein the tag is bound by an anti-epitope tag antibody, induces clearance of the toxin-bound VHH molecule from the body. In an embodiment, the binding of one or more epitope tags in an anti-TcdB VHH molecule by anti-epitope tag antibody(ies) may synergistically induce clearance of TcdB from the body following binding by the VHH or multimeric form thereof.
[0188] In an aspect, an anti-tag (i.e., anti-epitope tag) antibody may be administered to a subject who is also treated with or administered an anti-TcdB VHH or multimeric form thereof containing one or more epitope tags, or a pharmaceutical composition thereof. The anti-tag antibodies bind to the epitope tags of the anti-TcdB VHH, which, in turn, binds to one or more toxin B proteins, thereby forming a complex that is rapidly cleared from the body (Sepulveda, J. et al., 2010, Infect. Immun., 78(2):756-763; Mukherjee, J. et al., 2012, PLoS ONE, 7(1): e29941. PMCID: PMC3253120; http://doi.org/10.1371/journal.pone.0029941). In an embodiment, an anti-epitope tag monoclonal antibody of a specific isotype, for example IgG1, or a binding fragment or portion thereof that binds to the tag sequence, or a molecule containing its CDR components that bind to the tag sequence, may be provided to a subject who is also administered one or more anti-TcdB VHHs or a multimeric form thereof as described herein. The administration or co-administration of an anti-tag antibody advantageously enhances clearance from the body of a complex formed by toxin TcdB bound by anti-TcdB VHH or a multimeric form thereof, which is, in turn, bound by an anti-epitope tag antibody or binding portion thereof.
[0189] In certain embodiments, an anti-tag antibody may also effect or facilitate immunoglobulin effector functions. Anti-tag antibodies may include, for example, IgA, IgD, IgE, IgG, and IgM immunoglobulins and subtypes thereof. An immune response to an epitope tag included in an anti-TcdB VHH or multimeric form thereof may involve the elicitation of specific monoclonal antibodies and/or polyclonal antibodies that specifically bind to the tag. Immunoglobulin effector functions may involve, for example, interaction(s) between the Fc portion of the immunoglobulin and receptors or other protein molecules in a subject or cells thereof. Depending on the immunoglobulin type, the effector functions result in clearance of the disease agent (e.g., excretion, degradation, lysis or phagocytosis). In an embodiment, an anti-tag antibody of one immunoglobulin effector type binds to an anti-TcdB VHH or multimeric form thereof which comprises one or more epitope tags. In embodiments in which a multimeric form of an anti-TcdB VHH comprises at least one epitope tag, or two or more epitope tags, an anti-tag antibody, or binding portion thereof, binds to each of the tags of the multimeric molecule. In embodiments, the epitope tags may be the same or different in a given anti-TcdB VHH multimeric molecule. Without wishing to be bound by theory, the presence of more than one epitope tag bindable by an anti-epitope tag antibody, or binding portion thereof, in a multimeric form of an anti-TcdB VHH may increase the rate and/or level of clearance of toxin B bound to the anti-TcdB VHH multimer in a subject.
[0190] Suitable methods of producing or isolating antibody fragments having the requisite binding specificity and affinity for binding to an epitope tag include for example, methods which select recombinant antibody from a library or by PCR (e.g., U.S. Pat. Nos. 5,455,030 and 7,745,587 each of which is incorporated by reference herein in its entirety).
[0191] Functional fragments of antibodies, including fragments of chimeric, humanized, primatized, veneered, or single chain antibodies, can also be produced. Functional fragments or portions of the foregoing antibodies include those which are reactive with the toxin protein. For example, antibody fragments capable of binding to the toxin protein or a portion thereof, include, but not limited to, scFvs, Fabs, VHHs, Fv, Fab, Fab′ and F(ab′).sub.2. Such fragments can be produced by enzymatic cleavage or by recombinant techniques. For instance, papain or pepsin cleavage are used generate Fab or F(ab′).sub.2 antibody fragments, respectively. Antibody fragments are produced in a variety of truncated forms using antibody-encoding genes in which one or more stop codons has been introduced upstream of the natural stop site. For example, a chimeric gene encoding a F(ab′).sub.2 heavy chain peptide portion can be designed to include DNA sequences encoding the CH.sub.1 peptide domain and hinge region of an immunoglobulin heavy chain.
Pharmaceutical Compositions
[0192] Also featured herein are methods for treating or preventing pathologies and disease caused by toxin B produced by C. difficile following infection of a subject by C. difficile microorganisms. The methods include administering to a subject in need thereof an amount of an anti-TcdB VHH or multimeric anti-TcdB VHH binding molecule that is effective to specifically bind to and optimally neutralize C. difficile toxin B. In an embodiment, if an anti-TcdB VHH or multimeric anti-TcdB VHH binding molecule includes an epitope tag, an anti-epitope tag antibody may be administered to the subject. (see, e.g., WO 2019/094095A1, the contents of which is incorporated by reference herein in its entirety). In an embodiment, an anti-TcdB VHH or multimeric anti-TcdB VHH binding molecule is provided or used in a pharmaceutical composition.
[0193] Typically, a carrier or excipient is included in a composition as described herein, such as a pharmaceutically acceptable carrier or excipient, which includes, for example, sterile water, aqueous saline solution, aqueous buffered saline solutions, aqueous sucrose, dextrose, or mannose solutions, aqueous glycerol solutions, ethanol, calcium carbonate, albumin, starch, cellulose, silica gel, polyethylene glycol (PEG), dried skim milk, rice flour, magnesium stearate, and the like, or combinations thereof. The terms “pharmaceutically acceptable carrier” and a “carrier” refer to any generally acceptable excipient or drug delivery device that is relatively inert and non-toxic.
[0194] As used herein, the term “pharmaceutically acceptable carrier” includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's The Science and Practice of Pharmacy Ed. by LWW 21.sup.st EQ. PA, 2005 discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Carriers are selected to prolong dwell time for example following any route of administration, including IP, IV, subcutaneous, mucosal, sublingual, inhalation or other form of intranasal administration, or other route of administration.
[0195] Some examples of materials that can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.
[0196] The preparation of such compositions and solutions ensuring sterility, pH, isotonicity, and stability is effected according to protocols established in the art. Generally, a carrier or excipient is selected to minimize allergic and other undesirable effects, and to suit the particular route of administration, e.g., subcutaneous, intramuscular, intranasal, intravenous, oral, and the like.
[0197] In certain embodiments, these compositions optionally further comprise one or more additional therapeutic agents. In certain embodiments, the additional therapeutic agent(s) is/are selected from antibiotics particularly antibacterial compounds, anti-viral compounds, anti-fungals. In some embodiment, additional therapeutic agent(s) may include one or more of growth factors, anti-inflammatory agents, vasopressor agents, collagenase inhibitors, topical steroids, matrix metalloproteinase inhibitors, ascorbates, angiotensin II, angiotensin III, calreticulin, tetracyclines, fibronectin, collagen, thrombospondin, transforming growth factors (TGF), keratinocyte growth factor (KGF), fibroblast growth factor (FGF), insulin-like growth factors (IGF), epidermal growth factor (EGF), platelet derived growth factor (PDGF), neu differentiation factor (NDF), hepatocyte growth factor (HGF), and hyaluronic acid.
[0198] In an aspect, according to the methods of treatment described herein, immunization is promoted by contacting the subject with a pharmaceutical composition containing an anti-TcdB VHH or multimeric form thereof, as described herein. Thus, methods are provided for immunization, comprising administering to a subject in need thereof, such as a subject infected with C. diff. or a subject having CDI or symptoms thereof, a therapeutically effective amount of a pharmaceutical composition comprising an anti-TcdB VHH or multimeric form thereof as active agent for a time necessary to achieve the desired result. It will be appreciated that the methods encompass protectively administering a composition comprising an anti-TcdB VHH or multimeric form thereof as a preventive or therapeutic measure to ameliorate, reduce, abrogate, or diminish infection or the effects thereof by C. difficile, thus, minimizing complications associated with a slow development of immunity or response to infection (especially in compromised patients such as those who are nutritionally challenged, or at risk patients such as the elderly or infants).
[0199] A therapeutically effective dose refers to that amount of active agent which ameliorates at least one symptom or condition. Therapeutic efficacy and toxicity of active agents can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED.sub.50 (the dose that is therapeutically effective in 50% of the population) and LD.sub.50 (the dose that is lethal to 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD.sub.50/ED.sub.50. Pharmaceutical compositions which exhibit large therapeutic indices are especially useful. The data obtained from cell culture assays and from animal studies are used in formulating a range of dosages for human administration. By way of example, a therapeutic dose may be at least about 1 μg per kg, at least about 5, 10, 50, 100, 500 μg per kg, at least about 1 mg/kg, 5, 10, 50 or 100 mg/kg body weight of a composition or active component thereof per body weight of the subject, although the doses may be more or less depending on age, health status, history of prior infection, and immune status of the subject as would be known by one of skill in the art. Doses may be divided or unitary and may be administered once daily, or repeated at appropriate intervals.
Administration of Pharmaceutical Compositions
[0200] After formulation with an appropriate pharmaceutically acceptable carrier in a desired dosage, a pharmaceutical composition comprising an anti-TcdB VHH or multimeric form thereof, or an anti-TcdB VHH or multimeric form thereof, can be administered to humans and other mammals by routes known and practiced in the art.
