Compositions and methods relating to mutant clostridium difficile toxins
阅读说明：本技术 涉及突变体难辨梭菌毒素的组合物及其方法 (Compositions and methods relating to mutant clostridium difficile toxins ) 是由 M·K·希胡 A·S·安德森 R·G·K·唐纳德 K·U·扬森 N·K·卡利安 T·L·米 于 2012-04-20 设计创作，主要内容包括：在一方面,本发明涉及免疫原性组合物,其包含突变体难辨梭菌毒素A和/或突变体难辨梭菌毒素B。相对于相应的野生型难辨梭菌毒素,每种突变体毒素包括具有至少一个突变的葡萄糖基转移酶结构域和具有至少一个突变的半胱氨酸蛋白酶结构域。所述突变体毒素还可包含至少一个经化学交联的氨基酸。在另一方面,本发明涉及结合所述免疫原性组合物的抗体或其结合片段。在另外的方面,本发明涉及编码任意前述物质的分离的核苷酸序列,以及使用任意前述组合物的方法。(In one aspect, the invention relates to an immunogenic composition comprising a mutant clostridium difficile toxin a and/or a mutant clostridium difficile toxin B. Each mutant toxin includes a glucosyltransferase domain having at least one mutation and a cysteine protease domain having at least one mutation relative to the corresponding wild-type clostridium difficile toxin. The mutant toxin may also comprise at least one chemically cross-linked amino acid. In another aspect, the invention relates to antibodies or binding fragments thereof that bind to the immunogenic composition. In further aspects, the invention relates to isolated nucleotide sequences encoding any of the foregoing, as well as methods of using any of the foregoing compositions.)
1. A polypeptide comprising SEQ ID NO:6, wherein the methionine residue at position 1 is optionally absent.
2. A nucleic acid encoding the amino acid sequence of SEQ ID NO:6, wherein the methionine residue at position 1 is optionally absent.
3. An immunogenic composition for inducing neutralizing antibodies against Clostridium difficile (Clostridium difficile) toxin B, comprising an effective amount of the amino acid sequence of SEQ ID NO:6, wherein the methionine residue at position 1 is optionally absent.
4. The composition of claim 3, wherein the polypeptide comprises SEQ ID NO: 6.
5. the composition of claim 3, further comprising a carbohydrate.
6. The composition of claim 5, wherein the carbohydrate is selected from the group consisting of sorbitol, mannitol, starch, dextran, sucrose, trehalose, lactose and glucose.
7. The composition of claim 5, wherein the carbohydrate comprises sucrose.
8. The composition of claim 5, wherein the carbohydrate comprises trehalose.
9. The composition of any one of claims 3-8, further comprising a buffering agent.
10. The composition of claim 9, wherein the buffer is selected from the group consisting of phosphate (e.g., potassium phosphate, sodium phosphate) buffers; acetate (e.g., sodium acetate) buffer; succinate (e.g., sodium succinate) buffer; a glycine buffer; a histidine buffer; a carbonate buffer; tris (Tris hydroxymethyl aminomethane) buffer; and bicarbonate (e.g., ammonium bicarbonate) buffers.
11. The composition of any one of claims 3-9, wherein the buffer comprises Tris buffer.
12. The composition of any one of claims 3-9, wherein the buffering agent comprises histidine.
13. The composition of any one of claims 3-9, wherein the buffer comprises phosphate.
14. The composition of any one of claims 3-13, further comprising a surfactant.
15. The composition of claim 14, wherein the surfactant is selected from the group consisting of polyoxyethylene sorbitan ester surfactants; polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols, triethylene glycol monododecyl ether; t-octyl phenoxypolyethoxyethanol; and sorbitan esters.
16. The composition of claim 14, wherein the surfactant comprises polyoxyethylene sorbitan ester.
17. The composition of claim 14, wherein the surfactant comprises polyoxyethylene sorbitan monooleate.
18. The composition of claim 14, wherein the surfactant comprises polysorbate 80.
19. The composition of any one of claims 3-18, wherein the composition does not comprise formaldehyde.
20. The composition of any one of claims 3-19, wherein the composition comprises an adjuvant.
21. The composition of claim 20, wherein the adjuvant is selected from the group consisting of 3 De-O-acylated monophosphoryl lipid a (MPL)TM) (ii) a Aluminum hydroxide; an aluminum salt; aluminum phosphate; aluminum sulfate; a saponin; QS-21; immunostimulatory particles; polyglutamic acid; a polylysine; a CpG oligonucleotide; CpG oligodeoxynucleotide; and a polypeptide comprising SEQ ID NO:48 CpG oligonucleotide.
22. The composition of claim 20, wherein the adjuvant comprises aluminum hydroxide.
23. The composition of claim 20, wherein the adjuvant comprises aluminum sulfate.
24. The composition of claim 20, wherein the adjuvant comprises a CpG oligonucleotide.
25. The composition of claim 20, wherein said adjuvant comprises QS-21.
26. The composition of claim 20, wherein the adjuvant comprises an immunostimulatory particle.
27. The composition of any one of claims 3-26, wherein the composition is lyophilized.
28. The composition of any one of claims 3-26, wherein the composition is liquid.
29. An antibody or antigen-binding fragment thereof having neutralizing activity against clostridium difficile toxin B induced by the composition of claim 3, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 49 and 55, or SEQ ID NO: 60 and 66, or SEQ ID NO: 71 and 77, for a light chain and a heavy chain comprising SEQ ID NO: 6. optionally, the immunogenic composition wherein the methionine residue at position 1 is absent is specific.
30. An antibody or antigen-binding fragment thereof having neutralizing activity against clostridium difficile toxin B induced by the composition of claim 3, comprising the amino acid sequence of SEQ ID NO:51(CDR H1), SEQ ID NO:52(CDR H2) and SEQ ID NO:53(CDR H3) and the amino acid sequence of the heavy chain Complementarity Determining Region (CDR) shown in SEQ ID NO:57(CDR L1), SEQ ID NO:58(CDR L2) and SEQ ID NO:59(CDR L3).
31. An antibody or antigen-binding fragment thereof having neutralizing activity against clostridium difficile toxin B induced by the composition of claim 3, comprising the amino acid sequence of SEQ ID NO:62(CDR H1), SEQ ID NO:63(CDR H2) and SEQ ID NO: 64(CDR H3) and the amino acid sequence of the heavy chain Complementarity Determining Region (CDR) shown in SEQ ID NO:68(CDR L1), SEQ ID NO:69(CDR L2) and SEQ ID NO:70(CDR L3).
32. An antibody or antigen-binding fragment thereof having neutralizing activity against clostridium difficile toxin B induced by the composition of claim 3, comprising the amino acid sequence of SEQ ID NO:73(CDR H1), SEQ ID NO:74(CDR H2) and SEQ ID NO:75(CDR H3) and the amino acid sequence of the heavy chain Complementarity Determining Region (CDR) shown in SEQ ID NO:79(CDR L1), SEQ ID NO:80(CDR L2) and SEQ ID NO:81(CDR L3).
33. A composition having neutralizing activity against clostridium difficile toxin B comprising an effective amount of a combination of two or more antibodies, or antigen-binding fragments thereof, selected from any one of claims 30-32.
Technical Field
The present invention is directed to compositions and methods relating to mutant Clostridium difficile (Clostridium difficile) toxins.
Background
Clostridium difficile (c.difficile) is a gram-positive anaerobic bacterium that is associated with human gastrointestinal disease. If the natural intestinal flora is reduced by treatment with antibodies, colonization of clostridium difficile usually occurs in the colon. Through the secretion of glucosylated toxins, toxin a and toxin B (308 and 270kDa, respectively, which are the major virulence factors of clostridium difficile), infections can lead to antibiotic-associated diarrhea and sometimes pseudomembranous colitis.
Toxin a and toxin B are encoded by genes tcdA and tcdB, respectively, within the 19kb pathogenicity locus (PaLoc). This locus was replaced by an additional 115 base pair sequence in a non-pathogenic strain of C.difficile.
Both toxin a and toxin B are potent cytotoxins. These proteins are homologous glucosyltransferases that inactivate the small GTPases of the Rho/Rac/Ras family. The resulting disruption of signaling can cause loss of cell-cell junctions, actin cytoskeleton dysregulation, and/or apoptosis, resulting in profound secretory diarrhea (profounds secretary diarrhea) associated with Clostridium Difficile Infection (CDI).
In the last decade, the number and severity of clostridium difficile outbreaks in hospitals, nursing homes, and other long-term care facilities has increased dramatically. The major factors for this increase include the emergence of highly toxic pathogenic strains, increased use of antibiotics, improved detection methods, and increased exposure to airborne spores in health care facilities.
Metronidazole and vancomycin are currently accepted standards of care for treatment with Clostridium Difficile Associated Disease (CDAD) antibiotics. However, approximately 20% of patients receiving such treatment experience a recurrence of infection after a first phase of CDI, and up to approximately 50% of those patients have additional recurrences. Treatment of relapses is a very important challenge, and most relapses usually occur within one month of the previous phase.
Thus, there is a need for immunogenic and/or therapeutic compositions and methods thereof involving clostridium difficile.
Summary of The Invention
These and other objects are provided herein by the present invention.
In one aspect, the invention relates to an immunogenic composition comprising a mutant clostridium difficile toxin a. The mutant clostridium difficile toxin a comprises a glucosyltransferase domain having at least one mutation and a cysteine protease domain having at least one mutation, relative to the corresponding wild-type clostridium difficile toxin a. In one embodiment, at least one amino acid of the mutant clostridium difficile toxin a is chemically cross-linked.
In one aspect, the invention relates to an isolated polypeptide comprising the amino acid sequence set forth in
In one embodiment, at least one amino acid of the mutant clostridium difficile toxin is chemically cross-linked.
In one embodiment, the at least one amino acid is chemically crosslinked by formaldehyde, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinate, or a combination of EDC and NHS.
In one embodiment, the immunogenic composition is recognized by the respective antitoxin neutralizing antibody or binding fragment thereof
In one embodiment, the immunogenic composition exhibits reduced cytotoxicity relative to a corresponding wild-type clostridium difficile toxin.
In another aspect, the invention relates to an immunogenic composition comprising a mutant clostridium difficile toxin a comprising a glucosyltransferase domain having SEQ ID NO:29 with amino acid substitutions at positions 285 and 287 (relative to the corresponding wild-type clostridium difficile toxin a) and a cysteine protease domain having SEQ ID NO:32 with amino acid substitutions at position 158 (relative to the corresponding wild-type clostridium difficile toxin a), wherein at least one amino acid of the mutant clostridium difficile toxin a is chemically cross-linked.
In a further aspect, the invention relates to an immunogenic composition comprising a mutant clostridium difficile toxin a comprising SEQ ID NO:4, wherein at least one amino acid of the mutant clostridium difficile toxin a is chemically cross-linked.
In another aspect, the invention relates to an immunogenic composition comprising
In one aspect, the invention relates to an immunogenic composition comprising a mutant clostridium difficile toxin B. The mutant clostridium difficile toxin B comprises a glucosyltransferase domain having at least one mutation and a cysteine protease domain having at least one mutation, relative to the corresponding wild-type clostridium difficile toxin B.
In another aspect, the invention relates to an isolated polypeptide comprising the amino acid sequence set forth in
In another aspect, the invention relates to an immunogenic composition comprising a mutant clostridium difficile toxin B comprising a glucosyltransferase domain having SEQ ID NO:31 with amino acid substitutions at positions 286 and 288 (relative to the corresponding wild-type clostridium difficile toxin B), and a cysteine protease domain having SEQ ID NO:33 with amino acid substitutions at position 155 (relative to the corresponding wild-type clostridium difficile toxin B), wherein at least one amino acid of the mutant clostridium difficile toxin B is chemically cross-linked.
In a further aspect, the invention relates to an immunogenic composition comprising a mutant clostridium difficile toxin B comprising
In one aspect, the invention relates to an immunogenic composition comprising a mutant clostridium difficile toxin a comprising SEQ ID No. 4 and a mutant clostridium difficile toxin B comprising SEQ ID No. 6, wherein at least one amino acid of each of said mutant clostridium difficile toxins is chemically cross-linked.
In a further aspect, the invention relates to a recombinant cell or progeny thereof comprising a polynucleotide encoding any of the aforementioned mutant clostridium difficile toxins, wherein the cell lacks an endogenous polynucleotide encoding the toxin.
In another aspect, the invention relates to antibodies, or antibody binding fragments thereof, specific for immunogenic compositions comprising mutant clostridium difficile toxins.
In one aspect, the invention relates to a method of treating clostridium difficile infection in a mammal. The method comprises administering to the mammal an immunogenic composition comprising a mutant clostridium difficile toxin a comprising
In one aspect, the invention relates to a method of treating clostridium difficile infection in a mammal. The method comprises administering to the mammal an immunogenic composition comprising a mutant clostridium difficile toxin a comprising
In one aspect, the invention relates to a method of inducing an immune response to clostridium difficile infection in a mammal. The method comprises administering to the mammal an immunogenic composition comprising a mutant clostridium difficile toxin a comprising
In one aspect, the invention relates to a method of inducing an immune response to clostridium difficile infection in a mammal. The method comprises administering to the mammal an immunogenic composition comprising a mutant clostridium difficile toxin a comprising
In one embodiment, the method of treatment or the method of inducing an immune response is in a mammal in need thereof.
In one embodiment, the method of treatment or the method of inducing an immune response comprises a mammal that has had a recurrent clostridium difficile infection.
In one embodiment, the method of treatment or the method of inducing an immune response comprises parenteral administration of the composition.
In one embodiment, the method of treatment or method of inducing an immune response comprises an immunogenic composition further comprising an adjuvant.
In one embodiment, the adjuvant comprises aluminum hydroxide gel and CpG oligonucleotides. In another embodiment, the adjuvant comprises ISCOMATRIX.
In one embodiment, the isolated polypeptide comprises at least one aspartic acid residue side chain of the polypeptide or at least one glutamic acid residue side chain of the polypeptide chemically cross-linked via glycine.
In one embodiment, the isolated polypeptide comprises at least one crosslink between an aspartic acid residue side chain of the polypeptide and a lysine residue side chain of the polypeptide; and at least one crosslink between a glutamic acid residue side chain of said polypeptide and a lysine residue side chain of said polypeptide.
In one embodiment, the isolated polypeptide comprises a β -alanine moiety attached to the side chain of at least one lysine residue of the polypeptide.
In one embodiment, the isolated polypeptide comprises a glycine moiety attached to the side chain of an aspartic acid residue of the polypeptide or to the side chain of a glutamic acid residue of the polypeptide.
In one embodiment, the isolated polypeptide comprises the amino acid sequence set forth in
In one embodiment, the isolated polypeptide comprises the amino acid sequence set forth in
In one embodiment, the isolated polypeptide comprises a side chain of a second lysine residue attached to a side chain of an aspartic acid residue or to a side chain of a glutamic acid residue.
In one embodiment, the isolated polypeptide comprises an aspartate residue side chain or a glutamate residue side chain of the polypeptide linked to a glycine moiety.
In one embodiment, the isolated polypeptide has an EC of at least about 100 μ g/ml50。
In one aspect, the immunogenic composition comprises an isolated polypeptide having the amino acid sequence shown in SEQ ID NO. 4 (wherein the methionine residue at
In one embodiment, the polypeptide comprises at least one of any of a) a) at least one β -alanine moiety attached to a lysine side chain of the polypeptide; b) at least one crosslink between an aspartic acid residue side chain and a lysine residue side chain of the polypeptide; and c) at least one crosslink between a glutamic acid residue side chain and a lysine residue side chain of said polypeptide.
In one embodiment, the isolated polypeptide has an EC of at least about 100 μ g/ml50。
In one aspect, the immunogenic composition comprises an isolated polypeptide having the amino acid sequence shown in SEQ ID NO. 4 (wherein the methionine residue at
In one embodiment, the immunogenic composition comprises a second lysine residue side chain of SEQ ID No. 4 linked to an aspartic acid residue side chain or to a glutamic acid residue side chain, and wherein the second lysine residue of SEQ ID No. 6 is linked to an aspartic acid residue side chain or to a glutamic acid residue side chain.
In one embodiment, the immunogenic composition comprises an aspartic acid residue side chain or a glutamic acid residue side chain of a polypeptide having the amino acid sequence shown in SEQ ID NO 4 (wherein the methionine residue at
In one embodiment, the immunogenic composition comprises an aspartic acid residue side chain or a glutamic acid residue side chain of a polypeptide having the amino acid sequence shown in SEQ ID NO 6 (wherein the methionine residue at
In one embodiment, the isolated polypeptide has an EC of at least about 100 μ g/ml50。
In one aspect, the immunogenic composition comprises an isolated polypeptide having the amino acid sequence set forth in SEQ ID NO 84 and an isolated polypeptide having the amino acid sequence set forth in SEQ ID NO 86, wherein each polypeptide comprises a) at least one crosslink between an aspartic acid residue side chain of the polypeptide and a lysine residue side chain of the polypeptide; b) at least one crosslink between a glutamic acid residue side chain of said polypeptide and a lysine residue side chain of said polypeptide; c) a β -alanine moiety attached to the side chain of at least one lysine residue of said polypeptide; and d) a glycine moiety attached to at least one aspartic acid residue side chain of said polypeptide or at least one glutamic acid residue side chain of said polypeptide.
Brief Description of Drawings
FIG. 1 sequence alignment of wild type Clostridium difficile toxin A from strains 630, VPI10463, R20291, CD196 and mutant toxin A with SEQ ID NO. 4 using CLUSTALW alignment, default parameters.
FIG. 2 sequence alignment of wild type Clostridium difficile toxin A and mutant toxin B with SEQ ID NO 6 from strains 630, VPI10463, R20291, CD196 using CLUSTALW alignment, default parameters.
FIG. 3 is a diagram showing the identification of wild-type toxin-negative Clostridium difficile strains. For toxin a, media of 13 c.difficile strains were tested by ELISA. As shown therein, 7 strains expressed toxin a and 6 strains did not (strains 1351, 3232, 7322, 5036, 4811 and VPI 11186).
FIGS. 4A and B SDS-PAGE results, which illustrate the use of the triple mutant A (SEQ ID NO:4), the double mutant B (SEQ ID NO:5), and the triple mutant B (SEQ ID NO:6) in UDP-14Rac1 or RhoA GTPase was not glycosylated in an in vitro glucosylation assay of C-glucose; whereas 10. mu.g to 1ng of wild-type toxin B is glucosylated
FIG. 5 Western blot showing the elimination of cysteine protease activity in mutant toxins A and B (SEQ ID NOS: 4 and 6, respectively) compared to the cleaved fragments of wild-type toxins A and B (SEQ ID NOS: 1 and 2, respectively). See example 13.
FIG. 6 is a graph showing that triple mutant toxins A and B (SEQ ID NOS: 4 and 6, respectively) exhibit residual cytotoxicity when tested at high concentrations (e.g., about 100 μ g/ml) by performing in vitro cytotoxicity assays in IMR-90.
FIG. 7 shows a triple processEC of variant toxin B (SEQ ID NO:6) and seven-fold mutant toxin B (SEQ ID NO:8)50A graph with similar values.
FIG. 8 is a graph representing the results of in vitro cytotoxicity assays, in which ATP levels (RLU) are plotted against the increased concentration of triple mutant TcdA (SEQ ID NO:4) (upper part) and triple mutant TcdB (SEQ ID NO:6) (lower part). Residual cytotoxicity of mutant toxin a and B could be completely eliminated with neutralizing antibodies specific for mutant toxin a (upper part-pAb a and mAb A3-25+ a60-22) and mutant toxin B (lower part-pAb B).
FIG. 9 IMR-90 cell morphology 72 hours after treatment. Section a shows mock-treated control cells. Section B shows cell morphology after treatment with formalin inactivated mutant TcdB (SEQ ID NO: 6). Part C shows the cell morphology after treatment with EDC-inactivated mutant TcdB (SEQ ID NO: 6). Section D shows the cell morphology after treatment with wild-type toxin B (SEQ ID NO: 2). Section E shows the cell morphology after treatment with the triple mutant TcdB (SEQ ID NO: 6). Similar results were observed in TcdA treatment.
FIG. 10 is a graph showing the neutralizing antibody titers described in example 25 (study of muCdiff 2010-06).
FIG. 11 is a graph showing the neutralizing antibody titers described in example 26 (study of muCdiff 2010-07).
FIG. 12 shows graphs of neutralizing antibody responses against toxins A and B in hamsters after 4 immunizations, as described in example 27 (study of ham Clostridium difficile 2010-02).
FIG. 13 shows a graph of neutralizing antibody responses in hamsters after inoculation with chemically inactivated genetic mutant toxins and List Biological toxoid, as described in example 27 (study of ham Clostridium difficile 2010-02).
FIG. 14 survival curves of hamsters from three immunization groups compared to non-immunized controls, as described in example 28 (study of ham Clostridium difficile 2010-02, follow).
FIG. 15 is a graph showing the relative neutralizing antibody response in hamsters against different formulations of Clostridium difficile mutant toxins (study ham Clostridium difficile 2010-03), as described in example 29.
FIGS. 16A-B are graphs showing strong relative neutralizing antibody responses against chemically inactivated genetic mutant toxins A and B (SEQ ID NOS: 4 and 6, respectively) in cynomolgus monkeys, as described in example 30.
FIG. 17 amino acid sequences of the light (VL) and Heavy (HL) chain variable regions of the IgE of the A3-25 mAb. Signal peptide-highlighted; CDR-italics and underlined; constant regions-bold and underlined (complete sequence not shown).
FIG. 18 is a graph showing titration of individual toxin A monoclonal antibodies in a toxin neutralization assay using ATP levels (quantified by relative light units-RLU) as an indicator of cell viability. In contrast to toxin (4 xEC)50) In contrast, mAbs A80-29, A65-33, A60-22 and A3-25 had neutralizing effects on toxin A with increasing concentration (but not on positive rabbit antitoxin A control levels). mAbs A50-10, A56-33 and A58-46 did not neutralize toxin A. Cell-only controls were 1-1.5X106RLU。
FIG. 19 mapping of groups of 8 epitopes of toxin B mAb by BiaCore.
FIGS. 20A-C synergistic neutralizing Activity of combinations of toxin A mAbs addition of different dilutions of neutralizing antibodies A60-22, A65-33, and A80-29 to increasing concentrations of A3-25mAb synergistically increased neutralization of toxin A, regardless of dilution. Shows toxin A only (4 × EC)50) Control RLU of (1)<0.3x106) While the cell only control was 2-2.5x106RLU as shown in fig. 20B and 20C.
FIG. 21 synergistic neutralizing activity of combinations of toxin B mAbs neutralization of toxin B by mAbs 8-26, B60-2 and B59-3 is shown in FIG. 21A. Neutralization of toxin B was synergistically enhanced upon combining B8-26 with dilutions of B59-3 (fig. 21B).
FIG. 22 Western blot showing that expression of Rac1GTPase decreased from 24 to 96 hours in genetic mutant toxin B (SEQ ID NO:6) extracts, but not in wild type toxin B (SEQ ID NO:2) treated extracts. The blot also showed that Rac1 was glucosylated in toxin B-treated extracts, but not in genetically mutant toxin B-treated extracts.
FIGS. 23A-K represent in vitro cellsGraph of toxicity test results, in which ATP levels (RLU) are plotted against increasing concentrations of Clostridium difficile medium and hamster serum pool (■), crude toxin (culture harvest) from each strain and hamster serum pool (●), purified toxin (commercially available toxin from List Biologicals) and hamster serum pool (▲), crude toxin
FIG. 24 illustrates exemplary EDC/NHS inactivation of mutant Clostridium difficile toxins resulting in at least three possible types of modification, cross-linking, glycine adducts, and β -alanine adducts. Section a illustrates crosslinking. The carboxylic acid residues of the triple mutant toxin were activated by addition of EDC and NHS. The activated ester reacts with the primary amine to form a stable amide bond, resulting in intramolecular and intermolecular cross-linking. Section B illustrates the formation of a glycine adduct. After deactivation, the residual activated ester is quenched by addition of glycine to form a stable amide bond. The C section illustrates the formation of a beta-alanine adduct. The 3 moles of NHS can react with 1 mole of EDC to form activated beta-alanine. This in turn reacts with the primary amine to form a stable amide bond.
FIG. 25 illustrates the EDC/NHS inactivation of an exemplary mutant Clostridium difficile toxin resulting in at least one type of modification selected from the group consisting of (A) cross-linking, (B) glycine adduct, and (C) beta-alanine adduct.
Brief description of the sequences
SEQ ID NO 5 shows the amino acid sequence of the mutant TcdB with mutations at positions 286 and 288 compared to
SEQ ID NO 7 shows the amino acid sequence of mutant TcdA having mutations at positions 269, 272, 285, 287, 460, 462 and 700 compared to
SEQ ID NO 9 shows the DNA sequence encoding wild type Clostridium difficile 630 toxin A (TcdA).
SEQ ID NO 11 shows the DNA sequence encoding
SEQ ID NO. 12 shows the DNA sequence encoding SEQ ID NO. 4.
SEQ ID NO 13 shows the DNA sequence encoding SEQ ID NO 5.
SEQ ID NO. 14 shows the DNA sequence encoding SEQ ID NO. 6.
SEQ ID NO. 15 shows the amino acid sequence of wild type Clostridium difficile R20291 TcdA.
SEQ ID NO 17 shows the amino acid sequence of wild type Clostridium difficile CD196 TcdA.
SEQ ID NO 18 shows the DNA sequence encoding SEQ ID NO 17.
SEQ ID NO 19 shows the amino acid sequence of wild type Clostridium difficile VPI10463 TcdA.
SEQ ID NO 20 shows the DNA sequence encoding SEQ ID NO 19.
21 shows the amino acid sequence of wild type C.difficile R20291 TcdB.
SEQ ID NO. 22 shows the DNA sequence encoding SEQ ID NO. 21.
SEQ ID NO 23 shows the amino acid sequence of wild type Clostridium difficile CD196 TcdB.
SEQ ID NO. 24 shows the DNA sequence encoding SEQ ID NO. 23.
SEQ ID NO. 25 shows the amino acid sequence of wild type Clostridium difficile VPI10463 TcdB.
SEQ ID NO 26 shows the DNA sequence encoding SEQ ID NO 25.
The amino acid sequences of residues 101-293 of SEQ ID NO:1 are shown in SEQ ID NO: 28.
SEQ ID NO. 29 shows the amino acid sequence of residues 1-542 of SEQ ID NO. 1.
The amino acid sequences of residues 101-293 of SEQ ID NO:2 are shown in SEQ ID NO: 30.
SEQ ID NO 31 shows the amino acid sequence of residues 1-543 of
The amino acid sequence of residues 543-809 of SEQ ID NO:1 is shown in SEQ ID NO: 32.
The amino acid sequence of residue 544-767 of SEQ ID NO:2 is shown in SEQ ID NO: 33.
SEQ ID NO 34 shows the amino acid sequence of a mutant TcdA in which residues 101, 269, 272, 285, 287, 460, 462, 541, 542, 543, 589, 655, and 700 may be any amino acid.
SEQ ID NO 35 shows the amino acid sequence of mutant TcdB, where 102, 270, 273, 286, 288, 384, 461, 463, 520, 543, 544, 587, 600, 653, 698 and 751 can be any amino acid.
SEQ ID NO:36 shows the amino acid sequence of the variable light chain of the neutralizing antibody of Clostridium difficile TcdA (A3-25 mAb).
SEQ ID NO:37 shows the amino acid sequence of the variable heavy chain of the neutralizing antibody of Clostridium difficile TcdA (A3-25 mAb).
SEQ ID NO 38 shows the amino acid sequence of CDR1 of the variable light chain of the neutralizing antibody of Clostridium difficile TcdA (A3-25 mAb).
SEQ ID NO:39 shows the amino acid sequence of CDR2 of the variable light chain of the neutralizing antibody of Clostridium difficile TcdA (A3-25 mAb). .
40 shows the amino acid sequence of CDR3 of the variable light chain of the neutralizing antibody of Clostridium difficile TcdA (A3-25 mAb).
SEQ ID NO:41 shows the amino acid sequence of CDR1 of the variable heavy chain of the neutralizing antibody of Clostridium difficile TcdA (A3-25 mAb).
42 shows the amino acid sequence of CDR2 of the variable heavy chain of the neutralizing antibody of Clostridium difficile TcdA (A3-25 mAb).
SEQ ID NO 43 shows the amino acid sequence of CDR3 of the variable heavy chain of the neutralizing antibody of Clostridium difficile TcdA (A3-25 mAb).
SEQ ID NO. 44 shows the DNA sequence encoding SEQ ID NO. 3.
SEQ ID NO 45 shows the DNA sequence encoding
SEQ ID NO 46 shows the DNA sequence encoding SEQ ID NO 5.
SEQ ID NO 47 shows the DNA sequence encoding
The nucleotide sequence of the immunostimulatory oligonucleotide ODN CpG 2455 is shown in SEQ ID NO 48.
SEQ ID NO:49 shows the amino acid sequence of the variable heavy chain of the C.difficile TcdB neutralizing antibody (B8-26 mAb). SEQ ID NO:50 shows the amino acid sequence of the signal peptide of the variable heavy chain of the C.difficile TcdB neutralizing antibody (B8-26 mAb).
SEQ ID NO:51 shows the amino acid sequence of CDR1 of the variable heavy chain of the C.difficile TcdB neutralizing antibody (B8-26 mAb).
SEQ ID NO:52 shows the amino acid sequence of CDR2 of the variable heavy chain of the C.difficile TcdB neutralizing antibody (B8-26 mAb).
SEQ ID NO:53 shows the amino acid sequence of CDR3 of the variable heavy chain of the Clostridium difficile TcdB neutralizing antibody (B8-26 mAb).
SEQ ID NO:54 shows the amino acid sequence of the constant region of the variable heavy chain of the C.difficile TcdB neutralizing antibody (B8-26 mAb).
SEQ ID NO:55 shows the amino acid sequence of the variable light chain of the C.difficile TcdB neutralizing antibody (B8-26 mAb).
SEQ ID NO:56 shows the amino acid sequence of the signal peptide of the variable light chain of the C.difficile TcdB neutralizing antibody (B8-26 mAb).
SEQ ID NO:57 shows the amino acid sequence of CDR1 of the variable light chain of C.difficile TcdB neutralizing antibody (B8-26 mAb).
SEQ ID NO:58 shows the amino acid sequence of CDR2 of the variable light chain of C.difficile TcdB neutralizing antibody (B8-26 mAb).
SEQ ID NO 59 shows the amino acid sequence of CDR3 of the variable light chain of the C.difficile TcdB neutralizing antibody (B8-26 mAb).
SEQ ID NO:60 shows the amino acid sequence of the variable heavy chain of the C.difficile TcdB neutralizing antibody (B59-3 mAb).
SEQ ID NO 61 shows the amino acid sequence of the signal peptide of the variable heavy chain of the C.difficile TcdB neutralizing antibody (B59-3 mAb).
SEQ ID NO:62 shows the amino acid sequence of CDR1 of the variable heavy chain of the C.difficile TcdB neutralizing antibody (B59-3 mAb).
SEQ ID NO:63 shows the amino acid sequence of CDR2 of the variable heavy chain of the C.difficile TcdB neutralizing antibody (B59-3 mAb).
SEQ ID NO:64 shows the amino acid sequence of CDR3 of the variable heavy chain of the C.difficile TcdB neutralizing antibody (B59-3 mAb).
SEQ ID NO:65 shows the amino acid sequence of the constant region of the variable heavy chain of the C.difficile TcdB neutralizing antibody (B59-3 mAb).
SEQ ID NO:66 shows the amino acid sequence of the variable light chain of the C.difficile TcdB neutralizing antibody (B59-3 mAb).