[0201] The administration of an anti-TcdB VHH or multimeric form thereof, or a pharmaceutical composition comprising of an anti-TcdB VHH or multimeric form thereof, as a therapeutic for the treatment or prevention of disease or pathology caused by toxin B produced by C. difficile following infection may be by any suitable means that results in a concentration of the therapeutic that, combined with other components, if desired, is effective in ameliorating, reducing, eliminating, abating, or stabilizing disease, pathology, or the symptoms thereof in a subject. The therapeutic may be administered systemically, for example, formulated in a pharmaceutically-acceptable composition or buffer such as physiological saline.
[0202] Routes of administration include, for example and without limitation, subcutaneous, intravenous, intraperitoneal, intramuscular, intrathecal, intraperitoneal, or intradermal injections that provide continuous, sustained levels of the therapeutic in the subject. Other routes include, without limitation, gastrointestinal, esophageal, oral, rectal, intravaginal, etc. The amount of the therapeutic to be administered varies depending upon the manner of administration, the age and body weight of the subject, and with the clinical symptoms of the bacterial infection or associated disease, pathology, or symptoms. Generally, amounts will be in the range of those used for other agents used in the treatment of disease or pathology associated with C. difficile infection, although in certain instances, lower amounts may be suitable because of the increased range of protection and treatment afforded by the described anti-TcdB VHHs or multimeric forms thereof as therapeutics. A composition is administered at a dosage that ameliorates, decreases, diminishes, abates, alleviates, or eliminates the effects of the bacterial (microorganism) infection or disease (e.g., CID or the symptoms thereof) as determined by a method known to one skilled in the art.
[0203] In embodiments, a therapeutic or prophylactic treatment agent may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for parenteral (e.g., subcutaneous, intravenous, intramuscular, intrathecal, or intraperitoneal) administration route. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).
[0204] Pharmaceutical compositions may in some cases be formulated to release the active agent substantially immediately upon administration or at any predetermined time or time period after administration. The latter types of compositions are generally known as controlled release formulations, which include (i) formulations that create a substantially constant concentration of a therapeutic agent or drug within the body over an extended period of time; (ii) formulations that after a predetermined lag time create a substantially constant concentration of a therapeutic agent or drug within the body over an extended period of time; (iii) formulations that sustain action during a predetermined time period by maintaining a relatively, constant, effective level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic pattern); (iv) formulations that localize action by, e.g., spatial placement of a controlled release composition adjacent to or in contact with an organ, such as the gut or gastrointestinal system; (v) formulations that allow for convenient dosing, such that doses are administered, for example, once every one or two weeks; and (vi) formulations that target a disease using carriers or chemical derivatives to deliver the therapeutic agent or drug to a particular cell type. For some applications, controlled release formulations obviate the need for frequent dosing during the day to sustain a therapeutic level in plasma, serum, or blood. In an embodiment, one or more anti-TcdB VHHs or multimeric forms thereof may be formulated with one or more additional components for administration to a subject in need, e.g., patients who have contracted C. difficile infection and suffer from the serious repercussions of toxin production by these microorganisms.
[0205] Any of a number of strategies can be pursued in order to obtain controlled release of a therapeutic agent in which the rate of release outweighs the rate of metabolism of the therapeutic agent or drug in question. In one example, controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings. Thus, the therapeutic agent or drug may be formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the therapeutic agent or drug in a controlled manner. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes.
[0206] Compositions for parenteral or oral use may be provided in unit dosage forms (e.g., in single-dose ampules), or in vials containing several doses and in which a suitable preservative may be added (see below). The composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. Apart from the active agent (i.e., an anti-TcdB VHH or multimeric form thereof) that reduces or ameliorates a disease, pathology, or symptom thereof, such as CID, C. difficile-associated diarrhea (CDAD), pseudomembranous colitis (PMC), bowel inflammation, enterocytic detachment, alteration, disruption, or elimination of natural intestinal microflora, and/or paralytic ileus caused by C. difficile infection, the composition may include suitable parenterally acceptable carriers and/or excipients. In some cases, an active therapeutic agent(s) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release. Furthermore, the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and/or dispersing, agents.
[0207] In some embodiments, compositions comprising an anti-TcdB VHH or multimeric form thereof are sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like which do not deleteriously react with the active compounds. In some embodiments, an anti-TcdB VHH or multimeric form thereof are combined, where desired, with other active substances, e.g., enzyme inhibitors, to reduce metabolic degradation. An effective amount of a pharmaceutical composition can vary according to choice or type of anti-TcdB VHH or multimeric form thereof as described herein, the particular composition formulated, the mode of administration and the age, weight and physical health or overall condition of the patient, for example. In an embodiment, an effective amount of an anti-TcdB VHH or multimeric form thereof and/or anti-epitope tag antibody is an amount which is capable of reducing one or more symptoms of disease or pathology caused by C. diff. infection and the production of its toxins, namely, toxin B.
[0208] In certain embodiments, a composition includes one or more polynucleotide sequences that encode one or more anti-TcdB VHHs or multimeric forms thereof as described herein. In an embodiment, a polynucleotide sequence encoding an anti-TcdB VHH or multimeric form thereof is in the form of a DNA molecule or multimer. In some embodiments, the composition includes a plurality of nucleotide sequences each encoding an anti-TcdB VHH or multimeric form thereof, or any combination of anti-TcdB VHHs described herein, such that the anti-TcdB VHH antibodies or multimers thereof are expressed and produced in situ. In such compositions, a polynucleotide sequence is administered using any of a variety of delivery systems known to those of ordinary skill in the art, including eukaryotic, bacterial, viral vector nucleic acid expression systems, or Spirulina-based delivery as described infra. Suitable nucleic acid expression systems contain appropriate nucleotide sequences operably linked for expression in a patient (such as suitable promoter and termination signals). Bacterial delivery systems involve administration of a bacterium that secretes or expresses the polypeptide on its cell surface, e.g., probiotic E. coli as described infra. In an embodiment, a polynucleotide molecule encoding an anti-TcdB VHH or multimeric form thereof can be introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, lentivirus, or adenovirus associated virus (AAV)), which uses a non-pathogenic (defective), replication competent virus. Techniques for incorporating nucleic acid (DNA) into such expression systems are well known to and practiced by those of ordinary skill in the art. The nucleic acid (DNA) can also be “naked,” as described, for example, in Ulmer et al., 1993, Science, 259:1745-1749 and as reviewed by Cohen, 1993, Science 259:1691-1692. The uptake of naked DNA can be increased by the use of nanoparticles comprising DNA or coating the DNA onto biodegradable beads, which are efficiently transported into recipient cells.
Therapeutic Methods
[0209] Methods of treating disease, conditions, pathology and/or symptoms thereof associated with C. difficile infection are provided. The methods comprise administering a therapeutically effective amount of an anti-TcdB VHH or multimeric form thereof as described herein, or a pharmaceutical composition comprising these agents to a subject (e.g., a mammal such as a human). In an embodiment, the method is for treating a subject suffering from or susceptible to CDI, C. difficile-associated diarrhea (CDAD), pseudomembranous colitis (PMC), bowel inflammation, enterocytic detachment, alteration, disruption, or elimination of natural intestinal microflora, and/or paralytic ileus caused by C. difficile infection, or a symptom thereof. The method includes administering to the subject a therapeutically effective amount of an anti-TcdB VHH, multimeric form thereof, or composition thereof sufficient to treat the disease, illness, condition, disorder and/or symptom thereof, under conditions such that the disease or disorder and/or symptom thereof is treated.
[0210] The therapeutic methods include prophylactic as well as therapeutic treatment. In an embodiment, the treatment method includes administering a therapeutically effective amount of an anti-TcdB VHH or multimeric form thereof as described herein, or a pharmaceutical composition comprising these agents, before or during the time that a subject is administered one or more antibiotics, or a treatment involving a course of antibiotics, to treat a different bacterial disease or infection. As will be appreciated by the skilled practitioner in the art, antibiotic treatment can disturb, disrupt, or otherwise adversely affect the normal microflora, microfauna and microbiome in the GI tract of a subject, thereby allowing C. difficile bacteria to proliferate, or over-proliferate, causing CDI and other C. difficile promoted disease or disorders and symptoms thereof. Accordingly, providing a subject with an anti-TcdB VHH or multimeric form of the anti-TcdB VHHs provides a beneficially useful and practical prophylactic and/or therapeutic treatment regimen for a subject in need.
[0211] A subject or patient includes an animal, particularly a mammal, and more particularly, a human. Such an anti-TcdB VHH or multimeric form thereof as described herein, or a pharmaceutical composition comprising these agents, used as therapeutics in treatments will be suitably administered to subjects or patients suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof caused by or associated with infection by C. difficile microorganisms and toxin B produced by C. diff. Determination of patients who are “susceptible” or “at risk” can be made by any objective or subjective determination obtained by the use of a diagnostic test or based upon the opinion of a patient or a health care provider (e.g., genetic test, enzyme or protein marker, family history, and the like). Identifying a subject in need of such treatment can be in the judgment of a subject himself or herself, or of a health care/medical professional and can be subjective (e.g., opinion) or objective (e.g., measurable or quantifiable by a test or diagnostic method).
Methods of Delivery
[0212] In an embodiment, an anti-TcdB VHH or multimeric form thereof as described herein, or a pharmaceutical composition comprising these agents, can be administered to a subject in need of treatment for intoxication by a toxin B protein (TcdB). In an embodiment, a mixture of anti-TcdB VHHs or multimeric forms thereof can be administered to a subject in need of treatment for infection or intoxication by C. diff. In an embodiment, the anti-TcdB VHH or multimeric form thereof may include one or more epitope tag sequences to which anti-epitope tag antibody(ies) specifically bind. In another embodiment in which the anti-TcdB VHH or multimeric form thereof administered to a subject includes one or more epitope tag sequences, a specific anti-epitope tag antibody can also be administered to the subject.