67 shows the amino acid sequence of the signal peptide of the variable light chain of the C.difficile TcdB neutralizing antibody (B59-3 mAb).
SEQ ID NO 68 shows the amino acid sequence of CDR1 of the variable light chain of C.difficile TcdB neutralizing antibody (B59-3 mAb).
69 shows the amino acid sequence of CDR2 of the variable light chain of the C.difficile TcdB neutralizing antibody (B59-3 mAb).
SEQ ID NO:70 shows the amino acid sequence of CDR3 of the variable light chain of C.difficile TcdB neutralizing antibody (B59-3 mAb).
SEQ ID NO:71 shows the amino acid sequence of the variable heavy chain of the C.difficile TcdB neutralizing antibody (B9-30 mAb).
SEQ ID NO:72 shows the amino acid sequence of the signal peptide of the variable heavy chain of the C.difficile TcdB neutralizing antibody (B9-30 mAb).
SEQ ID NO:73 shows the amino acid sequence of CDR1 of the variable heavy chain of the C.difficile TcdB neutralizing antibody (B9-30 mAb).
SEQ ID NO:74 shows the amino acid sequence of CDR2 of the variable heavy chain of the C.difficile TcdB neutralizing antibody (B9-30 mAb).
SEQ ID NO:75 shows the amino acid sequence of CDR3 of the variable heavy chain of the C.difficile TcdB neutralizing antibody (B9-30 mAb).
SEQ ID NO:76 shows the amino acid sequence of the constant region of the variable heavy chain of the C.difficile TcdB neutralizing antibody (B9-30 mAb).
SEQ ID NO:77 shows the amino acid sequence of the variable light chain of the C.difficile TcdB neutralizing antibody (B9-30 mAb).
SEQ ID NO:78 shows the amino acid sequence of the signal peptide of the variable light chain of the C.difficile TcdB neutralizing antibody (B9-30 mAb).
SEQ ID NO:79 shows the amino acid sequence of CDR1 of the variable light chain of C.difficile TcdB neutralizing antibody (B9-30 mAb).
SEQ ID NO:80 shows the amino acid sequence of CDR2 of the variable light chain of C.difficile TcdB neutralizing antibody (B9-30 mAb).
SEQ ID NO:81 shows the amino acid sequence of CDR3 of the variable light chain of C.difficile TcdB neutralizing antibody (B9-30 mAb).
SEQ ID NO:82 shows the amino acid sequence of the mutant TcdB where the residues at
SEQ ID NO:83 shows the amino acid sequence of mutant TcdA having mutations at positions 269, 272, 285, 287, 460, 462 and 700 compared to SEQ ID NO:1 in which the methionine at
SEQ ID NO:84 shows the amino acid sequence of mutant Clostridium difficile toxin A with mutations at positions 285, 287 and 700 compared to SEQ ID NO:1, wherein the methionine at
SEQ ID NO:85 shows the amino acid sequence of mutant Clostridium difficile toxin B with mutations at
SEQ ID NO 86 shows the amino acid sequence of mutant Clostridium difficile toxin B with mutations at positions 286, 288 and 698 compared to
SEQ ID NO:87 shows the amino acid sequence of wild
88 shows the amino acid sequence of wild type Clostridium difficile 2004111 TcdA.
89 shows the amino acid sequence of wild type Clostridium difficile 2004118 TcdA.
SEQ ID NO 90 shows the amino acid sequence of wild type Clostridium difficile 2004205 TcdA.
91 shows the amino acid sequence of wild type Clostridium difficile 2004206 TcdA.
SEQ ID NO 92 shows the amino acid sequence of wild type Clostridium difficile 2005022 TcdA.
SEQ ID NO 93 shows the amino acid sequence of wild type Clostridium difficile 2005088 TcdA.
SEQ ID NO 94 shows the amino acid sequence of wild type Clostridium difficile 2005283 TcdA.
SEQ ID NO 95 shows the amino acid sequence of wild type Clostridium difficile 2005325 TcdA.
The amino acid sequence of wild type C.difficile 2005359TcdA is shown in SEQ ID NO 96.
SEQ ID NO:97 shows the amino acid sequence of wild type Clostridium difficile 2006017 TcdA.
98 shows the amino acid sequence of wild
SEQ ID NO 99 shows the amino acid sequence of wild type Clostridium difficile 2007217 TcdA.
100 shows the amino acid sequence of wild
101 shows the amino acid sequence of wild type Clostridium difficile 2007816 TcdA.
The amino acid sequence of wild type C.difficile 2007838TcdA is shown in SEQ ID NO 102.
103 shows the amino acid sequence of wild type c.difficile 2007858 TcdA.
104 shows the amino acid sequence of wild
105 shows the amino acid sequence of wild type clostridium difficile 2008222 TcdA.
106 shows the amino acid sequence of wild type Clostridium difficile 2009078 TcdA.
107 shows the amino acid sequence of wild type Clostridium difficile 2009087 TcdA.
108 shows the amino acid sequence of wild
109 shows the amino acid sequence of wild type C.difficile 2009292 TcdA.
110 shows the amino acid sequence of wild
111 shows the amino acid sequence of wild type C.difficile 2004111 TcdB.
SEQ ID NO:112 shows the amino acid sequence of wild type Clostridium difficile 2004118 TcdB.
113 shows the amino acid sequence of wild type C.difficile 2004205 TcdB.
SEQ ID NO 114 shows the amino acid sequence of wild type Clostridium difficile 2004206 TcdB.
SEQ ID NO:115 shows the amino acid sequence of wild type Clostridium difficile 2005022 TcdB.
SEQ ID NO:116 shows the amino acid sequence of wild type Clostridium difficile 2005088 TcdB.
SEQ ID NO 117 shows the amino acid sequence of wild type Clostridium difficile 2005283 TcdB.
The amino acid sequence of wild type C.difficile 2005325TcdB is shown in SEQ ID NO. 118.
119 shows the amino acid sequence of wild type C.difficile 2005359 TcdB.
SEQ ID NO 120 shows the amino acid sequence of wild type Clostridium difficile 2006017 TcdB.
SEQ ID NO. 121 shows the amino acid sequence of wild type Clostridium difficile 2006376 TcdB.
122 shows the amino acid sequence of the wild
SEQ ID NO 123 shows the amino acid sequence of wild type Clostridium difficile 2007217 TcdB.
SEQ ID NO:124 shows the amino acid sequence of wild
125 shows the amino acid sequence of wild type C.difficile 2007816 TcdB.
126 shows the amino acid sequence of wild type c.difficile 2007838 TcdB.
127 shows the amino acid sequence of wild type Clostridium difficile 2007858 TcdB.
The amino acid sequence of wild type C.difficile 2007886TcdB is shown in SEQ ID NO 128.
129 shows the amino acid sequence of wild type Clostridium difficile 2008222 TcdB.
130 shows the amino acid sequence of wild type C.difficile 2009078 TcdB.
131 shows the amino acid sequence of wild type Clostridium difficile 2009087 TcdB.
132 shows the amino acid sequence of wild
The amino acid sequence of wild type C.difficile 2009292TcdB is shown in SEQ ID NO. 133.
SEQ ID NO 134 shows the amino acid sequence of wild type Clostridium difficile 014 TcdA.
The amino acid sequence of wild type Clostridium difficile 015TcdA is shown in SEQ ID NO 135.
136 shows the amino acid sequence of wild type Clostridium difficile 020 TcdA.
137 shows the amino acid sequence of wild type clostridium difficile 023 TcdA.
138 shows the amino acid sequence of wild type Clostridium difficile 027 TcdA.
139 shows the amino acid sequence of wild type Clostridium difficile 029 TcdA.
140 shows the amino acid sequence of wild type C.difficile 046 TcdA.
141 shows the amino acid sequence of wild type Clostridium difficile 014 TcdB.
142 shows the amino acid sequence of wild type Clostridium difficile 015 TcdB.
143 shows the amino acid sequence of wild type Clostridium difficile 020 TcdB.
SEQ ID NO:144 shows the amino acid sequence of wild type Clostridium difficile 023 TcdB.
145 shows the amino acid sequence of wild type Clostridium difficile 027 TcdB.
SEQ ID NO 146 shows the amino acid sequence of wild type Clostridium difficile 029 TcdB.
147 shows the amino acid sequence of wild type Clostridium difficile 046 TcdB.
148 shows the amino acid sequence of wild type Clostridium difficile 001 TcdA.
149 shows the amino acid sequence of wild type Clostridium difficile 002 TcdA.
150 shows the amino acid sequence of wild type Clostridium difficile 003 TcdA.
SEQ ID NO 151 shows the amino acid sequence of wild type Clostridium difficile 004 TcdA.
SEQ ID NO 152 shows the amino acid sequence of wild type Clostridium difficile 070 TcdA.
153 shows the amino acid sequence of wild type Clostridium difficile 075 TcdA.
154 shows the amino acid sequence of wild type Clostridium difficile 077 TcdA.
155 shows the amino acid sequence of wild type Clostridium difficile 081 TcdA.
156 shows the amino acid sequence of wild type clostridium difficile 117 TcdA.
157 shows the amino acid sequence of wild type Clostridium difficile 131 TcdA.
158 shows the amino acid sequence of wild type Clostridium difficile 001 TcdB.
159 shows the amino acid sequence of wild type Clostridium difficile 002 TcdB.
160 shows the amino acid sequence of wild type Clostridium difficile 003 TcdB.
161 shows the amino acid sequence of wild type Clostridium difficile 004 TcdB.
SEQ ID NO 162 shows the amino acid sequence of wild type Clostridium difficile 070 TcdB.
163 shows the amino acid sequence of wild type Clostridium difficile 075 TcdB.
SEQ ID NO 164 shows the amino acid sequence of wild type Clostridium difficile 077 TcdB.
165 shows the amino acid sequence of wild type Clostridium difficile 081 TcdB.
166 shows the amino acid sequence of the wild type Clostridium difficile 117 TcdB.
167 shows the amino acid sequence of wild type Clostridium difficile 131 TcdB.
168 shows the amino acid sequence of wild type Clostridium difficile 053 TcdA.
The amino acid sequence of wild type C.difficile 078TcdA is shown in SEQ ID NO 169.
170 shows the amino acid sequence of wild type Clostridium difficile 087 TcdA.
171 shows the amino acid sequence of wild type Clostridium difficile 095 TcdA.
The amino acid sequence of wild type C.difficile 126TcdA is shown in SEQ ID NO: 172.
SEQ ID NO. 173 shows the amino acid sequence of wild type Clostridium difficile 053 TcdB.
SEQ ID NO:174 shows the amino acid sequence of wild type Clostridium difficile 078 TcdB.
SEQ ID NO 175 shows the amino acid sequence of wild type Clostridium difficile 087 TcdB.
176 shows the amino acid sequence of wild type Clostridium difficile 095 TcdB.
177 shows the amino acid sequence of 126TcdB of wild type C.difficile.
Detailed Description
The present inventors have surprisingly found (among other things) mutant clostridium difficile toxins a and B, and methods thereof. The mutant is characterized in part in that it is immunogenic and exhibits reduced cytotoxicity compared to the respective wild-type form of the toxin. The invention also relates to immunogenic portions thereof, biological equivalents thereof, and isolated polynucleotides comprising nucleic acid sequences encoding any of the foregoing.
The immunogenic compositions described herein unexpectedly demonstrate the ability to elicit novel neutralizing antibodies against clostridium difficile toxins, and which may have the ability to confer active and/or passive protection against clostridium difficile challenge. The novel antibodies are directed against various epitopes of toxin a and toxin B. The inventors have also found that a combination of at least two neutralizing monoclonal antibodies is capable of exhibiting an unexpected synergistic effect in the in vitro neutralization of toxin a and toxin B, respectively.
The compositions can be used to treat, prevent, reduce the risk of, reduce the occurrence, reduce the severity, and/or delay the onset of clostridium difficile infection, Clostridium Difficile Associated Disease (CDAD), syndrome, condition, symptom, and/or complication thereof in a mammal compared to a mammal not administered the compositions of the present invention described herein.
In addition, the present inventors have discovered recombinant spore-forming clostridium difficile cells capable of stably expressing mutant clostridium difficile toxin a and toxin B, and methods for producing the same.
Immunogenic compositions
In one aspect, the invention relates to an immunogenic composition comprising a mutant clostridium difficile toxin. The mutant clostridium difficile toxin comprises an amino acid sequence having at least one mutation in the glucosyltransferase domain and at least one mutation in the cysteine protease domain relative to the corresponding wild-type clostridium difficile toxin.
The term "wild-type" as used herein refers to the form found in nature. For example, a wild-type polypeptide or polynucleotide sequence is a sequence that exists in an organism that can be isolated from a source in nature and that has not been intentionally modified by man. The invention also relates to isolated polynucleotides comprising nucleic acid sequences encoding any of the above polypeptides. In addition, the present invention relates to the use of any of the above compositions in the treatment, prevention, reduction of risk, reduction of severity, reduction of incidence and/or delay of onset of clostridium difficile infection, clostridium difficile-associated disease, syndrome, disease condition, symptom and/or complication in a mammal compared to a mammal not administered the composition, and methods for preparing the compositions.
As used herein, "immunogenic composition" or "immunogen" refers to a composition that elicits an immune response in a mammal to which the composition is administered.
An "immune response" refers to the development of a beneficial humoral (antibody-mediated) and/or cellular (antigen-specific T cell or its secretory product) response against a clostridium difficile toxin in a recipient patient. The immune response may be humoral, cellular, or both.
The immune response may be an active response induced by administration of the immunogenic composition, an immunogen. Alternatively, the immune response may be a passive response induced by administration of antibodies or primed T cells.
The presence of a humoral (antibody-mediated) immune response can be determined, for example, by cell-based assays known in the art, such as neutralizing antibody assays, ELISA, and the like.
Cellular immune responses are typically caused by presentation of polypeptide epitopes associated with class I or class II MHC molecules to activate antigen-specific CD4+ T helper cells and/or CD8+ cytotoxic T cells. The response may also involve activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia, eosinophils or other components of the natural immunity. The presence of a cell-mediated immune response can be determined by proliferation assays (CD4+ T cells) or CTLs (cytotoxic T lymphocytes) known in the art.
In one embodiment, the immunogenic composition is a vaccine composition. As used herein, a "vaccine composition" is a composition that elicits an immune response in a mammal to which the composition is administered. The vaccine composition can protect the immunized mammal against subsequent challenge by an immunizing agent or an immunologically cross-reactive agent. Protection may be complete or partial for reduction of symptoms or infection compared to an unimmunized mammal under the same conditions.
The immunogenic compositions described herein are cross-reactive, meaning having the ability to elicit an effective immune response (e.g., a humoral immune response) against a toxin produced by another strain of clostridium difficile that is different from the strain from which the composition is derived. For example, the immunogenic compositions described herein (e.g., derived from clostridium difficile 630) can elicit cross-reactive antibodies that are capable of binding to toxins produced by various strains of clostridium difficile (e.g., toxins produced by clostridium difficile R20291 and VPI 10463). See, for example, example 37. Cross-reactivity is indicative of the cross-protective potential of bacterial immunogens and vice versa.
The term "cross-protection" as used herein refers to the ability of an immunogenic composition to induce an immune response that prevents or attenuates infection by different bacterial strains or species of the same genus. For example, the immunogenic compositions described herein (e.g., derived from clostridium difficile 630) can induce an effective immune response in a mammal to attenuate clostridium difficile infection by strains other than 630 (e.g., clostridium difficile R20291) in the mammal and/or to attenuate clostridium difficile disease by strains other than 630 (e.g., clostridium difficile R20291) in the mammal.
Exemplary mammals for which the immunogenic composition or immunogen elicits an immune response include any mammal, such as mice, hamsters, primates, and humans. In a preferred embodiment, the immunogenic composition or immunogen elicits an immune response in a human to which the composition is administered.
As described above, toxin A (TcdA) and toxin B (TcdB) are homologous glucosyltransferases which inactivate the small GTPases of the Rho/Rac/Ras family. The effect of TcdA and TcdB on mammalian target cells depends on a multi-step mechanism of receptor-mediated endocytosis, membrane translocation, autoproteolytic processing and the mono-glycosylation of gtpase. Many of these functional activities have been attributed to discrete regions in the primary sequence of the toxin, and imaging of the toxin has been performed to show that these molecules are structurally similar.
The wild-type gene of TcdA has about 8130 nucleotides which encodes an about 308-kDa reduced molecular weight protein with about 2710 amino acids. As used herein, wild-type clostridium difficile TcdA includes clostridium difficile TcdA from any wild-type clostridium difficile strain. Wild type Clostridium difficile TcdA may comprise a wild type Clostridium difficile TcdA amino acid sequence which is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, preferably about 98%, more preferably about 99% or most preferably about 100% identical to SEQ ID NO:1 (full length) when optimally aligned, for example by using the programs GAP or BESTFIT of predetermined GAP weight (GAP weight).
In a preferred embodiment, the wild-type C.difficile TcdA comprises the amino acid sequence shown in SEQ ID NO:1, which describes the wild-type amino acid sequence of the TcdA from C.difficile strain 630 (also disclosed as GenBank accession numbers YP-001087137.1 and/or CAJ 67494.1). Clostridium difficile strain 630 is known in the art as the PCR-ribotype 012 strain. SEQ ID NO 9 describes the wild-type gene for TcdA from Clostridium difficile strain 630, also disclosed as GenBank accession No. NC-009089.1.
Another example of a wild-type C.difficile TcdA comprises the amino acid sequence shown in SEQ ID NO 15, which describes the wild-type amino acid sequence of TcdA from C.difficile strain R20291 (also disclosed as GenBank accession number YP-003217088.1). Clostridium difficile strain R20291 is known in the art as a highly virulent strain and as a PCR-ribotype 027 strain. The amino acid sequence of TcdA from clostridium difficile strain R20291 is about 98% identical to SEQ ID No. 1. 16 describes the wild-type gene from Clostridium difficile strain R20291, also disclosed as GenBank accession No. NC-013316.1.
Another example of a wild-type C.difficile TcdA comprises the amino acid sequence shown in SEQ ID NO 17, which describes the wild-type amino acid sequence of TcdA from C.difficile strain CD196 (also disclosed as GenBank accession number CBA 61156.1). CD196 is a strain from the recent canadian outbreak and is known in the art as the PCR-ribotype 027 strain. The amino acid sequence of the TcdA from clostridium difficile strain CD196 was about 98% identical to SEQ ID No. 1 and about 100% identical to the TcdA from clostridium difficile strain R20291. 18 describes the wild type gene for TcdA from clostridium difficile strain CD196, also disclosed as GenBank accession No. FN 538970.1.
Additional examples of the amino acid sequence of wild-type C.difficile TcdA include SEQ ID NO 19, which describes the wild-type amino acid sequence of TcdA from C.difficile strain VPI10463 (also disclosed as GenBank accession number CAA 63564.1). The amino acid sequence of TcdA from clostridium difficile strain VPI10463 has about 100% (99.8%) identity with SEQ ID No. 1. SEQ ID NO 20 describes the wild type gene for TcdA from Clostridium difficile strain VPI10463, also disclosed as GenBank accession number X92982.1.
Further examples of wild-type clostridium difficile TcdA include TcdA from wild-type clostridium difficile strains, which are available from the centers for disease control and prevention (CDC, Atlanta, GA). The inventors have found that the amino acid sequence of the TcdA from the wild-type Clostridium difficile strain obtainable from CDC is at least about 99.3% -100% identical to amino acid residues 1-821 of SEQ ID NO:1 (TcdA from Clostridium difficile 630) when optimally aligned. See table 1.
The inventors have also found that the amino acid sequence of TcdA from a wild-type c.difficile strain may comprise at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to about 100% identity to SEQ ID NO:1 when optimally aligned (e.g., when optimally aligned over the full-length sequence).
Table 1: percent identity of amino acid residues 1-821 of TcdA from various wild-type Clostridium difficile strains obtained from CDC and amino acid residues 1-821 of SEQ ID NO:1 when optimally aligned
Thus, in one embodiment, a wild-type C.difficile TcdA amino acid sequence comprises a sequence of at least about 500, 600, 700 or 800 contiguous residues that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, preferably about 98%, more preferably about 99% or most preferably about 100% identical to a sequence of the same length between residues 1-900 of SEQ ID NO:1 when optimally aligned, for example by using the programs GAP or BESTFIT of predetermined GAP weights. Examples include the strains described above (e.g., R20291, CD196, etc.) as well as those listed in table 1.
In another embodiment, a wild-type Clostridium difficile TcdA amino acid sequence comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, preferably about 97%, preferably about 98%, more preferably about 99% or most preferably about 100% identical when optimally aligned to any sequence selected from SEQ ID NOS: 87-109. See Table 1-a.
The wild-type gene of TcdB has about 7098 nucleotides which encodes an about 270kDa reduced molecular weight protein of about 2366 amino acids. As used herein, wild-type clostridium difficile TcdB comprises clostridium difficile TcdB from any wild-type clostridium difficile strain. Wild-type clostridium difficile TcdB may comprise a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, preferably about 98%, more preferably about 99% or most preferably about 100% identical to SEQ ID No. 2 when optimally aligned, for example by using the programs GAP or BESTFIT of predetermined GAP weights. In a preferred embodiment, wild-type C.difficile TcdB comprises the amino acid sequence shown in SEQ ID NO:2, which describes the wild-type amino acid sequence of TcdB from C.difficile strain 630 (also disclosed as GenBank accession numbers YP-001087135.1 and/or CAJ 67492). 10 describes the wild-type gene for TcdB from clostridium difficile strain 630, also disclosed as GenBank accession No. NC _ 009089.1.
Another example of a wild-type C.difficile TcdB comprises the amino acid sequence shown in SEQ ID NO:21, which describes the wild-type amino acid sequence of TcdB from C.difficile strain R20291 (also disclosed as GenBank accession numbers YP-003217086.1 and/or CBE 02479.1). The amino acid sequence of TcdB from Clostridium difficile strain R20291 is about 92% identical to SEQ ID NO. 2. SEQ ID NO 22 describes the wild-type gene for TcdB from Clostridium difficile strain R20291, also disclosed as GenBank accession number NC-013316.1.
Another example of a wild-type C.difficile TcdB comprises the amino acid sequence shown in SEQ ID NO:23, which describes the wild-type amino acid sequence of TcdB from C.difficile strain CD196 (also disclosed as GenBank accession numbers YP-003213639.1 and/or CBA 61153.1). SEQ ID NO 24 describes the wild-type gene for TcdB from Clostridium difficile strain CD196, also disclosed as GenBank accession No. NC-013315.1. The amino acid sequence of TcdB from Clostridium difficile strain CD196 is about 92% identical to
Other examples of the amino acid sequence of wild type C.difficile TcdB comprise SEQ ID NO:25, which describes the wild type amino acid sequence of TcdB from C.difficile strain VPI10463 (also disclosed as GenBank accession numbers P18177 and/or CAA 37298). The amino acid sequence of TcdB from Clostridium difficile strain VPI10463 is 100% identical to
Other examples of wild-type C.difficile TcdB include TcdB from wild-type C.difficile strains, which are available from the centers for disease control and prevention (CDC, Atlanta, GA). The inventors have found that the amino acid sequence of TcdB from a wild-type Clostridium difficile strain obtainable from CDC is at least about 96% -100% identical to amino acid residues 1-821 of SEQ ID NO:2 (TcdB from Clostridium difficile 630) when optimally aligned. See table 2.
Table 2: percent identity of amino acid residues 1-821 of TcdB from various wild-type Clostridium difficile strains obtained from CDC and amino acid residues 1-821 of SEQ ID NO. 2 when optimally aligned
Thus, in one embodiment, the wild-type C.difficile TcdB amino acid sequence comprises a sequence of at least about 500, 600, 700, or 800 contiguous residues that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, preferably about 97%, preferably about 98%, more preferably about 99%, or most preferably about 100% identical to a sequence of the same length between residues 1-900 of SEQ ID NO. 2 when optimally aligned, e.g., by using the programs GAP or BESTFIT of predetermined GAP weights. Examples include the strains described above (e.g., R20291, CD196, etc.) as well as those listed in table 2.
In another embodiment, the wild-type C.difficile TcdB amino acid sequence comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, preferably about 97%, preferably about 98%, more preferably about 99% or most preferably about 100% identical when optimally aligned to any sequence selected from the group consisting of SEQ ID NO: 110-133. See table 2-a.
The genes for toxins a and B (tcdA and tcdB) are part of a 19.6-kb locus (pathogenic locus, PaLoc) which contains 3 additional small Open Reading Frames (ORFs) tcdD, tcdE and tcdC and can be considered useful for toxicity. PaLoc is known to be stable and conserved among toxigenic species. It is present at the same chromosomal integration site in all toxigenic strains analyzed so far. The pathogenic locus (PaLoc) is absent in the non-toxigenic strain. Thus, the wild-type clostridium difficile strain described herein is characterized by the presence of a pathogenic locus. Another preferred feature of the wild-type clostridium difficile strain described herein is the production of TcdA and TcdB.
In one embodiment, the wild-type clostridium difficile strain is a strain having a pathogenic locus that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, preferably about 98%, more preferably about 99% or most preferably about 100% identical to the pathogenic locus of clostridium difficile 630 or VPI 10463. The sequence of the total pathogenic locus of Clostridium difficile VPI10463 was registered in the EMBL database as sequence accession number X92982 and is also shown in SEQ ID NO 26. The strain in which PaLoc is identical to the reference strain VPI10463 is preferably referred to as
The glucosyltransferase domain is located at the N-terminus of the toxin. The glucosyltransferase activity of the toxin is associated with the cytotoxic function of the toxin. Without being bound by any mechanism or theory, it is believed that the glucosyltransferase activity in both toxins catalyzes the mono-glycosylation of small GTP-binding proteins in the Rho/Rac/Ras superfamily. Following glycosylation of these GTP-binding proteins, cellular physiology is significantly modified, allowing toxin-infected host cells to lose structural integrity and disruption of important signal transduction pathways. The Asp-Xaa-Asp (dxdd) motif associated with manganese,. Uridine Diphosphate (UDP) and glucose binding is a typical feature of glucosyltransferase domains. Without being bound by mechanism and theory, it is believed that residues important for catalytic activity, such as the DXD motif, do not change directly in TcdB from known "history" strains such as 630 and TcdB from highly toxic strains such as R20291. The DXD motif is located at residues 285-287 (numbering according to SEQ ID NO: 1) and 286-288 (numbering according to SEQ ID NO:2) of the TcdA of wild type C.difficile.
Global alignment algorithms (e.g., sequence analysis programs) are known in the art and can be used to optimally align two or more amino acid toxin sequences to determine whether the toxin comprises a particular identification sequence (e.g., DXD in the glucosyltransferase domain, DHC in the cysteine protease domain, etc., described below). The optimally aligned sequences are compared to the respective reference sequences (e.g., SEQ ID NO:1 for TcdA or SEQ ID NO:2 for TcdB) to determine the presence of the identifying motif. "optimal alignment" refers to an alignment that gives the highest percent identity score. Such alignment can be performed using known sequence analysis programs. In one embodiment, a CLUSTAL alignment (e.g., CLUSTALW) using predetermined parameters identifies a suitable wild-type toxin by comparing the query sequence to a reference sequence. The relative numbering of conserved amino acid residues is based on the residue numbering of reference amino acid sequences to indicate small insertions or deletions (e.g., 5 amino acids or less) in the aligned sequences.
The term "numbering according to …" as used herein refers to the numbering of the residues of a reference sequence when a given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the number or residue position of a given polymer is specified relative to the reference sequence rather than the actual numerical position of the residue in a given amino acid or polynucleotide sequence.
For example, a given amino acid sequence, e.g., of a highly toxic wild-type clostridium difficile strain, can be aligned with a reference sequence (e.g., of a historical wild-type clostridium difficile strain, e.g., 630) by introducing gaps, if necessary, to optimize the match between the two sequences. In these cases, the residue number in a given amino acid or polynucleotide is specified relative to the reference sequence being aligned, although gaps exist. As used herein, "reference sequence" refers to a defined sequence that is used as a basis for sequence comparison.
Unless otherwise indicated, all amino acid positions of TcdA herein refer to the numbering of
The glucosyltransferase domain of TcdA as used herein may start at
The glucosyltransferase domain of TcdB as used herein may start at
Without being bound by theory or mechanism, it is believed that the N-terminus of TcdA and/or TcdB is cleaved by the autoproteolytic process to allow translocation and release of the glucosyltransferase domain into the host cell cytosol, where it can interact with Rac/Ras/Rho gtpase. Wild-type clostridium difficile TcdA was confirmed to be cleaved between L542 and S543. Wild type C.difficile TcdB was shown to be cleaved between L543 and G544.
The cysteine protease domain is associated with the autocatalytic proteolytic activity of the toxin. The cysteine protease domain is located downstream of the glucosyltransferase domain and is characterized by the catalytic triad aspartate, histidine and cysteine (DHC), e.g. D589, H655 and C700 of the wild-type TcdA and D587, H653 and C698 of the wild-type TcdB. Without being bound by mechanism or theory, it is believed that the catalytic triad is conserved between the toxin from a "historical" strain, such as 630, and the TcdB from a highly toxic strain, such as R20291.
The cysteine protease domain of TcdA as used herein may start at exemplary residue 543 and may end at exemplary residues 809, 769, 768, or 767 of wild-type TcdA as in SEQ ID NO: 1. Any minimum residue position between residues 543 and 809 of wild-type TcdA can be combined with the maximum residue position to define the sequence of the cysteine protease domain, as long as the catalytic triad DHC motif region is comprised. For example, in one embodiment, the cysteine protease domain of the TcdA comprises SEQ ID NO:32 having the DHC motif regions located at residues 47, 113 and 158 of SEQ ID NO:32, which correspond to D589, H655 and C700, respectively, of a wild-type TcdA numbered according to SEQ ID NO: 1. 32 in SEQ ID NO is identical to residues 543-809 of
The cysteine protease domain of TcdB as used herein may begin at exemplary residue 544 of wild-type TcdB as set forth in SEQ ID NO. 2 and may end at exemplary residues 801, 767, 755, or 700. Any minimum residue position between residues 544 and 801 of the wild-type TcdB may be combined with the maximum residue position to define the sequence of the cysteine protease domain, as long as the catalytic triad DHC motif region is comprised. For example, in one embodiment, the cysteine protease domain of TcdB comprises SEQ ID NO 33 comprising the DHC motif region located at residues 44, 110 and 115 of SEQ ID NO 33, which corresponds to D587, H653 and C698, respectively, of wild-type TcdB numbered according to
In the present application, the immunogenic composition comprises a mutant clostridium difficile toxin. The term "mutant" as used herein refers to a molecule exhibiting a structure or sequence that differs from the corresponding wild-type structure or sequence, e.g. by having a cross-linking compared to the corresponding wild-type structure and/or by having at least one mutation compared to the corresponding wild-type sequence when optimally aligned, e.g. by using the programs GAP or BESTFIT of a predetermined GAP weight. The term "mutant" as used herein also encompasses molecules that exhibit different functional properties (e.g., lost glucosyltransferase and/or lost cysteine protease activity) than the corresponding wild-type molecule.
A Clostridium difficile toxin from any of the above-described wild-type strains can be used as a source for producing the mutant Clostridium difficile toxin. Preferably, clostridium difficile 630 is the source from which the mutant clostridium difficile toxin is produced.
Mutations may include substitutions, deletions, truncations or modifications of the wild type amino acid residue usually at that position. Preferably, the mutation is a non-conservative amino acid substitution. The invention also encompasses isolated polynucleotides comprising a nucleic acid sequence encoding any of the mutant toxins described herein.