[0213] In some embodiments, the administration of two or more anti-TcdB VHHs or multimeric forms thereof may increase the effectiveness of the therapy to treat infection by C. diff. and TcdB production and reduce the severity of one or more negative symptoms related to exposure of the subject to TcdB. In an embodiment, administering to a subject the anti-TcdB VHH or multimeric form thereof that includes one or more, e.g., two, epitope tag sequences may result in improved therapy, treatment, or protection against disease caused by C. diff. and its production of TcdB. In an embodiment, the epitope tag sites of the anti-TcdB VHH or multimeric form thereof are bound by a specific anti-tag antibody. The administration of an anti-TcdB VHH or multimeric form thereof as described herein, or a composition comprising the agent, and the administration of one or more anti-epitope tag antibodies may be performed simultaneously or sequentially in time. In an embodiment, an anti-TcdB VHH or multimeric form thereof is administered before, after, or at the same time as the administration of another anti-TcdB VHH or multimeric form thereof, or before administration of an anti-tag antibody, provided that the anti-TcdB VHH(s) or multimeric form(s) thereof and/or the anti-tag antibody(ies) are administered close enough in time to have the desired effect (e.g., before the anti-TcdB VHHs or multimeric forms thereof have been cleared by the body). Accordingly, “co-administration” embraces the administration of an anti-TcdB VHH or multimeric form thereof and a subsequent anti-TcdB VHH or multimeric form thereof, or the anti-tag antibody, at time points that will achieve effective treatment of disease caused by C. diff. toxin B, such as reduction in the level of toxin B and disease and symptoms associated with the presence of the toxin. The described methods are not limited by time intervals between which an anti-TcdB VHH or multimeric form thereof and/or the anti-tag antibody(ies) are administered; provided that these agents, or compositions containing these agents, are administered close enough in time to produce or achieve the desired effect. In an embodiment, only an anti-TcdB VHH or multimeric form thereof is administered to a subject in need thereof. In another embodiment, an anti-TcdB VHH or multimeric form thereof and an anti-epitope tag antibody are premixed and administered together, or are not premixed but are co-administered within minutes of each other. In other embodiments, the anti-TcdB VHH or multimeric form thereof and anti-epitope tag antibody(ies) are co-administered with other medications, drugs, compounds, or compositions suitable for treating the disease agent.
[0214] In yet other embodiments, an anti-TcdB VHH or multimeric form thereof, or a composition containing the agent(s), is administered to a subject prior to the potential risk of exposure to infection by toxin B-producing C. difficile in order to protect a subject from disease and symptoms caused by toxin B. For example, an anti-TcdB VHH or multimeric form thereof and/or anti-epitope tag antibody (“clearing antibody”) is administered minutes, hours or days prior to the risk of exposure to infection by C. difficile. Alternatively, an anti-TcdB VHH or multimeric form thereof is administered concomitantly with the risk of exposure of a subject to infectious C. difficile microorganisms and production of toxin B (i.e., the target of the anti-TcdB VHH or multimeric form thereof), or slightly after the risk of exposure. For example, an anti-TcdB VHH or multimeric form thereof is administered to a subject at the moment that the subject contacts, enters, or passes through an environment (e.g., room, hallway, building, and field) containing a risk of exposure to C. difficile microorganisms.
[0215] The methods described herein provide treating or protecting a subject from intoxication and disease (and the symptoms thereof) caused by the toxin B protein produced by C. difficile bacteria that have infected the subject. In accordance with the method, one or more anti-TcdB VHHs or multimeric forms thereof and, optionally, an anti-tag antibody(ies) as described herein are administered to the affected or at risk subject. Administration ameliorates, reduces, or alleviates the severity of one or more the symptoms of the C. diff.-induced disease or condition. The presence, absence, or severity of symptoms is measured, for example, using physical examination, tests and diagnostic procedures known and practiced in the art. In certain embodiments, the presence, absence and/or level of C. diff and/or its produced toxin B protein are measured using methods known and employed in the art. Symptoms or levels of the toxin B protein can be measured at one or more time points (e.g., before, during and after treatment, or any combination thereof) during the course of treatment with an anti-TcdB VHH or multimeric form thereof to determine if the treatment is effective. A decrease, reduction, or no change in the levels of the toxin B protein, or in the severity of symptoms associated therewith indicates that treatment is effective, and an increase in the level of toxin B, or in the severity of symptoms in a subject indicates that treatment is not effective. In various embodiments, the symptoms and levels of toxin B are measured using methods known and employed in the art. Specific, yet nonlimiting, symptoms that are monitored in subjects with C. difficile infection may include watery diarrhea, cramping/tenderness (mild symptoms); severe watery diarrhea, colitis, severe abdominal cramping/tenderness, rapid heart rate, fever, blood in stool, dehydration, loss of appetite and weight, swollen abdomen, kidney failure and increase leukocyte count (severe symptoms). Treatment or protection from intoxication by toxin B is assessed as increased survival and reduction, alleviation, or prevention of symptoms. Methods, compositions and kits involving the use of the anti-TcdB VHHs or multimeric forms thereof described herein decrease and alleviate the symptoms of C. diff. infection and toxin B production and also improve survival from exposure to the C. diff. bacteria themselves.
Modes of Delivery of Anti-TcdB VHHs for Treatment of CDI
[0216] The anti-TcdB VHHs or multimeric forms thereof described herein can serve as effective parenteral therapeutics, e.g., in several animal models and in patients. These anti-TcdB VHHs or multimeric forms may substantially improve functional stability in the harsh proteolytic conditions of the GI tract. A number of different modes of delivery may be employed to administer these VHH molecules, such as enteric delivery, intravenous or subcutaneous delivery. In addition, suitable oral delivery modes and systems as described herein may circumvent or overcome loss of protein stability associated with the provision of protein therapeutics in the environment of the GI tract. In embodiments, the anti-TcdB VHHs and multimeric forms thereof are employed in oral, enteric, or parenteral delivery systems and offer more stable therapeutics for treating TcdB intoxication and CDI caused by C. difficile infection.
Spirulina-Based Oral or Enteric Treatment of CDI
[0217] In an aspect, the anti-TcdB VHHs or multimeric forms thereof described herein are delivered orally or enterically using genetically modified cyanobacteria or Spirulina, a blue-green alga, as described, for example, in U.S. Pat. No. 10,131,870 (Lumen Biosciences) and WO 2016/040499 A1, the contents of which are incorporated by reference herein in their entireties. In an embodiment, Spirulina algae are non-naturally occurring, stable transformants comprising at least one introduced targeted nucleotide mutation in their genome. In embodiments, the Spirulina is Arthrospira platensis or Arthrospira maxima. In some embodiments, the Spirulina is Arthrospira platensis NIES-39 or Arthrospira sp. PCC 8005. Spirulina modified to contain one or more stable, targeted mutations are suitable for molecularly engineering to harbor and express polynucleotides encoding the anti-TcdB binding polypeptides (anti-TcdB VHHs) as described herein and for producing and manufacturing products for therapeutic use. In an embodiment, the Spirulina organisms are engineered to express protease stable anti-TcdB VHHs. In an embodiment, such engineered Spirulina organisms are orally delivered to a subject in need for the treatment of CDI in the subject. In another embodiment, such engineered Spirulina organisms are enterically delivered to a subject in need for the treatment of CDI in the subject. Protease-stable anti-TcdB VHHs that neutralize TcdB toxin may thus be provided to the subject. Engineered Spirulina have been demonstrated be capable of expressing functional VHHs directed against an enteric pathogen at high levels (e.g., >1% dry mass); therapeutic efficacy of such a VHH product expressed by engineered Spirulina has also be shown.
Probiotic E. coli Engineered to Secrete or Express TcdB-Binding VHH
[0218] The human gastrointestinal (GI) tract contains a complex symbiotic microbiota that is estimated to comprise more than 40,000 species and in some regions more than 10.sup.11 organisms. The microbiota of the GI tract aids in maintaining immune homeostasis in the gut-associated lymphoid tissues, optimizing nutritional uptake, and supporting gut development. The entire intestinal epithelium is overlaid with a thick mucus layer, which, in conjunction with an effective immune system, keep the enormous bacterial load strictly sequestered on the luminal side of the gut, thus preventing penetration across the epithelial barrier. (See, e.g., K. Gronbach et al., 2010, Infect Immun, 78(7):3036-3046).
[0219] The importance of the cross talk between the microbiota, intestinal epithelial cells, and the innate and adaptive components of the immune system is shown by a variety of intestinal pathological conditions, including Crohn's disease, ulcerative colitis, pouchitis, irritable bowel syndrome, necrotizing enterocolitis (NEC), and CDI. Immature or genetically compromised immunity results in exaggerated intestinal inflammation or disruption or altered composition of the intestinal mucosa, which, in turn, disturbs the homeostasis between a human host and its intestinal microbial symbionts. Pathological events, such as infection by pathogens, e.g., C. difficile, change the relative balance between beneficial and aggressive enteric symbionts, convert beneficial bacteria into pathogens, or select for new opportunistic pathogens. A qualitatively and quantitatively changed gastrointestinal microbiota, often termed small bowel bacterial overgrowth (SIBO) or dysbiosis, may contribute substantially to local chronic inflammation in a vicious cycle and provoke bacterial translocation that leads to fatal sepsis.
[0220] The foregoing provides the rationale for selective therapeutic manipulation of the abnormal microbiota by probiotics as therapeutics for the intestinal diseases. In general, probiotics are viable microorganisms with beneficial physiological or therapeutic activities. Various in vitro and animal studies with probiotics, including Escherichia coli strain Nissle 1917, have demonstrated the capacity of probiotics to reduce intestinal inflammation, to strengthen the intestinal barrier against pathogens, to increase the host innate immune functions, or to prevent adherent and invasive E. coli strains from adhering to and invading human intestinal epithelial cells. (Id.) Limited clinical trials using E. coli strain Nissle 1917 or other microorganisms have suggested that this therapeutic strategy can be efficacious in patients with GI tract conditions such as chronic idiopathic inflammatory bowel diseases (IBD), irritable bowel syndrome (IBS), and NEC.
[0221] In an embodiment, strains of the probiotic E. coli Nissle are molecularly engineered to express and secrete functional anti-TcdB VHH polypeptides, or to express the polypeptides produced by these microorganism on their surface. Such anti-TcdB VHH-producing probiotic microorganisms provide an oral therapeutic or treatment modality for delivering the anti-TcdB VHHs to the GI tract to treat C. difficile pathologies and their symptoms, such as CDI. By way of example, E. coli Nissle strains were engineered to express VHH polypeptides that neutralized Shiga toxin; these microorganisms were shown to establish in the GI tract of animal models where they continued to produce the anti-Shiga toxin VHHs. In the same manner, the anti-TcdB binding and/or neutralizing VHHs described herein and expressed by E. coli Nissle strains may be utilized to provide a safe oral therapy for CDI.
Encapsulated Delivery of Anti-TcdB VHH
[0222] In another embodiment, encapsulated delivery and packaging technologies that allow orally administered protein therapeutics to pass safely through the stomach and release in the small or large intestine may be used to deliver anti-TcdB VHHs as described herein in the treatment of C. difficile infection, CDI and symptoms thereof. Encapsulation and enteric coating techniques and processes commonly known and used in the art are suitable for delivering anti-TcdB VHHs. In embodiments, nanoparticle-based delivery of drugs and biologics and enteric coating of nanoparticles have been described by J. K. Patra et al., 2018, J. Nanobiotech, 16, Art. No. 71 (doi.org/10.1186/s12951-018-0392-8); US Publication No. 20200129444, the contents of which are incorporated by reference herein. Nanoparticles engineered to deliver protease-resistant, TcdB-binding and/or neutralizing VHHs may be introduced into the colons of animal models. The animals may be assessed using methods and protocols known and used in the art to determine that these animals are protected from CDI pathology.
mRNA Delivery of TcdB VHH
[0223] In an embodiment, a polynucleotide encoding an anti-TcdB VHHs or multimeric form thereof as described herein, in particular, mRNA, in the form of lipid nanoparticles is used to deliver these anti-TcdB binding agents and produce effective and long-lasting antibody titers in subjects who are passively immunized with the mRNA-nanoparticles. In a particular embodiment, the mRNA, which is otherwise unmodified, may be codon optimized to afford efficient expression of an anti-TcdB VHH or multimeric form thereof from the transcribed mRNA. It has been reported that exogenous mRNA has the ability to instruct cells to produce VHHs, as well as other types of antibodies. See, M. Thran et al., 2017, EMBO Mol. Medicine, online publication no. DOI 10.15252/emmm.201707678. The advantages of using mRNA for passive immunization are appreciated by those in the art. (See, M. Thran et al., Id.). mRNA-based approaches for therapeutics may be safer and more cost effective compared with DNA-based approaches. Because mRNA does not integrate into a host's DNA and is more transient in nature, mRNA-based protein expression is considered to be easier to control for protein expression.
Substantially Identical Amino Acid and Nucleotide Sequences for VHHs
[0224] There is a large body of information in the literature supporting the fact that closely related antibody (Ab) sequences are capable of performing the same binding and therapeutic functions such that this is now generally accepted by those with ordinary skill in the art of immunological sciences. The creation of Abs with small numbers of amino acid sequence variations occurs naturally within mammals and some other animal species during the process of ‘affinity maturation’ in which Ab-producing cells that bind a newly encountered antigen (Ag) are expanded, and their progeny cells contain random mutations within portions of the Ab coding DNA that results in new, related Ab sequences. The cells expressing Abs that have gained improved binding properties for the new Ag are then selected and expanded, thereby increasing the amount of the improved antibody in the animal. This process continues through multiple generations of mutation and selection until Abs with greatly improved binding properties result, thus providing, for example, better immunity against pathogens expressing or possessing the new Ag. The process of Ab affinity maturation demonstrates that related, yet not identical, Ab amino acid sequences can possess similar target binding properties and perform similar therapeutic functions in vivo.
[0225] Example 1 herein provides anti-TcdB VHH antibodies having related sequences that perform similar functions and provide similar therapeutic benefits. The Abs described herein are heavy-chain only, single domain VHH antibodies, which are generated in camelid alpacas, which have been reported to be convenient sources of camelid VHH antibodies (See, e.g., Maass, D. R. et al., 2007, J. Immunol. Methods, 324:13-25). Briefly, alpacas are immunized with a selected C. difficile toxin B antigen (TcdB Ag) multiple times to permit the animal to undergo affinity maturation of the anti-TcdB VHHs that are produced. Anti-TcdB VHHs are then isolated and the encoding DNA selected for expression of soluble VHHs that bind TcdB Ag and have potential therapeutic or diagnostic properties. During this process, many examples of closely related anti-TcdB binding VHHs are isolated, which are distinctive, and which are presumably intermediates that result from the affinity maturation process which occurs during anti-TcdB VHH production in alpaca lymphocytes. These related anti-TcdB VHHs are screened for binding to TcdB Ag, and the most promising members of homology groups of TcdB-binding VHHs are identified and become lead candidates for further development.
[0226] Similar to all mammalian antibodies, VHHs comprise four, well-conserved ‘framework’ regions (FRs) which are important in forming the antibody structure. Between the FRs (FR1, FR2, FR3 and FR4) are three much less well-conserved CDRs or hypervariable regions (CDR1, CDR2 and CDR3) which principally interact with and bind to antigenic determinants or epitopes on antigens (Ags), such as TcdB. The CDR sequences vary widely so as to interact and bind to epitopes of Ags. The third CDR, CDR3, is generally the longest in sequence and is most diverse of the CDRs within VHHs, both in size and sequence. By way of nonlimiting example, CDR3 in VHHs can range in size from about 7 to about 28 amino acid residues. The CDR3 regions of VHHs generated in the same alpacas and selected for binding to a common target Ag are highly similar in size (number of amino acids comprising CDR3) and can vary in their amino acid identities. Without being bound by theory, VHHs and CDR3 regions that bind to the same TcdB target Ag are considered to have resulted from affinity maturation of a common precursor VHH within the animal and are classified as a ‘homology group’. Individual VHHs within a homology group are classified by their binding to a target Ag, and the members of the VHH homology group are able to ‘compete’ with each other for binding to the Ag, thus demonstrating that they bind to the same region on the target Ag. In VHH molecules, the CDRs (CDR1, CDR2 and CD3) play a role in the ability of a VHH to bind to the target Ag, e.g., TcdB, in conjunction with CDR1 and CDR2.
[0227] Since the FRs are critical for maintaining the structure of a VHH and the positioning of the CDRs for binding to the target Ag, the FRs of VHHs typically do not vary extensively in sequence. However, some VHH FR amino acid sequence variation is permissible, particularly in cases in which an amino acid substitution involves the replacement or substitution of one amino acid with another amino acid having similar properties (e.g., similarity in being charged or uncharged), i.e., a conservative substitution. Such conservative changes in FRs can often be found naturally within VHHs that have undergone affinity maturation in an animal. Similar to the case with FRs, VHH CDRs also typically do not vary extensively in amino acid sequence or type so as not to compromise their ability to specifically bind to Ag. As would be appreciated by one skilled in the art, an estimation of the extent of amino acid sequence variation that can be tolerated within VHHs without compromising their Ag binding ability can be made by observing the variation that occurs naturally within affinity-matured homology groups of VHHs isolated from the same types of animals and which bind to the same Ag.
[0228] An illustrative example of VHH sequence relatedness and the retention of common antigen binding properties is shown in
[0229] Another example evidencing that VHH sequence variation is acceptable within related VHHs having the same Ag binding characteristics is described in Tremblay et al., 2013, Infect Immun 81:4592-4603. In this report, 11 VHH sequences comprise a large homology group with closely related CDR3 sequences, and the unusual property of cross-specific binding to two different Shiga toxins, Stx1 and Stx2. Two of the more distantly related VHH members of this homology group are characterized as having common Ag binding characteristics. These two related VHHs were found to have 32 amino acid changes in the total VHH sequence of 120 or 121 residues. Thus, a 26% variation in amino acid sequence did not adversely affect the common Ag binding properties of the VHH proteins.