As used herein, a "non-conservative" amino acid substitution refers to a change from one type of amino acid to another according to Table 3 below.
Examples of non-conservative amino acid substitutions include those in which an aspartic acid residue (Asp, D) is replaced by an alanine residue (Ala, A). Other examples include replacing aspartic acid residues (Asp, D) with asparagine residues (Asn, N), and replacing arginine (Arg, R), glutamic acid (Glu, E), lysine (Lys, K), and/or histidine (His, H) residues with alanine residues (Ala, a).
Conservative substitutions refer to amino acid exchanges between the same classes, for example according to table 3.
Mutant toxins of the invention can be prepared by techniques known in the art for making mutations, such as site-directed mutagenesis, mutagenesis using a mutagen (e.g., UV light), and the like. Preferably site-directed mutagenesis is used. Alternatively, a nucleic acid molecule having a target sequence can be synthesized directly. Such chemical synthesis methods are known in the art.
In the present invention, the mutant clostridium difficile toxin comprises at least 1 mutation in the glucosyltransferase domain relative to the corresponding wild-type clostridium difficile toxin. In one embodiment, the glucosyltransferase domain comprises at least 2 mutations. Preferably, the mutation reduces or eliminates the glucosyltransferase activity of the toxin as compared to the glucosyltransferase activity of the corresponding wild-type clostridium difficile toxin.
Exemplary amino acid residues in the glucosyltransferase domain of TcdA that can be mutated comprise at least one of the following or any combination thereof (numbering according to SEQ ID NO:1 compared to wild-type clostridium difficile TcdA): w101, D269, R272, D285, D287, E460, R462, S541 and L542.
Exemplary mutations in the glucosyltransferase domain of TcdA comprise at least one of the following or any combination thereof (as compared to wild-type clostridium difficile TcdA): W101A, D269A, R272A, D285A, D287A, E460A, R462A, S541A and L542G. In another preferred embodiment, the glucosyltransferase domain of TcdA comprises (compared to wild-type clostridium difficile TcdA) D285A and D287A mutations.
Exemplary amino acid residues in the glucosyltransferase domain of TcdB that can be mutated comprise at least one of the following or any combination thereof (numbering according to SEQ ID NO:2 compared to wild type clostridium difficile TcdB): w102, D270, R273, D286, D288, N384, D461, K463, W520 and L543.
Exemplary mutations in the glucosyltransferase domain of TcdB at least one of the following or any combination thereof (compared to wild-type clostridium difficile TcdB): W102A, D270A, D270N, R273A, D286A, D288A, N384A, D461A, D461R, K463A, K463E, W520A, and L543A. In a preferred embodiment, the glucosyltransferase domain of TcdB comprises (as compared to wild type clostridium difficile TcdB) L543A. In another preferred embodiment, the glucosyltransferase domain of TcdB comprises (compared to wild type clostridium difficile TcdB) the D286A and D288A mutations.
Any of the mutations described above may be combined with a mutation in the cysteine protease domain. In the present invention, the mutant clostridium difficile toxin comprises at least 1 mutation in the cysteine protease domain relative to the corresponding wild-type clostridium difficile toxin. Preferably, the mutation reduces or eliminates the cysteine protease activity of the toxin as compared to the cysteine protease activity of the corresponding wild-type clostridium difficile toxin.
Exemplary amino acid residues in the cysteine protease domain of the TcdA that can be mutated comprise at least one of the following or any combination thereof (numbering according to SEQ ID NO:1 compared to the wild-type clostridium difficile TcdA): s543, D589, H655 and C700. Exemplary mutations in the glucosyltransferase domain of TcdA comprise at least one of the following or any combination thereof (as compared to wild-type clostridium difficile TcdA): S543A, D589A, D589N, H655A, C700A. In a preferred embodiment, the cysteine protease domain of TcdA comprises a C700A mutation (compared to the wild-type clostridium difficile TcdA).
Exemplary amino acid residues in the cysteine protease domain of the TcdB that may be mutated comprise at least one of the following or any combination thereof (numbering according to SEQ ID NO:2 as compared to the wild-type Clostridium difficile TcdB): g544, D587, H653 and C698. In a preferred embodiment, the cysteine protease domain of TcdB comprises the C698A mutation (compared to wild-type clostridium difficile TcdB). Other amino acid residues in the cysteine protease domain of TcdB that may be mutated comprise (compared to wild-type TcdB) K600 and/or R751. Exemplary mutations include K600E and/or R751E.
Thus, the mutant clostridium difficile toxins of the present invention have a mutated glucosyltransferase domain relative to the corresponding wild-type clostridium difficile toxin and a mutated cysteine protease domain relative to the corresponding wild-type clostridium difficile toxin.
An exemplary mutant clostridium difficile TcdA comprises a glucosyltransferase domain comprising SEQ ID NO:29 with amino acid substitutions at positions 285 and 287 relative to the corresponding wild-type clostridium difficile toxin a; and a cysteine protease domain comprising SEQ ID NO 32 with an amino acid substitution at position 158 relative to a corresponding wild-type Clostridium difficile toxin A. For example, such a mutant Clostridium difficile TcdA comprises the amino acid sequence shown in
Other examples of mutant Clostridium difficile toxin A comprise the amino acid sequence shown in SEQ ID NO. 7, having mutations with D269A, R272A, D285A, D287A, E460A, R462A, and C700A of SEQ ID NO. 1, wherein the initial methionine is optionally absent. In another embodiment, the mutant Clostridium difficile toxin A comprises the amino acid sequence shown in SEQ ID NO: 83.
Another exemplary mutant TcdA comprises SEQ ID NO:34, wherein the residues at positions 101, 269, 272, 285, 287, 460, 462, 541, 542, 543, 589, 655 and 700 may be any amino acid.
In some embodiments, the mutant clostridium difficile toxin exhibits reduced or eliminated autoproteolytic processing as compared to the corresponding wild-type clostridium difficile toxin. For example, the mutant clostridium difficile TcdA may comprise a mutation at one of the following residues or any combination thereof (compared to the corresponding wild-type clostridium difficile TcdA): s541, L542, and/or S543. Preferably, the mutant clostridium difficile TcdA comprises at least one of the following mutations or any combination thereof (compared to the corresponding wild-type clostridium difficile TcdA): S541A, L542G, and S543A.
Another exemplary mutant clostridium difficile TcdA comprises (compared to the corresponding wild-type clostridium difficile TcdA) S541A, L542, S543 and C700 mutations.
An exemplary mutant clostridium difficile toxin B comprises a glucosyltransferase domain comprising SEQ ID NO:31 having amino acid substitutions at positions 286 and 288 relative to a corresponding wild-type clostridium difficile toxin B, and a cysteine protease domain comprising SEQ ID NO:33 having an amino acid substitution at position 155 relative to a corresponding wild-type clostridium difficile toxin B. For example, such a mutant Clostridium difficile TcdB comprises the amino acid sequence shown in
Further examples of a mutant Clostridium difficile TcdB comprise the amino acid sequence shown in
Another exemplary mutant TcdB comprises SEQ ID NO 35, wherein the residues at positions 101, 269, 272, 285, 287, 460, 462, 541, 542, 543, 589, 655 and 700 may be any amino acid.
As another example, the mutant clostridium difficile TcdB may comprise a mutation at position 543 and/or 544 compared to the corresponding wild-type clostridium difficile TcdB. Preferably, the mutant clostridium difficile TcdB comprises (compared to the corresponding wild-type clostridium difficile TcdB) L543 and/or G544 mutations. More preferably, the mutant clostridium difficile TcdB comprises (as compared to the corresponding wild-type clostridium difficile TcdB) the L543G and/or G544A mutations.
Another exemplary mutant clostridium difficile TcdB comprises (as compared to the corresponding wild-type clostridium difficile TcdB) L543G, G544A and C698 mutations.
In one aspect, the invention relates to an isolated polypeptide having a mutation at any position of amino acid residues 1-1500 numbered according to
In another aspect, the invention relates to an isolated polypeptide having a mutation at any position of amino acid residues 1-1500 numbered according to SEQ ID NO:1 to define an exemplary mutant Clostridium difficile toxin A. For example, in one embodiment, the isolated polypeptide comprises a mutation between amino acid residues 832 and 992 of SEQ ID NO. 1. Exemplary positions of the mutation include positions 972 and 978 numbered according to SEQ ID NO: 1. Preferably, the mutation between residues 832 and 992 is a substitution. In one embodiment, the mutation is a non-conservative substitution, wherein an asp (d) and/or glu (e) amino acid residue is acidified without being replaced by a neutralizing amino acid residue, such as lysine (K), arginine (R), and histidine (H). Exemplary mutations include D972K, D972R, D972H, D978K, D978R, D978H of SEQ ID No. 1 to define mutant clostridium difficile toxin a.
The polypeptides of the invention may comprise an initial methionine residue, in some cases due to host cell mediated processes. Depending on, for example, recombinant production methods and/or fermentation or growth conditions of the host cell, it is known in the art that the N-terminal methionine encoded by the translation initiation codon may be removed from the polypeptide post-translationally in the cell, or the N-terminal methionine may remain in the isolated polypeptide.
Thus, in one aspect, the invention relates to an isolated polypeptide comprising the amino acid sequence shown in SEQ ID NO. 4, wherein the initial methionine (at position 1) is optionally absent. In one embodiment, the initial methionine of SEQ ID NO. 4 is absent. In one aspect, the invention relates to an isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO:84, which is identical to SEQ ID NO:4, but in the absence of the initial methionine.
In another aspect, the isolated polypeptide comprises the amino acid sequence set forth in
In a further aspect, the isolated polypeptide comprises the amino acid sequence set forth in SEQ ID NO 7, wherein the initial methionine (at position 1) is optionally absent. In one embodiment, the invention relates to an isolated polypeptide comprising the amino acid sequence shown as SEQ ID NO 83, which is identical to SEQ ID NO 7, but without the initial methionine. In another aspect, the isolated polypeptide comprises the amino acid sequence set forth in
In one aspect, the invention relates to an immunogenic composition comprising
In another aspect, the invention relates to an immunogenic composition comprising SEQ ID NO 83. In one aspect, the invention relates to an immunogenic composition comprising SEQ ID NO: 84. In one aspect, the invention relates to an immunogenic composition comprising SEQ ID NO 85. In another aspect, the invention relates to an immunogenic composition comprising SEQ ID NO 86.
In addition to generating an immune response in a mammal, the immunogenic compositions described herein have reduced cytotoxicity as compared to a corresponding wild-type clostridium difficile toxin. Preferably, the immunogenic composition is safe for administration in mammals and has a minimum (e.g., about 6-8 log) as compared to the respective wild-type toxin10Reduced) to no cytotoxicity.
The term cytotoxicity as used herein is a term understood in the art and refers to the state of apoptotic cell death and/or wherein one or more of the cells is normally biochemically or biologically abnormally damaged as compared to the same cell under identical conditions but in the absence of a cytotoxic agent. Toxicity can be quantified, for example, by the amount of material required to induce 50% cell death (i.e., the respective EC) in a cell or mammal50Or ED50) Or quantified by other methods known in the art.
Assays indicative of cytotoxicity are known in the art, such as cell rounding assays (see, e.g., Kuehne et al Nature.2010Oct 7; 467(7316): 711-3). The effects of TcdA and TcdB cause cells to round (e.g., lose morphology) and die, and such phenomena can be observed by light microscopy. See, for example, fig. 9.
Other exemplary cytotoxicity assays known in the art include those using [ alpha ], [ beta ], [14C]Glucose-labeled Ras phosphoscreen imaging assay-related glycosylation assays (as described in Busch et al, J Biol chem.1998Jul 31; 273(31): 19566-72) and preferably cytotoxicity assays as described in the examples below, wherein EC is50Can refer to a preferably human diploid fibroblast cell (e.g., IMR90 cell (ATCC CCL-186) as compared to the same cell under the same conditions in the absence of toxinTM) The concentration of immunogenic composition exhibiting a cytopathic effect (CPE) of at least about 50% in the cells of (a). In vitro cytotoxicity assays can also be used to evaluate the presence of a preferred human diploid fibroblast (e.g., IMR90 cell (ATCC CCL-186) as compared to the same cell under the same conditions in the absence of toxinTM) Inhibits at least about 50% of the cytopathic effect induced by the wild-type Clostridium difficile toxin in the cell of (a)(CPE) concentration of the composition. Other exemplary cytotoxicity assays include Doern et al, J Clin microbiol.1992aug; 30(8) 2042-6. Cytotoxicity can also be determined by measuring ATP levels in cells treated with the toxin. For example, luciferase-based substrates such as
In one embodiment, the cytotoxicity of the immunogenic composition is reduced by at least about 1000, 2000, 3000, 4000, 5000-, 6000-, 7000-, 8000-, 9000-, 10000-, 11000-, 12000-, 13000-fold, 14000-fold, 15000-fold or more as compared to the corresponding wild-type clostridium difficile toxin. See, e.g., table 20.
In another embodiment, the cytotoxicity of an immunogenic composition is reduced by at least about 2-log as compared to the corresponding wild-type toxin under the same conditions10More preferably about 3-log10And most preferably about 4-log10Or more. E.g., as measured by standard cytopathic effect (CPE), with EC50Has a value of at least about 10-12EC of mutant Clostridium difficile TcdB compared to the exemplary wild type Clostridium difficile TcdB at g/ml50May be about 10-9g/ml. See, e.g., tables 7A, 7B, 8A and 8B of the examples section below.
In another embodiment, the EC of the cytotoxicity of the mutant clostridium difficile toxin as measured by an in vitro cytotoxicity assay, e.g., as described herein50At least about 50. mu.g/ml, 100. mu.g/ml, 200. mu.g/ml, 300. mu.g/ml, 400. mu.g/ml, 500. mu.g/ml, 600. mu.g/ml, 700. mu.g/ml, 800. mu.g/ml, 900. mu.g/ml, 1000. mu.g/ml or more. Thus, in preferred embodiments, the immunogenic compositions and mutant toxins are biologically safe for administration to a mammal.
Without being bound by mechanism or theory, TcdA having D285 and D287 mutations compared to wild-type TcdA and TcdB having D286 and D288 mutations compared to wild-type TcdB are expected to be deficient in glycosyltransferase activity and therefore deficient in inducing a cytopathic effect. In addition, toxins with mutations in the DHC motif are expected to be deficient in autocatalytic processing and therefore do not have any cytotoxic effect.
However, the inventors have surprisingly found that the exemplary mutant TcdA having SEQ ID NO:4 and the exemplary mutant TcdB having SEQ ID NO:6 unexpectedly exhibit cytotoxicity (albeit significantly reduced compared to wild-type Clostridium difficile 630 toxin) despite exhibiting dysfunctional glucosyltransferase activity and dysfunctional cysteine protease activity. Without being bound by mechanism or theory, it is believed that the mutant toxin is cytotoxic by a novel mechanism. However, the exemplary mutant TcdA having SEQ ID NO. 4 and the exemplary mutant TcdB having SEQ ID NO. 6 were unexpectedly immunogenic. See the examples below.
Although chemical cross-linking of the wild-type toxin may not inactivate the toxin, the present inventors further discovered that chemically cross-linking at least one amino acid of the mutant toxin further reduces the cytotoxicity of the mutant toxin compared to the same mutant toxin lacking the chemical cross-linking and compared to the corresponding wild-type toxin. Preferably, the mutant toxin is purified prior to contact with the chemical cross-linking agent.
Moreover, while chemical cross-linkers may alter useful epitopes, the inventors have surprisingly discovered that genetically modified mutant clostridium difficile toxins having at least one chemically cross-linked amino acid result in immunogenic compositions that elicit a variety of neutralizing antibodies or binding fragments thereof. Thus, the epitopes associated with neutralizing antibody molecules are unexpectedly retained after chemical cross-linking.