Kits
[0230] Provided herein are kits for the treatment or prevention of C. difficile infection or disease. In some embodiments, the kit includes an effective amount of one or more anti-TcdB VHHs or multimeric forms thereof as described herein, in unit dosage form. In an embodiment, the kit further contains an anti-epitope tag antibody, in unit dosage form. In other embodiments, the kit includes a therapeutic or prophylactic composition containing an effective amount of one or more anti-TcdB VHHs or multimeric forms thereof, in unit dosage form. In still other embodiments, the kit includes a therapeutic or prophylactic composition containing an effective amount of one or more anti-TcdB VHHs or multimeric forms thereof, and an anti-epitope tag antibody, in unit dosage form. In some embodiments, the kit comprises a device, e.g., an automated or implantable device for subcutaneous delivery; an implantable drug-eluting device, or a nebulizer or metered-dose inhaler, for dispersal of the composition or a sterile container which contains a pharmaceutical composition. Non-limiting examples of containers include boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
[0231] If desired, a pharmaceutical composition is provided together with instructions for administering the pharmaceutical composition containing one or more anti-TcdB VHHs or multimeric forms thereof, or one or more anti-TcdB VHHs or multimeric forms thereof and an anti-epitope tag antibody, to a subject having or at risk of contracting or developing an infection or disease or pathology, and/or the symptoms thereof, associated with infection by C. difficile and toxin B production by C. difficile. The instructions will generally include information about the use of the composition for the treatment or prevention of an infection and intoxication by the C. difficile bacteria and toxin B proteins that they produce. In other embodiments, the instructions include at least one of the following: description of the therapeutic/prophylactic agent; dosage schedule and administration for treatment or prevention of infection, disease or symptoms thereof caused by one or more of C. difficile bacteria and/or toxin B proteins that they produce; precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
[0232] In another aspect, a kit is provided for treating a subject exposed to, intoxication by, or at risk for exposure to or intoxication by C. difficile toxin B, in which the kit includes a pharmaceutical composition for treating a subject at risk for exposure to or who is exposed to C. diff and/or toxin B produced by C. diff., and the pharmaceutical composition includes at least one recombinant anti-TcdB VHH or multimeric form thereof, such that the anti-TcdB VHH or multimeric form thereof neutralizes C. diff. TcdB, thereby treating the subject for exposure to the C. diff disease agent; a container; and, instructions for use. In various embodiments, the instructions for use include instructions for a method for treating a subject at risk for exposure to, exposed to, or intoxicated by the C. diff disease agent using the pharmaceutical composition.
[0233] The practice of the present aspects and embodiments described herein employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the aspects and embodiments described herein, and, as such, may be considered in making and practicing the same.
[0234] Particularly useful techniques for particular embodiments will be discussed in the Examples that follow.
EXAMPLES
Example 1—Anti-C. difficile Toxin B (TcdB)-Binding VHHs
[0235] Presented in Example 1 are the amino acid and encoding polynucleotide (nucleic acid) sequences of C. difficile toxin B (TcdB)-binding VHH polypeptides (anti-TcdB VHHs) as described herein. The amino acids comprising the Complementarity Determining Regions (CDRs) of each of the anti-TcdB VHHs are designated in each VHH polypeptide as follows: CDR1 is designated by a single underline; CDR2 is designated by a double underline; and CDR3 is designated in bold with a single underline.
TABLE-US-00004 JZS-A2a (XAF-1) VHH amino acid sequence (SEQ ID NO: 1) QVQLAESGGGLVQAGGSLRLSCAASGSVYTFMGWYRQAPGKTRELVAGISGGTITKYADS VKGRFIISRDSPKNTIYLQMNELKVEDTGVYYCNAGDTIAQAMGTRRFPFDRWGQGTQVT VAS JZS-A2a (XAF-1) polynucleotide sequence (SEQ ID NO: 2) CAGGTGCAGCTCGCGGAGTCGGGAGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTC TCCTGTGCAGCCTCTGGAAGCGTGTATACTTTTATGGGCTGGTACCGCCAGGCTCCAGGG AAGACGCGCGAATTGGTCGCAGGGATTTCAGGTGGTACGATCACAAAATATGCAGACTCC GTGAAGGGCCGATTCATCATCTCCAGAGACAGCCCCAAGAACACAATCTATCTGCAAATG AACGAGCTAAAAGTTGAAGACACAGGCGTGTATTACTGTAATGCAGGCGACACCATTGCA CAGGCTATGGGGACGCGGAGGTTTCCGTTCGACCGCTGGGGCCAGGGGACCCAGGTCACC GTCGCCTCA JZS-B2 (XAF-4) VHH amino acid sequence (SEQ ID NO: 3) QVQLAESGGGLVQAGGSLRLSCVGSGTSFPRNYMGWYRQAPGKQRELVAAISHDGNVEYA DSVKGRFTISRGNFVNTVALQMNSLKSEDTAVYYCKLVTLRRDEYWGQGTQVTVSS JZS-B2 (XAF-4) polynucleotide sequence (SEQ ID NO: 4) CAGGTGCAGCTCGCGGAGTCGGGAGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTC TCCTGTGCAGCCTCTGGAAGCGTGTATACTTTTATGGGCTGGTACCGCCAGGCTCCAGGG AAGACGCGCGAATTGGTCGCAGGGATTTCAGGTGGTACGATCACAAAATATGCAGACTCC GTGAAGGGCCGATTCATCATCTCCAGAGACAGCCCCAAGAACACAATCTATCTGCAAATG AACGAGCTAAAAGTTGAAGACACAGGCGTGTATTACTGTAATGCAGGCGACACCATTGCA CAGGCTATGGGGACGCGGAGGTTTCCGTTCGACCGCTGGGGCCAGGGGACCCAGGTCACC GTCGCCTCA JZS-B9 (XAF-5) VHH amino acid sequence (SEQ ID NO: 5) QLQLVESGGGLVQPGGSLRLSCTSARFSLINYAIGWFRQAPGQKREGVSLLTSGGATYYA DSARDRFTISRDNARNTVYLQMNSLKPEDTAVYSCAAGPYSRTLVSRWKVGDGMEYWGKG TLVTVSS JZS-B9 (XAF-5) polynucleotide sequence (SEQ ID NO: 6) CAGTTGCAGCTCGTGGAGTCTGGGGGAGGCCTGGTGCAGCCTGGGGGTTCTCTGAGACTC TCCTGTACATCTGCGAGATTCTCTTTGATTAATTATGCCATAGGCTGGTTCCGCCAGGCC CCAGGACAGAAGCGCGAGGGGGTCTCACTACTTACTAGTGGTGGTGCTACATACTATGCT GACTCCGCGAGGGACCGATTCACCATCTCCAGAGACAACGCCAGGAACACGGTGTATTTG CAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTCGTGTGCAGCAGGGCCCTAT TCCCGAACCTTAGTCTCACGTTGGAAGGTGGGGGACGGCATGGAGTACTGGGGCAAAGGG ACCCTAGTCACCGTCTCCTCA JZS-C2 (XAF-6) VHH amino acid sequence (SEQ ID NO: 7) QVQLAESGGGLVQPGGSLRLSCAASGFTSNSYYIGWFRQAPGKGREAVSSISSSGGSPNY ANAVKGRFTITRDNANNTVYLQMDNLKPEDTAVYYCAASKFPLTTMASNRYHYWGQGTQV TVSS JZS-C2 (XAF-6) polynucleotide sequence (SEQ ID NO: 8) CAGGTGCAGCTCGCGGAGTCGGGCGGAGGCTTGGTGCAGCCTGGGGGTTCTCTGAGACTC TCCTGTGCAGCCTCTGGATTCACTTCGAATTCTTATTACATAGGCTGGTTCCGCCAGGCC CCAGGGAAGGGGCGCGAGGCGGTCTCAAGTATTAGTAGTAGTGGAGGTAGCCCTAACTAT GCGAATGCCGTGAAGGGCCGATTCACCATAACCAGAGACAACGCCAACAACACGGTCTAT CTGCAAATGGACAACCTGAAACCTGAGGACACGGCCGTTTATTACTGCGCAGCATCGAAG TTTCCGCTAACCACTATGGCGTCCAACCGATATCATTACTGGGGTCAGGGGACCCAGGTC ACCGTCTCCTCA JZS-C10 (XAF-9) VHH amino acid sequence (SEQ ID NO: 9) QLQLVETGGLVQAGGSLRLSCVGSGRGPGINVMGWYRQAPGTERELVATWQTGGTTNYAD SVKGRFTISRDNLKNTVSLQMDSLKPEDTAVYYCYLKKWRDEYWGQGTQVTVSS JZS-C10 (XAF-9) polynucleotide sequence (SEQ ID NO: 10) CAGTTGCAGCTGGTGGAGACGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCC TGTGTAGGCTCTGGAAGAGGCCCCGGGATCAATGTCATGGGCTGGTACCGCCAGGCTCCA GGGACTGAGCGCGAGTTGGTCGCAACTTGGCAAACCGGTGGTACCACAAACTATGCAGAC TCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACCTCAAGAACACGGTGTCGCTGCAA ATGGACAGTCTGAAACCTGAGGACACAGCCGTCTATTACTGCTATCTGAAAAAATGGAGA GATGAGTATTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA JZS-E4 (XAF-10) VHH amino acid sequence (SEQ ID NO: 11) QVQLVESGGGLAQAGGSLRLSCAASGSSFSMNVMGWYRQPPGKQRELVATIRSDGITNYA ESVKGRFTISRDNVKNTVHLEMNRLKAEDTAVYYCFHGRARTGNNADLGSWGQGTQVTVS S JZS-E4 (XAF-10) polynucleotide sequence (SEQ ID NO: 12) CAGGTGCAGCTGGTGGAGTCGGGTGGAGGCTTGGCGCAGGCTGGGGGGTCTCTGAGACTC TCCTGTGCAGCCTCTGGAAGTAGCTTCAGCATGAATGTCATGGGCTGGTACCGCCAGCCT CCAGGGAAGCAGCGCGAGTTGGTCGCGACTATTCGTAGTGATGGTATCACAAACTATGCA GAGTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGTCAAGAACACAGTGCATCTG GAAATGAACAGGCTGAAAGCTGAAGACACAGCCGTATATTACTGCTTTCATGGCCGGGCC CGCACAGGGAATAATGCTGACTTGGGTTCTTGGGGCCAGGGGACCCAGGTCACCGTCTCC TCG JZS-E6 (XAF-11) VHH amino acid sequence (SEQ ID NO: 13) QVQLAETGGGLVQAGGSLRLSCAASGRLSERIFMISTMAWYRQVPGKQRELVAEISRLGR ANYSDSVTDRFIISRDNTKNTVDLQMNSLKPEDTAVYYCNLKPFVDNYRGPGTQVTVSS JZS-E6 (XAF-11) polynucleotide sequence (SEQ ID NO: 14) CAGGTGCAGCTGGCGGAGACGGGTGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTC TCCTGTGCAGCTTCGGGAAGGCTCTCGGAAAGGATCTTCATGATCAGTACGATGGCCTGG TACCGCCAGGTTCCAGGGAAGCAGCGCGAGTTGGTCGCAGAAATTTCGCGACTTGGTAGG GCCAACTATTCAGACTCCGTGACGGACCGATTCATCATCTCCAGAGACAACACCAAGAAC ACGGTGGATCTACAAATGAACAGCCTGAAGCCTGAGGACACAGCCGTCTATTACTGCAAT CTTAAACCCTTCGTCGACAACTACCGGGGCCCGGGGACCCAGGTCACCGTCTCCTCT JZS-F6 (XAF-12) VHH amino acid sequence (SEQ ID NO: 15) QLQLAESGGGLVQPGGSLRLSCAASGITFSNVAMSWVRQAPGKGLEWVSTISTGGSSTSY LDSVKSRFTISRDNAKKTVYLQMNSLKPEDTAVYYCVKGPKYSATIRRPEGQGTQVTVSS JZS-F6 (XAF-12) polynucleotide sequence (SEQ ID NO: 16) CAGTTGCAGCTCGCGGAGTCCGGCGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTC TCCTGTGCAGCCTCTGGAATCACCTTCAGTAACGTTGCCATGAGCTGGGTCCGCCAGGCT CCAGGAAAGGGGCTCGAGTGGGTCTCAACTATTAGTACGGGCGGTAGTAGTACAAGCTAT TTAGACTCCGTGAAGAGCCGATTGAGCATCTCCAGAGACAACGCCAAGAAGACGGTGTAT CTGCAAATGAACAGTCTGAAACCTGAGGACACGGCCGTGTATTACTGTGTAAAAGGGCCC AAGTATTCCGCTACAATCCGTCGTCCTGAGGGCCAGGGGACCCAGGTCACCGTCTCCTCA JZS-H4 (XAF-13) VHH amino acid sequence (SEQ ID NO: 17) QLQLVESGGGLVQPGGSLRLSCVASGFNFSVQIMSWVRQAPGKGLEWVSAISTGGASKSY ADFAKGRFTISRDNAKNTLYLQMNSLQLEDTAVYFCSKGPRTWINSSPRGQGTQVTVSS JZS-H4 (XAF-13) polynucleotide sequence (SEQ ID NO: 18) CAGTTGCAGCTCGTGGAGTCCGGTGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTC TCCTGTGTAGCCTCTGGATTCAACTTCAGTGTGCAGATTATGAGCTGGGTCCGCCAGGCT CCAGGAAAGGGGCTCGAGTGGGTCTCAGCTATTAGTACTGGTGGCGCTAGCAAAAGTTAT GCAGACTTCGCGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTAT CTGCAAATGAACAGCCTACAACTCGAGGACACGGCCGTGTATTTTTGTTCTAAGGGTCCG AGGACTTGGATCAATTCTAGTCCCCGGGGCCAGGGGACCCAGGTCACCGTCTCCTCG JZS-H9 (XAF-14) VHH amino acid sequence (SEQ ID NO: 19) QVQLVESGGGLVQPGGSLTINCTVSGTAFSLDTMTWYRQAPGKQRELAADISSSGASNYL ASVKGRFTISRDNAKSALYLQMNSLKPEDTGTYYCYRGRVRGVWPLDSGMMYWGKGTLVT VSS JZS-H9 (XAF-14) polynucleotide sequence (SEQ ID NO: 20) CAGGTGCAGCTGGTGGAGTCCGGTGGAGGCTTGGTGCAGCCTGGGGGCTCTCTGACAATC AACTGTACAGTCTCTGGAACCGCGTTCAGTCTCGATACCATGACCTGGTACCGCCAGGCT CCAGGGAAGCAGCGCGAGTTGGCCGCCGATATTAGTAGTAGTGGTGCCTCAAACTATTTA GCCTCCGTGAAGGGCCGATTCACCATCTCCAGAGATAACGCCAAGAGCGCTCTGTATCTG CAAATGAACAGCCTGAAACCTGAGGACACAGGCACATATTATTGCTATAGAGGGCGAGTG CGGGGAGTCTGGCCGTTGGACAGCGGCATGATGTACTGGGGTAAAGGGACCTTGGTCACC GTCTCCTCA JZT-F7 (XAG-1) VHH amino acid sequence (SEQ ID NO: 21) QVQLVETGGGLVQAGGSLRLSCAASGSILSSMGWYRQAPGNQREFVASISRTGATDYADS VAGRFIISRDRGKNTVLALQMDSLKPEDTAVYYCNAGLGMGDPRRPGPWWGQGTQVTVSS JZT-F7 (XAG-1) polynucleotide sequence (SEQ ID NO: 22) CAGGTGCAGCTCGTGGAGACTGGAGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTC TCCTGTGCAGCCTCTGGAAGCATCCTCAGTTCCATGGGCTGGTACCGCCAGGCTCCAGGA AACCAGCGCGAGTTCGTCGCGTCTATTTCTCGTACTGGGGCGACGGACTATGCAGACTCC GTGGCGGGCCGATTCATCATCTCTAGGGACAGAGGCAAGAACACGGTATTAGCGCTGCAA ATGGACAGCCTGAAACCTGAGGACACAGCCGTCTATTACTGTAATGCAGGATTAGGAATG GGGGACCCGCGACGGCCCGGTCCGTGGTGGGGCCAGGGAACCCAGGTCACCGTCTCCTCA JZT-G9 (XAG-7) VHH amino acid sequence (SEQ ID NO: 23) QVQLAETGGGLVQAGGSLRLSCVSSERNPGINAMGWYRQAPGSQRELVAVWQTGGSLSYA DSVKGRFTISRDNLKNTVYLQMNSLKPEDAAVYYCYLKKWRDQYWGQGTRVTVSS JZT-G9 (XAG-7) polynucleotide sequence (SEQ ID NO: 24) CAGGTGCAGCTCGCGGAGACTGGTGGAGGCTTGGTTCAGGCAGGCGGGTCTCTGAGACTC TCCTGTGTAAGCTCTGAAAGAAATCCCGGGATCAATGCCATGGGCTGGTATCGCCAGGCT CCAGGGAGTCAGCGCGAGTTGGTCGCAGTTTGGCAAACCGGTGGTAGCCTAAGCTATGCA GACTCCGTGAAGGGTCGATTCACCATCTCCAGAGACAACCTCAAGAACACGGTGTATCTG CAAATGAACAGTCTGAAACCTGAGGAGGCAGCCGTCTATTATTGTTATCTGAAAAAGTGG AGAGATCAGTATTGGGGCCAGGGGACCCGGGTCACCGTCTCCTCA
[0236] Framework region (FR) sequences of the representative C. difficile toxin B (TcdB)-binding VHH polypeptides (anti-TcdB VHHs) described herein are presented below. Without intending to be limited by theory, in some cases, amino acid residue(s) close or directly adjacent to amino acid residue(s) at the boundaries of FR/CDR regions may be included in the FR sequence or in the CDR sequence, or such residues may vary, e.g., amino acid substitutions such as conservative substitutions, among the VHH polypeptides without adversely affecting the TcdB binding or function of the VHH polypeptides.
TABLE-US-00005 JZS-A2a (XAF-1): FR1: QVQLAESGGGLVQAGGSLRLSCAAS; FR2: MGWYRQAPGKTRELVAGI; FR3: YADSVKGRFIISRDSPKNTIYLQMNELKVEDTGVYYC; FR4: WGQGTQVTVAS. JZS-B2 (XAF-4): FR1: QVQLAESGGGLVQAGGSLRLSCVGS; FR2: MGWYRQAPGKQRELVAAI; FR3: YADSVKGRFTISRGNFVNTVALQMNSLKSEDTAVYYC; FR4: WGQGTQVTVSS. JZS-B9 (XAF-5): FR1: QLQLVESGGGLVQPGGSLRLSCTSA; FR2: IGWFRQAPGQKREGVSLL; FR3: YADSARDRFTISRDNARNTVYLQMNSLKPEDTAVYSC; FR4: WGKGTLVTVSS. JZS-C2 (XAF-6): FR1: QVQLAESGGGLVQPGGSLRLSCAAS; FR2: IGWFRQAPGKGREAVSSI; FR3: YANAVKGRFTITRDNANNTVYLQMDNLKPEDTAVYYC; FR4: WGQGTQVTVSS. JZS-C10 (XAF-9): FR1: QLQLVETGGLVQAGGSLRLSCVGS; FR2: MGWYRQAPGTERELVATW; FR3: YADSVKGRFTISRDNLKNTVSLQMDSLKPEDTAVYYC; FR4: WGQGTQVTVSS. JZS-E4 (XAF-10): FR1: QVQLVESGGGLAQAGGSLRLSCAAS; FR2: MGWYRQPPGKQRELVATI; FR3: YAESVKGRFTISRDNVKNTVHLEMNRLKAEDTAVYYC; FR4: WGQGTQVTVSS. JZS-E6 (XAF-11): FR1: QVQLAETGGGLVQAGGSLRLSCAAS; FR2: MAWYRQVPGKQRELVAEI; FR3: YSDSVTDRFIISRDNTKNTVDLQMNSLKPEDTAVYYC; FR4: GPGTQVTVSS. JZS-F6 (XAF-12): FR1: QLQLAESGGGLVQPGGSLRLSCAAS; FR2: MSWVRQAPGKGLEWVSTI; FR3: YLDSVKSRFTISRDNAKKTVYLQMNSLKPEDTAVYYC; FR4: GQGTQVTVSS. JZS-H4 (XAF-13): FR1: QLQLVESGGGLVQPGGSLRLSCVAS; FR2: MSWVRQAPGKGLEWVSAI; FR3: YADFAKGRFTISRDNAKNTLYLQMNSLQLEDTAVYFC; FR4: GQGTQVTVSS. JZS-H9 (XAF-14): FR1: QVQLVESGGGLVQPGGSLTINCTVS; FR2: MTWYRQAPGKQRELAADI; FR3: YLASVKGRFTISRDNAKSALYLQMNSLKPEDTGTYYC; FR4: WGKGTLVTVSS. JZT-F7 (XAG-1): FR1: QVQLVETGGGLVQAGGSLRLSCAAS; FR2: MGWYRQAPGNQREFVASI; FR3: YADSVAGRFIISRDRGKNTVLALQMDSLKPEDTAVYYC; FR4: WGQGTQVTVSS. JZT-G9 (XAG-7): FR1: QVQLAETGGGLVQAGGSLRLSCVSS; FR2: MGWYRQAPGSQRELVAV; FR3: YADSVKGRFTISRDNLKNTVYLQMNSLKPEDAAVYYC; FR4: WGQGTRVTVSS.
Example 2—VHH-Display Library Preparation from Genes Expressed in Immunized Camelids (Alpacas)
[0237] In a general protocol, three alpacas were immunized with TcdB (100 μg) by five successive multi-site subcutaneous (SC) injections at three week intervals. For the first immunization, the adjuvant was alum/CpG and subsequent immunizations used alum. All alpacas achieved ELISA anti-TcdB titers of 1:1,000,000. Blood was obtained from the alpacas for peripheral blood lymphocyte (PBL) preparation seven days after the fifth immunization, and RNA was extracted using the RNEASY kit (Qiagen, Valencia, Calif.). VHH-display phage libraries were prepared as described in Maass, D. R., et al., 2007 Internal J Parasitology, 37, 953-962 and Tremblay, J. M., et al., 2010 Toxicon 56, 990-998.
[0238] In a particular aspect, an alpaca that had been immunized with TcdB immunogen 10 years earlier was twice boosted with 100 ug of TcdB immunogen. Following these immunizations, immune peripheral blood lymphocytes (PBL) were obtained from the animal, and a VHH display library was produced from the immune PBLs. This anti-TcdB VHH display library was called JZM-3 and had a complexity of 1.1×10′. Another anti-TcdB VHH display library, called JYH-3, was made by combining mRNA obtained from immune PBLs isolated from two other immunized alpacas. The two anti-TcdB VHH display libraries were subjected to a panning technique. (See, e.g., Mukherjee, J., et al., 2012 PLoS ONE, 7, e299411; Maass, D. R., et al., 2007 Internal J Parasitology, 37, 953-962 and Tremblay, J. M., et al., 2010 Toxicon 56, 990-998). Most toxins, particularly large toxins such as those produced by C. difficile, become partially denatured on plastic. Because VHH-binding is highly dependent on protein conformation, anti-TcdB VHH libraries were extensively panned on antibody-captured TcdB to capture and select VHHs that bound to conformationally native TcdB protein. A VHH heterodimer containing two TcdB-neutralizing VHHs, called 5D and E3 (plasmid JXW-2) was used as a reference.
[0239] Twenty-five (25) clonally-unrelated, unique anti-TcdB VHH antibody families were generated from the two libraries screened by panning. Based on early screening data including binding and toxin neutralization data, 20 VHHs were selected for expression, purification and characterization. Additional panning and screening for TcdB-neutralizing VHHs was performed to employ capture panning with improved libraries to identify anti-TcdB VHHs that bound to neutralizing epitopes of TcdB. In addition, assays were carried out to identify TcdB-neutralizing VHHs that were resistant to resident proteases in the gastrointestinal tract (GI proteases). Such GI protease resistant anti-TcdB VHHs advantageously provide therapeutics for enteric delivery and effectiveness, for example, by employing Spirulina for delivery.
[0240] Following additional characterizations, 12 out of the 20 anti-TcdB VHHs that demonstrated the highest apparent affinity for TcdB and/or the highest potency for TcdB-neutralization were selected as representatives for further use and development. The protein sequences and encoding DNA sequences of the 12 anti-TcdB VHH antibodies are provided supra.
[0241] The anti-TcdB VHH were purified and their TcdB-neutralization potencies were examined. Briefly, to evaluate the neutralization activities of these VHHs, a dilution series of the individual VHHs were incubated with Vero cells in culture, and the cells were then intoxicated with TcdB. The rounding effects of toxin exposure following culturing the cells with anti-TcdB VHHs were assessed at various times after toxin was added to the cells. As shown in
[0242] In summary, several hundred individual clones were screened for TcdB-binding; positive clones were characterized; and all new and unique TcdB-binding VHH coding DNAs were re-cloned into an expression vector for soluble protein expression and purification. The purified, quantified VHHs were characterized for their potencies to neutralize TcdB in a cell-based toxicity assay. The more potent neutralizing anti-TcdB VHHs were screened for their resistance to GI tract proteases using assays developed for enteric pharmacokinetic studies. Because panning on native TcdB was employed, as well as a more complex VHH display library prepared from optimally immunized alpacas, it was expected that ten or more unique new VHHs capable of binding TcdB with high affinity (sub-nM affinities) would be identified, and subsequently selected for use.
Example 3—Binding of Anti-TcdB VHHs to TcdB
[0243] TcdB binding assays were performed to assess the binding activity of representative anti-TcdB VHH antibodies as described herein and in Yang, Z. et al., 2014, J Infect Disease, 210(6):964-972; doi:10.1093/infdis/jiu196. In addition, the neutralization activity of the representative anti-TcdB VHH antibodies was assessed in neutralization assays as described herein and in Yang, Z. et al., 2014, Id. In particular, the anti-TcdB VHH antibodies were assayed for specific TcdB binding affinity (EC.sub.50) and neutralizing potency (IC.sub.50). The results are presented in Table 4 below. The anti-TcdB antibodies were found to bind to TcdB with varying affinities and/or neutralize TcdB activity with different potencies. In Table 4, the affinity and potency values are designated as “apparent” (rather than “intrinsic”), because they were determined experimentally using appropriate assays as commonly known and practiced in the art.
TABLE-US-00006 TABLE 4 Apparent Apparent VHH Vector affinity, potency, name name (EC.sub.50) (IC.sub.50) JZS-A2a XAF-1 >25 nM >25 nM JZS-B2 XAF-4 0.3 nM >125 nM JZS-B9 XAF-5 0.2 nM 1 nM JZS-C2 XAF-6 3 nM 0.5 nM JZS-C10 XAF-9 0.1 nM 0.5 nM JZS-E4 XAF-10 2 nM 1 nM JZS-E6 XAF-11 0.8 nM 1 nM JZS-F6 XAF-12 0.1 nM 0.5 nM JZS-H4 XAF-13 0.2 nM 0.5 nM JZX-H9 XAF-14 1 nM 15 nM JZT-F7 XAG-1 3 nM 1 nM JZT-G9 XAG-7 0.1 nM 3 nM
Example 4—Neutralization of TcdB by Anti-TcdB VHHs
[0244] Neutralization assays as described in Example 5 infra (and as in Yang, Z. et al., 2014, J Infect Disease, 210(6):964-972; doi:10.1093/infdis/jiu196) were performed to assess the neutralization activity and apparent potency of the representative anti-TcdB VHHs described herein. The results of the assays are presented in Table 4 above and in
Example 5—Materials and Methods
Enzyme Linked Immunosorbent Assay (ELISA)
[0245] EIA/RIA 96 well high binding plates (Corning Costar) coated with 0.5-5 μg/ml of recombinantly produced toxin, e.g., rTcdB, overnight at 4° C. were used for immuno-binding assays (ELISA). Plates were washed 3 times with 1×PBS+0.1% Tween, followed by washing 3 times with 1×PBS. Washed plates were blocked (4-5% non-fat dry milk in 1×PBS+0.1% Tween) for 1 hour at room temperature (RT) with rocking. Serially diluted (1:5) VHH-TcdB binding molecules targeting C. difficile toxin B, diluted in blocking solution, were incubated for 1 hour at RT with rocking and washed as above. Equivalent control samples were spiked with a known amount of an irrelevant VHH for use as an internal standard.
[0246] Binding of the VHHs to recombinant toxin B coating the wells was detected at A450 nm using horse radish peroxidase (HRP)-labeled anti-E-tag antibody and an ELISA reader. Bound HRP was detected using 3,3′,5,5′-tetramethylbenzidine (TMB substrate, Sigma) and values were plotted as a function of the input VHH concentration. Illustratively, the plates were incubated with goat anti-E-tag-HRP conjugated antibody (Bethyl labs) diluted 1:5000 in blocking solution for 1 hour at RT with rocking and were washed as above before adding TMB microwell peroxidase substrate (KPL) to develop (incubated for 10-40 min). Development was stopped with 1M H2504 and the plates were read at 450 nm on an ELx808 Ultra Microplate Reader (Bio-Tek instruments), (Mukherjee, J. et al., 2012, PloS ONE 7:e29941). VHH levels in unknown samples were determined by comparison of their signals to those of internal standards as previously described (Mukherjee, J. et al., 2014, PLoS One 9:e106422; Sheoran, A S et al., 2015, Infect Immun, 83:286-291; Moayeri, M. et al., 2016, Clin Vaccine Immunol, doi:10.1128/cvi.00611-15; Sponseller, J K et al., 2014, J Infect Dis, doi:10.1093/infdis/jiu605; Tzipori, S. et al., 1995, Infect Immun, 63:3621-3627). EC.sub.50 values were calculated for the VHH concentration that secreted in a signal equal to 50% of the maximum signal.