Cross-linking (also referred to herein as "chemical deactivation" or "deactivation") is a process of chemically linking two or more molecules by covalent bonds. The terms "crosslinking reagent", "crosslinking agent" and "crosslinking species" refer to molecules that are capable of reacting with and/or chemically linking to specific functional groups (primary amines, sulfhydryls, carboxyls, carbonyls, etc.) on peptides, polypeptides and/or proteins. In one embodiment, the molecule may comprise two or more reactive termini capable of reacting with and/or chemically linking to a specific functional group (primary amine, thiol, carboxyl, carbonyl, etc.) on the peptide, polypeptide, and/or protein. Preferably, the chemical crosslinking agent is water soluble. In another preferred embodiment, the chemical crosslinking agent is a heterobifunctional crosslinking species. In another embodiment, the chemical crosslinking agent is not a difunctional crosslinking species. Chemical crosslinking agents are known in the art.
In a preferred embodiment, the crosslinking agent is a 0-length crosslinking agent. "0 length" refers to a crosslinking agent that mediates or produces direct crosslinking between functional groups of two molecules. For example, in the cross-linking of two polypeptides, a 0-length cross-linking substance can result in the formation of a bridge or cross-link between the carboxyl group of the amino acid side chain of one polypeptide and the amino group of the other polypeptide without the introduction of exogenous substances. The 0-length crosslinker can catalyze, for example, the formation of an ester linkage between the hydroxyl and carboxyl moieties and/or an amide linkage between the carboxyl and primary amino moieties.
Exemplary suitable chemical crosslinkers include formaldehyde; formalin; acetaldehyde; propionaldehyde; water-soluble carbodiimides (RN ═ C ═ NR ') including 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC), 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride, 3- (2-morpholinyl- (4-ethyl) carbodiimide N-methyl-p-toluenesulphonic acid 1-cyclohexyl ester (CMC), N ' -Dicyclohexylcarbodiimide (DCC) and N, N ' -Diisopropylcarbodiimide (DIC) and derivatives thereof, and N-hydroxysuccinimide (NHS), benzaldehyde, and/or UDP-dialdehyde.
Preferably, the crosslinking agent is EDC. When the mutant clostridium difficile toxin polypeptide is chemically modified with EDC (e.g., by contacting the polypeptide with EDC), in one embodiment, the polypeptide includes (a) at least one crosslink between the side chain of an aspartic acid residue of the polypeptide and the side chain of a glutamic acid residue of the polypeptide. In one embodiment, the polypeptide comprises (b) at least one crosslink between a glutamic acid residue side chain of the polypeptide and a lysine residue side chain of the polypeptide. In one embodiment, the polypeptide comprises (C) at least one cross-link between a carboxyl group at the C-terminus of the polypeptide and an amino group at the N-terminus of the polypeptide. In one embodiment, the polypeptide comprises (d) at least one crosslink between a carboxyl group at the C-terminus of the polypeptide and a lysine residue side chain of the polypeptide. In one embodiment, the polypeptide comprises (e) at least one crosslink between an aspartic acid residue side chain of the polypeptide and a lysine residue side chain of a second isolated polypeptide. In one embodiment, the polypeptide comprises (f) at least one crosslink between a glutamic acid residue side chain of the polypeptide and a lysine residue side chain of a second isolated polypeptide. In one embodiment, the polypeptide comprises (g) at least one crosslink between a carboxyl group at the C-terminus of the polypeptide and an amino group at the N-terminus of a second isolated polypeptide. In one embodiment, the polypeptide comprises (h) at least one crosslink between a carboxyl group at the C-terminus of the polypeptide and a lysine residue side chain of a second isolated polypeptide. See, for example, fig. 24 and 25.
"second isolated polypeptide" refers to any isolated polypeptide present during the reaction with EDC. In one embodiment, the second isolated polypeptide is a mutant clostridium difficile toxin polypeptide having the same sequence as the first isolated polypeptide. In another embodiment, the second isolated polypeptide is a mutant clostridium difficile toxin polypeptide having a different sequence than the first isolated polypeptide.
In one embodiment, the polypeptide comprises at least 2 modifications selected from the group consisting of (a) - (d). In exemplary embodiments, the polypeptide comprises (a) at least one crosslink between an aspartic acid residue side chain of the polypeptide and a glutamic acid residue side chain of the polypeptide; and (b) at least one crosslink between a glutamic acid residue side chain of said polypeptide and a lysine residue side chain of said polypeptide. In a further embodiment, the polypeptide comprises at least 3 modifications selected from the group consisting of (a) - (d). In further embodiments, the polypeptide comprises (a), (b), (c), and (d) modifications.
When more than 1 mutant polypeptide is present during chemical modification by EDC, in one embodiment, the resulting composition comprises at least one of any of (a) - (h) modifications. In one embodiment, the composition comprises at least 2 modifications selected from the group consisting of modifications (a) - (h). In further embodiments, the composition comprises at least 3 modifications selected from the group consisting of modifications (a) - (h). In further embodiments, the composition comprises at least 4 modifications selected from the group consisting of modifications (a) - (h). In another embodiment, the composition comprises at least one of each of the (a) - (h) modifications.
In exemplary embodiments, the resulting composition comprises (a) at least one crosslink between an aspartic acid residue side chain of the polypeptide and a glutamic acid residue side chain of the polypeptide; and (b) at least one crosslink between a glutamic acid residue side chain of said polypeptide and a lysine residue side chain of said polypeptide. In one embodiment, the composition further comprises (C) at least one crosslink between a carboxyl group at the C-terminus of the polypeptide and an amino group at the N-terminus of the polypeptide; and (d) at least one crosslink between a carboxyl group at the C-terminus of said polypeptide and a lysine residue side chain of said polypeptide.
In further exemplary embodiments, the resulting composition comprises (e) at least one crosslink between an aspartic acid residue side chain of the polypeptide and a lysine residue side chain of a second isolated polypeptide; (f) at least one crosslink between a glutamic acid residue side chain of the polypeptide and a lysine residue side chain of a second isolated polypeptide; (g) at least one crosslink between a carboxyl group at the C-terminus of the polypeptide and an amino group at the N-terminus of a second isolated polypeptide; and (h) at least one crosslink between a carboxyl group at the C-terminus of the polypeptide and a lysine residue side chain of a second isolated polypeptide.
In further exemplary embodiments, the resulting composition comprises (a) at least one crosslink between an aspartic acid residue side chain of the polypeptide and a glutamic acid residue side chain of the polypeptide; (b) at least one crosslink between a glutamic acid residue side chain of said polypeptide and a lysine residue side chain of said polypeptide; (e) at least one crosslink between an aspartic acid residue side chain of the polypeptide and a lysine residue side chain of a second isolated polypeptide; and (f) at least one crosslink between a glutamic acid residue side chain of the polypeptide and a lysine residue side chain of a second isolated polypeptide.
In a preferred embodiment, the chemical crosslinking agent comprises formaldehyde, more preferably, a reagent comprising formaldehyde in the absence of lysine. Glycine with a primary amine and other suitable compounds can be used as quenchers in the crosslinking reaction. Thus, in another preferred embodiment, the chemical agent comprises formaldehyde and glycine is used.
In another preferred embodiment, the chemical crosslinking agent comprises EDC and NHS. As known in the art, NHS may be included in EDC coupling methods. However, the inventors have surprisingly found that NHS can facilitate further reduction of the cytotoxicity of the mutant clostridium difficile toxin compared to the corresponding wild-type toxin, compared to the genetically mutated toxin and compared to the genetically mutated toxin chemically cross-linked by EDC. See, for example, example 22. Thus, without being bound by mechanism or theory, mutant toxin polypeptides having a beta-alanine moiety attached to the side chain of at least one lysine residue of the polypeptide (e.g., resulting from the reaction of the mutant toxin polypeptide, EDC, and NHS) can facilitate further reduction in cytotoxicity of the mutant toxin as compared to, for example, a clostridium difficile toxin (wild-type or mutant) in which the beta-alanine moiety is not present.
The use of EDC and/or NHS may also include the use of glycine or other suitable compounds with primary amines as quenchers. Any compound having a primary amine may be used as a quencher, such as glycine methyl ester and alanine. In a preferred embodiment, the quencher compound is a non-polymeric hydrophilic primary amine. Examples of non-polymeric hydrophilic primary amines include, for example, amino sugars, amino alcohols, and amino polyols. Specific examples of non-polymeric hydrophilic primary amines include glycine, ethanolamine, glucosamine, amine-functionalized polyethylene glycols, and amine-functionalized ethylene glycol oligomers.
In one aspect, the present invention relates to mutant clostridium difficile toxin polypeptides having at least one amino acid side chain chemically modified with EDC and a non-polymeric hydrophilic primary amine, preferably glycine. The resulting glycine adduct (e.g., from triple mutant toxin treated with EDC, NHS and quenched with glycine) may facilitate reducing cytotoxicity of the mutant toxin as compared to the corresponding wild-type toxin.
In one embodiment, when the mutant clostridium difficile toxin polypeptide is chemically modified with EDC and glycine, the polypeptide comprises at least one of the modifications when the polypeptide is modified with EDC (e.g., at least one of any of the above (a) - (h) modifications) and at least one of the following exemplary modifications: (i) a glycine moiety linked to a carboxyl moiety at the C-terminus of the polypeptide; (j) a glycine moiety attached to the side chain of at least one aspartic acid residue of said polypeptide; and (k) a glycine moiety attached to the side chain of at least one glutamic acid residue of the polypeptide. See, for example, fig. 24 and 25.
In one embodiment, at least one amino acid of the mutant clostridium difficile TcdA is chemically cross-linked and/or at least one amino acid of the mutant clostridium difficile TcdB is chemically cross-linked. In another embodiment, at least one amino acid of
As another example, at least one amino acid may be chemically cross-linked by a reagent comprising NHS. The NHS ester activated cross-linking species can be reacted with a primary amine (e.g., at the N-terminus of each polypeptide chain and/or in the side chain of a lysine residue) to create an amide bond.
In another embodiment, at least one amino acid may be chemically crosslinked by reagents including EDC and NHS. For example, in one embodiment, the invention relates to an isolated polypeptide having an amino acid sequence set forth in SEQ ID No. 4, wherein the methionine residue at
When the mutant clostridium difficile toxin polypeptide is chemically modified (e.g., by contact) with EDC and NHS, in one embodiment, the polypeptide comprises at least one modification of the polypeptide when EDC is modified (e.g., at least one of any of the above (a) - (h) modifications) and (l) a β -alanine moiety attached to the side chain of at least one lysine residue of the polypeptide.
In another aspect, the present invention relates to a mutant clostridium difficile toxin polypeptide, wherein said polypeptide comprises at least one amino acid side chain chemically modified with EDC, NHS and a non-polymeric hydrophilic primary amine, preferably glycine. In one embodiment, the polypeptide comprises at least one modification of the polypeptide when modified with EDC (e.g., at least one of any of the above (a) - (h)), at least one modification of the polypeptide when modified with glycine (e.g., at least one of any of the above (i) - (k)), and (l) a β -alanine moiety attached to the side chain of at least one lysine residue of the polypeptide. See, for example, fig. 24 and 25.
In one aspect, the invention relates to a mutant clostridium difficile toxin polypeptide, wherein the side chain of at least one lysine residue of the polypeptide is linked to a β -alanine moiety. In one embodiment, the side chain of the second lysine residue of the polypeptide is linked to the side chain of an aspartic acid residue and/or to the side chain of a glutamic acid residue. A "second" lysine residue of a polypeptide includes a lysine residue of the polypeptide not linked to a β -alanine moiety. The side chain of aspartic acid and/or the side chain of glutamic acid to which the second lysine residue is attached may be the side chain of aspartic acid and/or the side chain of glutamic acid of the polypeptide to form an intramolecular cross-link, or may be the side chain of aspartic acid and/or the side chain of glutamic acid of the second polypeptide to form an intermolecular cross-link. In another embodiment, the side chain of at least one aspartic acid residue and/or the side chain of at least one glutamic acid residue of the polypeptide is attached to a glycine moiety. The aspartic acid residue and/or glutamic acid residue attached to the glycine moiety is not attached to a lysine residue.
As another example of a chemically cross-linked mutant clostridium difficile toxin polypeptide, at least one amino acid can be chemically cross-linked by an agent comprising formaldehyde. Formaldehyde can react with the N-terminal amino acid residue as well as the side chains of arginine, cysteine, histidine and lysine. Formaldehyde and glycine can form schiff base adducts that can be attached to the N-terminal primary amino group, arginine and tyrosine residues, and to a lesser extent to asparagine, glutamine, histidine and tryptophan residues.
The chemical cross-linking agent is believed to reduce the cytotoxicity of the toxin, e.g., as measured by an in vitro cytotoxicity assay or by animal toxicity, if the treated toxin has less toxicity (e.g., about 100%, 99%, 95%, 90%, 80%, 75%, 60%, 50%, 25%, or 10% less toxicity) than the untreated toxin under the same conditions.
Preferably, the chemical cross-linking agent reduces the cytotoxicity of the mutant Clostridium difficile toxin by at least about 2-log relative to the mutant toxin under the same conditions but in the absence of the chemical cross-linking agent10More preferably about a 3-log10And most preferably about 4-log10Or more. The chemical cross-linking agent preferably reduces cytotoxicity of the mutant toxin by at least about 5-log as compared to the wild-type toxin10About 6-log10About 7-log10About 8-log10Or more.
In another preferred embodiment, chemically inactivated mutant Clostridium difficile toxins exhibit an EC of greater than or at least about 50 μ g/ml, 100 μ g/ml, 200 μ g/ml, 300 μ g/ml, 400 μ g/ml, 500 μ g/ml, 600 μ g/ml, 700 μ g/ml, 800 μ g/ml, 900 μ g/ml, 1000 μ g/ml or greater, e.g., as measured by in vitro cytotoxicity assays such as those described herein50The value is obtained.
The reaction conditions for contacting the mutant toxin with the chemical crosslinking agent are within the experience of one skilled in the art and may vary depending on the agent used. However, the present inventors have surprisingly found optimal reaction conditions for contacting a mutant clostridium difficile toxin polypeptide with a chemical cross-linker while maintaining a functional epitope and reducing the cytotoxicity of the mutant toxin compared to the corresponding wild-type toxin.
Preferably, the conditions are selected to contact the mutant toxin with the cross-linking agent, wherein the mutant toxin has a minimum concentration of about 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0mg/ml to a maximum concentration of about 3.0, 2.5, 2.0, 1.5, or 1.25 mg/ml. Any minimum value can be combined with any maximum value to define a suitable reactive concentration range for the mutant toxin. Most preferably, the reactive concentration of the mutant toxin is about 1.0-1.25 mg/ml.
In one embodiment, the minimum concentration of the reagent used in the reaction is about 1mM, 2mM, 3mM, 4mM, 5mM, 10mM, 15mM, 20mM, 30mM, 40mM, or 50mM, and the maximum concentration is about 100mM, 90mM, 80mM, 70mM, 60mM, or 50 mM. Any minimum value may be combined with any maximum value to define a suitable concentration range for the chemical agent of the reaction.
In preferred embodiments where the reagent comprises formaldehyde, the concentration used is preferably any concentration from about 2mM to 80mM, most preferably about 40 mM. In another preferred embodiment where the reagent comprises EDC, the concentration used is preferably any concentration from about 1.3mM to about 13mM, more preferably about 2mM to 3mM, and most preferably about 2.6 mM.
Exemplary reaction times for contacting the mutant toxin with the chemical crosslinking agent include a minimum of about 0.5, 1, 2, 3, 4, 5, 6, 12, 24, 36, 48, or 60 hours, and a maximum of about 14 days, 12 days, 10 days, 7 days, 5 days, 3 days, 2 days, 1 day, or 12 hours. Any minimum value may be combined with any maximum value to define a suitable range of reaction times.
In a preferred embodiment, the step of contacting the mutant toxin with the chemical cross-linking agent is performed for a time sufficient to allow the cytotoxicity of the mutant clostridium difficile toxin to be compared in a standard in vitro cytotoxicity assay to the same mutant toxin in the absence of the cross-linking agentReduction of EC into suitable human cells, such as IMR-90 cells50Values of at least about 1000. mu.g/ml. More preferably, the reacting step is carried out for a time sufficient to reduce the cytotoxicity of the mutant toxin to an EC suitable for use in human cells50Values of at least about 1000. mu.g/ml are at least 2 times and most preferably at least 3 times or more. In one embodiment, the reaction time is no more than about 168 hours (or 7 days).
For example, in one embodiment where the agent comprises formaldehyde, the mutant toxin is preferably contacted with the agent for about 12 hours, which is demonstrated to be an exemplary time period sufficient to reduce the cytotoxicity of the mutant clostridium difficile toxin in a standard in vitro cytotoxicity assay to an EC in a suitable human cell, such as an IMR-90 cell, as compared to the same mutant toxin in the absence of a cross-linking agent50Values of at least about 1000. mu.g/ml. In a more preferred embodiment, the reaction is carried out for about 48 hours, which is at least about 3 times the sufficient reaction time period. In such embodiments, the reaction time is preferably no more than about 72 hours.
In another embodiment where the agent comprises EDC, the mutant toxin is preferably contacted with the agent for about 0.5 hours, more preferably at least about 1 hour, or most preferably about 2 hours. In such embodiments, the reaction time is preferably no more than about 6 hours.
Exemplary phs at which the mutant toxin is contacted with the chemical crosslinking agent include a minimum of about pH 5.5, 6.0, 6.5, 7.0, or 7.5, and a maximum of about pH 8.5, 8.0, 7.5, 7.0, or 6.5. Any minimum value may be combined with any maximum value to define a suitable range of pH. Preferably, the reaction is carried out at a pH of 6.5 to 7.5, preferably pH 7.0.
Exemplary temperatures at which the mutant toxin is contacted with the chemical cross-linking agent include a minimum of about 2 ℃, 4 ℃, 10 ℃,20 ℃,25 ℃, or 37 ℃ and a maximum of about 40 ℃, 37 ℃, 30 ℃, 27 ℃,25 ℃, or 20 ℃. Any minimum value may be combined with any maximum value to define a suitable range for the reaction temperature. Preferably, the reaction is carried out at about 20 ℃ to 30 ℃, most preferably 25 ℃.
The immunogenic composition may comprise a mutant clostridium difficile toxin (a or B). Thus, the immunogenic composition can be in separate vials (e.g., a separate vial for a composition comprising mutant clostridium difficile toxin a and a separate vial for a composition comprising mutant clostridium difficile toxin B) in a formulation or kit. The immunogenic compositions may be used simultaneously, sequentially or separately.
In another embodiment, the above immunogenic composition may comprise two mutant clostridium difficile toxins (a and B). Any combination of the mutant clostridium difficile toxin a and the mutant clostridium difficile toxin B described can be combined into an immunogenic composition. Thus, the immunogenic composition can be combined in a single vial (e.g., a single vial comprising a composition comprising the mutant clostridium difficile TcdA and a composition comprising the mutant clostridium difficile TcdB). Preferably, the immunogenic composition comprises a mutant clostridium difficile TcdA and a mutant clostridium difficile TcdB.
For example, in one embodiment, the immunogenic composition comprises SEQ ID NO. 4 and SEQ ID NO. 6, wherein at least one amino acid of each of SEQ ID NO. 4 and SEQ ID NO. 6 is chemically cross-linked. In another embodiment, the immunogenic composition comprises a mutant clostridium difficile toxin a comprising SEQ ID No. 4 or SEQ ID No. 7 and a mutant clostridium difficile toxin B comprising SEQ ID No. 6 or SEQ ID No. 8, wherein at least one amino acid of each of said mutant clostridium difficile toxins is chemically cross-linked.
In another embodiment, the immunogenic composition comprises any sequence selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 84 and SEQ ID NO. 83 and any sequence selected from the group consisting of SEQ ID NO. 6, SEQ ID NO. 86 and SEQ ID NO. 85. In another embodiment, the immunogenic composition comprises SEQ ID NO:84 and the immunogenic composition comprises SEQ ID NO: 86. In another embodiment, the immunogenic composition comprises SEQ ID NO 83 and the immunogenic composition comprises SEQ ID NO 85. In another embodiment, the immunogenic composition comprises SEQ ID NO 84, SEQ ID NO 83, SEQ ID NO 86 and SEQ ID NO 85.
It is to be understood that any of the compositions of the present invention, e.g., immunogenic compositions comprising mutant toxin a and/or mutant toxin B, can be used in combination in different proportions or amounts for therapeutic effect. For example, the mutant clostridium difficile TcdA and the mutant clostridium difficile TcdB may be present in the immunogenic composition in a ratio in the range of 0.1:10 to 10:0.1, a: B. In another embodiment, for example, the mutant clostridium difficile TcdB and the mutant clostridium difficile TcdA may be present in the immunogenic composition in a ratio in the range of 0.1:10 to 10:0.1, B: a. In a preferred embodiment, the ratio is such that the composition comprises a greater total amount of mutant TcdB than the total amount of mutant TcdA.
In one aspect, the immunogenic composition is capable of binding a neutralizing antibody or binding fragment thereof. Preferably, the neutralizing antibody or binding fragment thereof is a neutralizing antibody or binding fragment thereof described below. In an exemplary embodiment, the immunogenic composition is capable of binding an antitoxin a antibody or antigen-binding fragment thereof, wherein the antitoxin a antibody or binding fragment thereof comprises a variable light chain having the amino acid sequence of SEQ ID No. 36 and a variable heavy chain having the amino acid sequence of SEQ ID No. 37. For example, the immunogenic composition may comprise a mutant Clostridium difficile TcdA, SEQ ID NO:4 or SEQ ID NO: 7. As another example, the immunogenic composition can comprise SEQ ID NO 84 or SEQ ID NO 83.
In further exemplary embodiments, the immunogenic composition is capable of binding an antitoxin B antibody or antigen-binding fragment thereof, wherein the antitoxin B antibody or binding fragment thereof comprises a variable light chain of B8-26 and a variable heavy chain of B8-26. For example, the immunogenic composition may comprise mutant Clostridium difficile TcdB,
Recombinant cell
In another aspect, the invention relates to a recombinant cell or progeny thereof. In one embodiment, the cell or progeny thereof comprises a polynucleotide encoding a mutant clostridium difficile TcdA and/or a mutant clostridium difficile TcdB.
In another embodiment, the recombinant cell or progeny thereof comprises a nucleic acid sequence encoding a polypeptide having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, preferably about 98%, more preferably about 99% or most preferably about 100% identity to any of SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 7 or SEQ ID NO. 8 when optimally aligned, e.g., by using the programs GAP or BESTFIT of predetermined GAP weights.
In another embodiment, the recombinant cell or progeny thereof comprises a nucleic acid sequence encoding a polypeptide having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, preferably about 98%, more preferably about 99% or most preferably about 100% identity to any of SEQ ID NO 84, SEQ ID NO 86, SEQ ID NO 83 or SEQ ID NO 85 when optimally aligned, e.g., by using the programs GAP or BESTFIT of predetermined GAP weights.
In further embodiments, the recombinant cell or progeny thereof comprises a nucleic acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% preferably about 98%, more preferably about 99% or most preferably about 100% identical to any of SEQ ID NO 11,
The recombinant cell may be derived from any cell that is useful for the recombinant production of a polypeptide of the invention. For example, prokaryotic or eukaryotic cells. Preferably, the recombinant cell is derived from any cell suitable for expression of a heterologous nucleic acid sequence of greater than about 5000, 6000, preferably about 7000, and more preferably about 8000 nucleotides or longer. The prokaryotic host cell may be any gram-negative or gram-positive bacterium. In exemplary embodiments, the prokaryotic host cell lacks endogenous polynucleotides encoding toxins and/or spores.
Gram-negative bacteria include, but are not limited to, Campylobacter (Campylobacter), Escherichia coli (E.coli), Flavobacterium (Flavobacterium), Fusobacterium (Fusobacterium), Helicobacter (Helicobacter), Clavibacterium (Ilyobacter), Neisseria (Neisseria), Pseudomonas (Pseudomonas), Salmonella (Salmonella), and Ureaplasma (Ureapasma). For example, the recombinant cells can be derived from Pseudomonas fluorescens (Pseudomonas fluorescens) cells, as described in paragraphs [0201] - [0230] of U.S. patent application publication 2010013762, which is incorporated herein by reference.
Gram-positive bacteria include, but are not limited to, Bacillus (Bacillus), Clostridium (Clostridium), Enterococcus (Enterococcus), Geobacillus (Geobacillus), Lactobacillus (Lactobacillus), Lactococcus (Lactococcus), marine Bacillus (Oceanobacillus), Staphylococcus (Staphylococcus), Streptococcus (Streptococcus), and Streptomyces (Streptomyces). Preferably, the cell is derived from a clostridium difficile cell.
The inventors identified strains of wild-type clostridium difficile that lack endogenous polynucleotides encoding clostridium difficile toxins. Strains lacking the endogenous toxin a and B genes include the following strains available from the American Type Culture Collection (ATCC): clostridium difficile 1351(ATCC 43593)TM) Clostridium difficile 3232(ATCC BAA-1801)TM) Clostridium difficile 7322(ATCC 43601)TM) Clostridium difficile 5036(ATCC 43603)TM) Clostridium difficile 4811(ATCC 43602)TM) And Clostridium difficile VPI11186(ATCC 700057)TM)。
Thus, in one embodiment, the recombinant clostridium difficile cell is derived from a strain as described herein. Preferably, the recombinant clostridium difficile cell or progeny thereof is derived from a strain selected from the group consisting of: clostridium difficile 1351, clostridium difficile 5036 and clostridium difficile VPI 11186. More preferably, the recombinant Clostridium difficile cell or progeny thereof is derived from a Clostridium difficile VPI11186 cell.
In a preferred embodiment, the sporulation gene of the recombinant clostridium difficile cell or progeny thereof is inactivated. Spores can be infectious, highly resistant, and promote the maintenance of clostridium difficile in an aerobic environment outside the host. Spores can also promote the survival of clostridium difficile in a host during antimicrobial therapy. Thus, clostridium difficile cells lacking the sporulation gene can be used to produce safe immunogenic compositions for administration to mammals. In addition, the use of such cells promotes safety during preparation, for example, the safety of protecting equipment, future products, and personnel.
For targeting disordersExamples of viable sporulation genes include spo0A, spoIIE, sigmaE、σGAnd σK. Preferably, the spo0A gene is inactivated.
Methods for inactivating clostridium difficile sporulation genes are known in the art. For example, sporulation genes can be inactivated by targeted insertion of a selectable marker, such as an antibiotic resistance marker. See, e.g., Heap et al, JMicrobiol methods.2010jan; 80(1) 49-55; heap et al, j.microbiol.methods,2007 Sept; 70(3) 452, 464; and Underwood et al, J bacteriol.2009dec; 191(23):7296-305. See also, for example, Minton et al, WO2007/148091, entitled "DNA Molecules and Methods," pages 33-66 of which are incorporated herein by reference in their entirety, or the corresponding U.S. publication Nos.
Methods for producing mutant clostridium difficile toxins
In one aspect, the invention relates to methods of producing mutant clostridium difficile toxins. In one embodiment, the method comprises culturing any of the recombinant cells described above or progeny thereof under suitable conditions to express the polypeptide.
In another embodiment, the method comprises culturing the recombinant cell or progeny thereof under suitable conditions to express a polynucleotide encoding a mutant clostridium difficile toxin, wherein the cell comprises a polynucleotide encoding a mutant clostridium difficile toxin, and wherein the mutant comprises a glucosyltransferase domain having at least one mutation and a cysteine protease domain having at least one mutation (relative to the corresponding wild-type clostridium difficile toxin). In one embodiment, the cell lacks an endogenous polynucleotide encoding a toxin.
In additional embodiments, the method comprises culturing a recombinant clostridium difficile cell or progeny thereof under suitable conditions to express a polynucleotide encoding a mutant clostridium difficile toxin, wherein the cell comprises a polynucleotide encoding the mutant clostridium difficile toxin and the cell lacks an endogenous polynucleotide encoding the toxin.
In another aspect, the invention relates to a method of producing a mutant clostridium difficile toxin. The method comprises the steps of (a) contacting a clostridium difficile cell with a recombinant escherichia coli cell, wherein the clostridium difficile cell lacks an endogenous polynucleotide encoding a clostridium difficile toxin and the escherichia coli cell comprises a polynucleotide encoding a mutant clostridium difficile toxin; (b) culturing the clostridium difficile cell and the escherichia coli cell under suitable conditions for transferring the polynucleotide from the escherichia coli cell to the clostridium difficile cell; (c) selecting a clostridium difficile cell comprising a polynucleotide encoding a mutant clostridium difficile toxin; (d) culturing the clostridium difficile cell of step (c) under suitable conditions to express the polynucleotide; and (e) isolating the mutant clostridium difficile toxin.
In the methods of the invention, the recombinant E.coli cell comprises a heterologous polynucleotide encoding a mutant Clostridium difficile toxin described herein. The polynucleotide may be DNA or RNA. In an exemplary embodiment, the polynucleotide encoding the mutant clostridium difficile toxin is codon optimized for e. Methods for codon optimizing polynucleotides are well known in the art.
In one embodiment, the polynucleotide comprises a nucleic acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a polynucleotide encoding a mutant clostridium difficile TcdA described herein. Exemplary polynucleotides encoding mutant Clostridium difficile toxin A include SEQ ID NO 11,
In another embodiment, the polynucleotide comprises a nucleic acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a polynucleotide encoding a mutant clostridium difficile TcdB as described herein. Exemplary polynucleotides encoding mutant Clostridium difficile toxin B include SEQ ID NO 13,
In one embodiment, the E.coli cell comprising the heterologous polynucleotide is an E.coli cell that stably harbors (host) the heterologous polynucleotide encoding the mutant Clostridium difficile toxin. Exemplary E.coli cells include cells selected from the group consisting of: MAX
The method of the present invention further comprises the steps of: culturing the clostridium difficile cell and the escherichia coli cell under suitable conditions to transfer the polynucleotide from the escherichia coli cell to the clostridium difficile cell results in a recombinant clostridium difficile cell. In a preferred embodiment, the culture conditions are conditions suitable for transferring the polynucleotide from an E.coli cell (donor cell) into a Clostridium difficile cell (recipient cell) and resulting in genetically stable inheritance.
Most preferably, the culture conditions are suitable for bacterial conjugation, which is known in the art. "conjugation" refers to the specific process of transferring a polynucleotide, wherein unidirectional transfer of the polynucleotide (e.g., a bacterial plasmid) from one bacterial cell (i.e., a "donor") to another (i.e., a "recipient") occurs. The conjugation process involves donor cell-to-recipient cell contact. Preferably, the donor E.coli cells are E.coli CA434 cells.
Exemplary suitable (conjugation) conditions for transferring a polynucleotide from an E.coli cell to a Clostridium difficile cell include growing a Clostridium difficile liquid culture in brain heart extract broth (BHI; Oxoid) or Schaedlers anaerobic broth (SAB; Oxoid). In another embodiment, a solid clostridium difficile culture can be grown on Fresh Blood Agar (FBA) or BHI agar. Preferably, Clostridium difficile grows in an anaerobic environment at 37 ℃ (e.g., 80% N)2，10％CO2And 10% H2[vol/vol]). In one embodiment, the suitable conditions include aerobic growth of E.coli in Luria-Bertani (LB) broth or on LB agar at 37 ℃. For conjugative transfer to C.difficile, exemplary suitable conditions include anaerobic growth of E.coli on FBA. Antibiotics may be included in the liquid or solid media, as is known in the art. Examples of such antibiotics include cycloserine (250. mu.g/ml), cefoxitin (8. mu.g/ml), chloramphenicol (12.5. mu.g/ml), thiamphenicol (15. mu.g/ml) and erythromycin (5. mu.g/ml).