Neutralization Assay
[0247] Vero cells (ATCC) at a concentration of 2.4×10.sup.4 cells/100 μl of medium (DMEM high glucose+1 mM sodium pyruvate, 2 mM L-glutamine, 50 U/ml and 50 μg/ml Pen/Strep pH 7.4 (HyClone)) were plated in 96-well plates overnight for 90-95% confluency, prior to addition of anti-TcdB VHH in serial dilutions (in media) (1:5) or from 100 μg/ml-1.0 fg/ml and toxic levels of TcdB, such as 0.01 ng/ml to 1 ng/ml TcdB, in a 24 hour cytotoxicity/cell rounding assay (Yang, Z. et al., 2014, J Infect Disease, 210(6):964-972; doi:10.1093/infdis/jiu196). After 24 hours, toxicity to the cells was assessed by quantifying the percent (%) of cells in the plates that became rounded in each of the wells. Apparent IC.sub.50 values for the anti-TcdB VHH polypeptides were determined.
Computational Analysis
[0248] In general, data are analyzed using GraphPad Prism software version 6. All error bars refer to standard deviations. ELISA data are analyzed using nonlinear regression.
Animal Experiments
[0249] Animal experiments involving the anti-TcdB VHHs as described herein are conducted at the Department of Infectious Disease and Global Health, Tufts Cummings School of Veterinary Medicine (North Grafton, USA) in conformance with Tufts University IACUC Protocol #G2016-74. Six- to eight-week-old female CD1 mice (Charles River Labs, Wilmington, USA) are randomized based on body weight and receive single intravenous injections of LNP-formulated mRNA into the tail vein. Blood is sampled by retro-orbital bleeding at defined times, and VHH accumulation in sera is measured as described by J. M. Tremblay et al., 2013, Infec. Immun., 81:4592-4603; Mukherjee et al., 2014, PLoS ONE 9e106422. In all studies, animals are housed under standard and humane conditions with a standard commercial rodent diet and tap water provided to the animals ad libitum.
[0250] All experiments involving animals are performed under protocols approved by Tufts University and National Institute of Allergy and Infectious Diseases (NIAID) Animal Care and Use Committees. Work with alpacas was performed at Tufts under approved protocol Tuskegee University School of Veterinary Medicine (TUSVM) and Institutional Animal Care and Use Committee (IACUC) Protocol #G2015-49. Mouse studies are performed at NIAID under approved protocols LPD8E and LPD9E.
Mouse Systemic Toxin Challenge
[0251] For in vivo studies using mice, 6 or 8 week old C57BL/6 or Balb/cJ female mice (Jackson Laboratories, Bar Harbor, Me.) are intraperitoneally (IP) injected with a single dose of VHH, e.g., anti-C. diff. TcdB VHH (50 μg/mouse), 1 hour prior to IP injection of TcdB (100 or 200 ng/mouse). Mice are monitored for signs and symptoms of toxemia (including; lethargy, depression, anorexia, dehydration, ruffled coat, and hunched posture), e.g., over a predetermined time period, e.g., 10 days. Moribund mice are euthanized following IACUC-approved removal criteria.
Mouse CDI Challenge
[0252] In vivo experiments using mice are conducted to mimic the human condition of C. difficile infection (CDI) and disease and to facilitate colonization with C. difficile. For these experiments, ten 6 week old, C57BL/6 female mice receive filter sterilized antibiotics (kanamycin, gentamycin, colistin, metranidozole, and vancomycin) in drinking water for 5 days followed by 2 days of water alone. After 2 days of drinking the supplemented water, the mice receive one (100 μl) intraperitoneal (IP) injection of clindamycin (2 mg/ml). One day later, mice are orally challenged (Chen, X. et al., 2008, Gastroenterology, 135:1984-1992) with 10.sup.6 spores of an NAPI/027/BI C. difficile strain, designated strain UK6 (Killgore, G. et al., 2008, J. Clin Microbiol, 46:431-437) only (control group), or are inoculated with spores and administered anti-TcdB VHH (25-50 μg/mouse) or dimeric or multimeric forms thereof, at 4, 24 and 48 hours post-challenge (treated group). Blood is collected at 72, 96 and 120 hours post-challenge to determine VHH titers. Animals administered anti-TcdB VHH are expected to be less prone to C. difficile-associated disease or severe disease, symptoms and outcomes, and to have increased survival (less morbidity and death) compared with control animals that did not receive anti-TcdB VHH. In some experiments, the animals receive a prophylactic dose of anti-TcdB VHH, or dimeric or multimeric forms thereof, prior to inoculation with spores so as to prevent C. difficile-associated disease and/or to reduce the severity of disease and symptoms thereof, and/or to improve survival.
Hamster CDI Challenge
[0253] In vivo experiments using hamsters are conducted to mimic the human condition of C. difficile infection and disease and to facilitate colonization with C. difficile. For these experiments, male Golden Syrian hamsters (110-135 g) are administered clindamycin (30 mg/kg) via oral gavage for 5 days prior to oral inoculation with 1000 C. difficile strain UK6 spores. Infected control hamsters are administered clindamycin, are inoculated with UK6 spores and are given sterile PBS, by intraperitoneal (IP) administration, 2 times per day for the duration of the experiment. Anti-TcdB VHH-treated hamsters are administered clindamycin, inoculated with spores and are given purified anti-TcdB VHH (1 mg/kg), or dimeric or multimeric forms thereof, by IP administration, 2 times a day for the duration of the experiment. A blood sample is collected at time of euthanasia for detection of anti-TcdB VHH in serum. Necropsies are performed on euthanized animals and tissues are collected for histopathologic examination. Animals administered anti-TcdB VHH are expected to be less prone to C. difficile-associated disease or severe disease, symptoms and outcomes, and to have increased survival (less morbidity and death) compared with control animals that did not receive anti-TcdB VHH. In some experiments, the animals receive a prophylactic dose of anti-TcdB VHH, or dimeric or multimeric forms thereof, prior to inoculation with spores so as to prevent C. difficile-associated disease and/or to reduce the severity of disease and symptoms thereof, and/or to improve survival.
Pig CDI Challenge
[0254] Pigs have been demonstrated to mimic C. difficile infection, colonization and disease as experienced by humans. For in vivo experiments using pigs, thirty gnotobiotic piglets are derived via Caesarean section and maintained in sterile isolators for the duration of the experiment (Tzipori, S. et al., 1995, Infect Immun, 63:3621-3627). Five groups of piglets are orally inoculated with 10.sup.6 C. difficile UK6 spores (group 1-5) and group 6 was the uninfected control group. Group 1 (n=3) receives anti-TcdB VHH (1 mg/pig) 4 hours prior to, and Group 2 (n=3) 18 hours post oral inoculation with spores. After the initial dose, the treated groups receive 2 doses of anti-TcdB VHH (1 mg/pig) per day either via IP or intra muscular (IM) administration for the duration of the experiment. The anti-TcdB VHH treated group (Group 3; n=9) is given 1.0×10″ viral particles by IV administration one day prior to oral inoculation with 10.sup.6 C. difficile UK6 spores and 3 days post infection. Group 4 (n=6) receives buffer as control, given 4 hours prior to oral inoculation with 10.sup.6 C. difficile UK6 spores and at 24 hour post inoculation, and then every 12 hours until the termination of the experiment. Group 5 is given control adenovirus expressing an unrelated VHH (n=6), (1.0×10.sup.11 viral particles) by IV administration one day prior to oral inoculation with 10.sup.6 C. difficile UK6 spores and 3 days post infection. Group 6 (n=3) is uninfected. Fecal samples are collected from all piglets for bacterial culture, and blood samples are collected 1-3 times (when possible) during the experiment and at the time of euthanasia to determine anti-TcdB VHH titers. Necropsies are performed on all animals and tissues are collected for histopathologic examination. Animals administered anti-TcdB VHH are expected to be less prone to C. difficile-associated disease or severe disease, symptoms and outcomes, and to have increased survival (less morbidity and death) compared with control animals that did not receive anti-TcdB VHH. In some experiments, the animals receive a prophylactic dose of anti-TcdB VHH, or dimeric or multimeric forms thereof, prior to inoculation with spores so as to prevent C. difficile-associated disease and/or to reduce the severity of disease and symptoms thereof, and/or to improve survival.
Histology
[0255] Tissue samples from C. difficile colonized animals are collected during necropsy and preserved in 10% neutral buffered formalin. Formalin fixed samples are embedded in paraffin, sectioned at 5 μm, and stained using hematoxylin and eosin using routine histochemical techniques at TCSVM Histopathology Service Laboratory. Light microscopic examination and lesion evaluation are performed by a board-certified veterinary pathologist (GB) with results reported for severity (minimal, mild, moderate, marked), epithelial ulceration, luminal contents, and quantification (Sponseller, J K et al., 2014, J Infect Dis, doi:10.1093/infdis/jiu605). Briefly, a quantitative assessment of colitis severity is performed by counting neutrophilic foci in colon sections from each sample. Foci are observed between colonic crypts in the lamina propria in 10 random fields with ×20 magnification.
[0256] All publications, patents, published patent applications and sequence database entries mentioned and disclosed herein are hereby incorporated by reference in their entireties as if each individual publication or patent were specifically and individually indicated to be incorporated by reference.