The method of the invention further comprises the step of selecting the resulting recombinant clostridium difficile cell comprising a polynucleotide encoding a mutant clostridium difficile toxin. In exemplary embodiments, the recombinant clostridium difficile cell is a receptor that has been conjugated to a polynucleotide encoding a mutant clostridium difficile toxin from a recombinant escherichia coli cell.
The methods of the invention include the step of culturing the recombinant cell or progeny thereof under conditions suitable for expression of the polynucleotide encoding the mutant clostridium difficile toxin, which results in production of the mutant clostridium difficile toxin. Suitable conditions for recombinant cell expression of the polynucleotide include culture conditions suitable for growth of a C.difficile cell, which are known in the art. For example, suitable conditions may include culturing Clostridium difficile transformants in brain heart extract broth (BHI; Oxoid) or Schaedlers anaerobic broth (SAB; Oxoid). In another embodiment, a solid clostridium difficile culture can be grown on FBA or BHI agar. Preferably, Clostridium difficile grows in an anaerobic environment (e.g., 80% N) at 37 ℃2，10％CO2And 10% H2[vol/vol])。
In one embodiment, the method of the invention comprises the step of isolating the resulting mutant clostridium difficile toxin. Methods for isolating proteins from clostridium difficile are known in the art.
In another embodiment, the method comprises the step of purifying the resulting mutant clostridium difficile toxin. Methods for purifying polypeptides, such as chromatography, are known in the art.
In exemplary embodiments, the method further comprises the step of contacting the isolated mutant clostridium difficile toxin with a chemical cross-linking agent as described above. Preferably, the crosslinking agent comprises formaldehyde, ethyl-3- (3-dimethylaminopropyl) carbodiimide, or a combination of EDC and NHS. Exemplary reaction conditions are described above and in the examples section below.
In another aspect, the invention relates to an immunogenic composition comprising a mutant clostridium difficile toxin described herein produced by any method, preferably by a method described above.
Antibodies
Surprisingly, the immunogenic compositions of the invention described above elicit novel antibodies in vivo, indicating that the immunogenic compositions comprise native structures (e.g., preserved epitopes) that are preserved by the respective wild-type clostridium difficile toxin and that the immunogenic compositions comprise epitopes. Antibodies raised against a toxin of one strain of clostridium difficile may be capable of binding to the corresponding toxin produced by another strain of clostridium difficile. That is, antibodies and binding fragments thereof can be "cross-reactive," which refers to the ability to react with similar antigenic sites on toxins produced by various strains of clostridium difficile. Cross-reactivity also includes the ability of an antibody to react with or bind to an antigen that does not stimulate its production, i.e., a reaction between an antigen and an antibody raised against a different but similar antigen.
In one aspect, the present inventors have surprisingly found monoclonal antibodies having a neutralizing effect on clostridium difficile toxin and methods of making the same. The antibodies of the invention can neutralize clostridium difficile toxin cytotoxicity in vitro, inhibit the binding of clostridium difficile toxin to mammalian cells, and/or can neutralize clostridium difficile toxin enterotoxicity in vivo. The present invention also relates to isolated polynucleotides comprising a nucleotide sequence encoding any of the above polypeptides. In addition, the present invention relates to the use of any of the above compositions in the treatment, prevention, reduction of risk, reduction of severity, reduction of incidence and/or delay of onset of clostridium difficile infection, clostridium difficile-associated disease, syndrome, disease condition, symptom and/or complication in a mammal compared to a mammal not administered the composition, and methods for preparing the compositions.
The inventors have further found that a combination of at least two neutralizing monoclonal antibodies may show an unexpected synergistic effect in neutralizing TcdA or TcdB, respectively. Antitoxin antibodies or binding fragments thereof can be used to inhibit clostridium difficile infection.
An "antibody" is a protein comprising at least one or two heavy (H) chain variable regions (abbreviated herein as VH) and at least one or two light (L) chain variable regions (abbreviated herein as VL). VH and VL can be further subdivided into hypervariable regions, termed "complementarity determining regions" ("CDRs"), interspersed with more conserved regions termed "framework regions" (FRs). The extent of the framework regions and CDRs has been precisely defined (see Kabat, E.A., et al. sequences of Proteins of immunological interest, Fifth Edition, U.S. department of Health and Human Services, NIHPubtilization No.91-3242,1991, and Chothia, C.et al., J.mol.biol.196: 901-. The term "antibody" includes intact immunoglobulins of the IgA, IgG, IgE, IgD, IgM class (and subclasses thereof), wherein the light chains of the immunoglobulin may be of the kappa or lambda type.
The antibody molecule may be full-length (e.g., an IgG1 or IgG4 antibody). Antibodies can be of various isotypes, including IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA1, IgA2, IgD, or IgE. In a preferred embodiment, the antibody is of the IgG isotype, e.g.,
In another embodiment, an antibody molecule includes an "antigen-binding fragment" or "binding fragment," which, as used herein, refers to the portion of an antibody that specifically binds to a toxin of clostridium difficile (e.g., toxin a). Binding fragments are, for example, molecules in which one or more immunoglobulin chains are not full length but specifically bind a toxin.
Examples of binding moieties encompassed within the term "binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) f (ab')2A fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bond in the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) fv fragments, consisting of the VL and VH domains of a single arm of an antibody; (v) dAb fragments (Ward et al, Nature 341:544-546,1989) which consist of a VH domain; and (vi) an isolated Complementarity Determining Region (CDR) having sufficient framework to specifically bind to an antigen binding portion such as a variable region. Binding fragments of light chain variable regions and binding fragments of heavy chain variable regions, such as the two domains of an Fv fragment, VL and VH, can be joined by a synthetic linker that makes them a single protein chain using recombinant methods, in which the VL and VH pair to form monovalent molecules (known as single chain Fv (scFv), see, e.g., Bird et al (1988) Science 242: 423-. Such single chain antibodies are also encompassed within the term "binding fragment" of an antibody. These antibody portions are obtained using techniques known in the art, and the portions are screened for use in the same manner as whole antibodies.
As used herein, an antibody that "specifically binds" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide is an antibody that binds to the particular polypeptide or an epitope on a particular polypeptide but does not substantially bind to any other polypeptide or polypeptide epitope. For example, when referring to a biomolecule (e.g., a protein, a nucleic acid, an antibody, etc.) that "specifically" binds to a target, the biomolecule binds its target molecule in a heterogeneous population of molecules that includes the target, and does not bind other molecules in significant amounts, as measured under specified conditions (e.g., immunoassay conditions in the case of antibodies). Binding between an antibody and its target determines the presence of the target in the heterogeneous population of molecules. For example, "specifically binds" or "specifically binds" means that the antibody or binding fragment thereof binds to the wild-type and/or mutant toxin of clostridium difficile with at least twice the affinity for the non-specific antigen.
In exemplary embodiments, the antibody is a chimeric antibody. Chimeric antibodies can be produced by recombinant DNA techniques known in the art. For example, a gene encoding the Fc constant region of a mouse (or other species) monoclonal antibody molecule may be digested with restriction enzymes to remove the region encoding mouse Fc and replace the equivalent portion of the gene encoding human Fc constant region. Chimeric antibodies can also be produced by recombinant DNA techniques, in which DNA encoding the mouse variable region can be linked to DNA encoding the human constant region.
In further exemplary embodiments, the antibody or binding fragment thereof is humanized by methods known in the art. For example, once a mouse antibody is obtained, the CDRs of the antibody can be replaced with at least a portion of the human CDRs. Humanized antibodies can also be generated by replacing the sequences of the mouse Fv variable region that are not directly involved in antigen binding with equivalent sequences of human Fv variable regions. General methods for generating humanized antibodies are known in the art.
For example, monoclonal antibodies directed against Clostridium difficile TcdA or Clostridium difficile TcdB can also be produced by tagging techniques such as hybridoma technology (see, e.g., Kohler and Milstein,1975, Nature,256: 495-. Briefly, immortalized cell lines are fused to lymphocytes from a mammal immunized with clostridium difficile TcdA, clostridium difficile TcdB, or a mutant clostridium difficile toxin as described herein, and the culture supernatants of the resulting hybridoma cells are screened to identify hybridomas that produce monoclonal antibodies that bind clostridium difficile TcdA or clostridium difficile TcdB. Typically, the immortalized cell lines are derived from lymphocytes of the same mammal. Hybridoma cells producing the monoclonal antibodies of the invention are detected by screening hybridoma culture supernatants for antibodies that bind to clostridium difficile TcdA or clostridium difficile TcdB using an assay such as ELISA. Human hybridomas can be prepared in a similar manner.
Instead of generating antibodies by immunization and selection, the antibodies of the invention can also be identified by screening recombinant combinatorial immunoglobulin libraries with clostridium difficile TcdA, clostridium difficile TcdB, or mutant clostridium difficile toxins as described herein. The recombinant antibody library may be, for example, a scFv library or a Fab library. Furthermore, the antibodies described herein can also be used in competition binding studies to identify additional anti-TcdA or anti-TcdB antibodies and binding fragments thereof. For example, additional anti-TcdA or anti-TcdB antibodies and binding fragments thereof can be identified by screening a human antibody library and identifying molecules in the library that compete with the antibodies described herein in a competitive binding assay.
In addition, antibodies encompassed by the present invention include recombinant antibodies, which can be produced by utilizing phage display methods known in the art. In phage display methods, phage can be used to display antigen binding domains expressed from a pool (repotorene) or antibody library (e.g., human or mouse). Phage expressing an antigen binding domain that binds to an immunogen described herein (e.g., a mutant clostridium difficile toxin) can be selected or identified with an antigen, such as a labeled antigen.
Antibodies and binding fragments thereof, in which specific amino acids have been substituted, deleted or added, are also within the scope of the present invention. Preferably, preferred antibodies have amino acid substitutions in the framework regions to improve binding to the antigen. For example, a selected small number of acceptor framework residues of the immunoglobulin chain may be replaced by the corresponding donor amino acid. Preferred positions for substitution comprise amino acid residues in the vicinity of the CDRs, or which are capable of interacting with the CDRs. Criteria for selecting amino acids from donors are described in U.S. Pat. No.5,585,089 (e.g., columns 12-16). The acceptor framework may be the mature human antibody framework sequence or a consensus sequence thereof.
As used herein, "neutralizing antibody or binding fragment thereof" refers to the respective antibody or binding fragment thereof that binds to a pathogen (e.g., clostridium difficile TcdA or TcdB) and reduces infectivity and/or activity (e.g., reduces cytotoxicity) of the pathogen in a mammal and/or cell culture as compared to the pathogen under the same conditions in the absence of the neutralizing antibody or binding fragment thereof. In one embodiment, the neutralizing antibody or binding fragment thereof is capable of neutralizing at least about 70%, 75%, 80%, 85%, 90%, 95%, 99% or more of the biological activity of a pathogen as compared to the pathogen under the same conditions in the absence of said neutralizing antibody or binding fragment thereof.
The term "antitoxin antibody or binding fragment thereof" as used herein refers to an antibody or binding fragment thereof that binds to a respective clostridium difficile toxin (e.g., clostridium difficile toxin a or toxin B). For example, an antitoxin a antibody or binding fragment thereof to an antibody or binding fragment thereof that binds TcdA.
The antibodies or binding fragments thereof described herein can be produced in any wild-type and/or transgenic mammal, including, for example, mice, humans, rabbits, and goats.
When the above immunogenic composition is one that has been previously administered to a population, e.g., for vaccination, the antibody response generated in an individual can be used to neutralize toxins from the same strain as well as from strains that do not stimulate production of the antibody. See, e.g., example 37, which shows the cross-reactivity of immunogenic compositions between toxins from 630 strain and from various wild-type c.
In one aspect, the invention relates to an antibody or binding fragment thereof specific for clostridium difficile TcdA. Monoclonal antibodies that specifically bind TcdA include a65-33, a60-22, a80-29 and/or preferably A3-25.
In one aspect, the invention relates to antibodies or binding fragments thereof specific for TcdA from any wild-type clostridium difficile strain, such as those described above, e.g. specific for SEQ ID No. 1. In another aspect, the invention relates to antibodies or binding fragments thereof specific for the immunogenic compositions described above. For example, in one embodiment, the antibody or binding fragment thereof is specific for an immunogenic composition comprising SEQ ID NO. 4 or SEQ ID NO. 7. In another embodiment, the antibody or binding fragment thereof is specific for an immunogenic composition comprising SEQ ID NO. 4 or SEQ ID NO. 7, wherein at least one amino acid of SEQ ID NO. 4 or SEQ ID NO. 7 is crosslinked with formaldehyde, EDC, NHS, or a combination of EDC and NHS. In another embodiment, the antibody or binding fragment thereof is specific for an immunogenic composition comprising SEQ ID NO:84 or SEQ ID NO: 83.
Antibodies or binding fragments thereof having variable heavy and variable light chain regions that are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, preferably about 98%, more preferably about 99% or most preferably about 100% identical to the variable heavy and light chain regions of a65-33, a60-22, a80-29 and/or preferably A3-25 may also bind TcdA.
In one embodiment, the antibody or antigen-binding fragment thereof comprises a variable heavy chain region comprising an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the variable heavy chain region amino acid sequence of A3-25 as set forth in SEQ ID NO: 37.
In another embodiment, the antibody or antigen-binding fragment thereof comprises a variable light chain region comprising an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the variable light chain region amino acid sequence of A3-25 as set forth in SEQ ID NO: 36.
In another aspect, an antibody or antigen-binding fragment thereof comprises a variable heavy chain region comprising an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the variable heavy chain region amino acid sequence set forth in SEQ ID NO. 37; and comprises a variable light chain region comprising an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the variable light chain region amino acid sequence set forth in SEQ ID NO: 36.
In another embodiment, a Complementarity Determining Region (CDR) antibody or binding fragment thereof having a variable heavy chain and/or variable light chain of A65-33, A60-22, A80-29, and/or preferably A3-25 can bind TcdA. The CDRs of the variable heavy chain region of A3-25 are shown in Table 4 below.
The CDRs of the variable light chain region of A3-25 are shown in Table 5 below.
In one embodiment, the antibody or binding fragment thereof comprises the amino acid sequences of the heavy chain Complementarity Determining Regions (CDRs) shown as SEQ ID NOs 41(CDR H1), 42(CDR H2), and 43(CDR H3); and/or comprises the amino acid sequences of the light chain CDRs shown in SEQ ID NOs: 38(CDRL1), 39(CDR L2) and 40(CDR L3).
In an exemplary embodiment, an antibody or binding fragment thereof specific for Clostridium difficile toxin A specifically binds to an epitope in an N-terminal region of TcdA, e.g., an epitope between amino acids 1-1256 of TcdA numbered according to SEQ ID NO: 1.
In a preferred embodiment, the antibody or binding fragment thereof specific for Clostridium difficile toxin A specifically binds to an epitope in the C-terminal region of toxin A, e.g.between amino acids 1832-2710 of the TcdA numbered according to SEQ ID NO: 1. Examples include A3-25, A65-33, A60-22, A80-29.
In another embodiment, the antibody or binding fragment thereof specific for Clostridium difficile toxin A specifically binds to an epitope in the "translocation" region of Clostridium difficile toxin A, e.g. an epitope preferably comprising residue 956-1128 of the TcdA numbered according to SEQ ID NO:1, e.g. an epitope between amino acids 659-1832 of the TcdA numbered according to SEQ ID NO: 1.
In another aspect, the invention relates to an antibody or binding fragment thereof specific for clostridium difficile TcdB. For example, the antibody or binding fragment thereof may be specific for TcdB from any wild-type Clostridium difficile strain, such as those described above, e.g., specific for SEQ ID NO. 2. In another aspect, the invention relates to antibodies or binding fragments thereof specific for the above immunogenic compositions. For example, in one embodiment, the antibody or binding fragment thereof is specific for an immunogenic composition comprising
In another embodiment, the antibody or binding fragment thereof is specific for an immunogenic composition comprising
Monoclonal antibodies that specifically bind TcdB include antibodies produced by the B2-31, B5-40, B70-2, B6-30, B9-30, B59-3, B60-2, B56-6, and/or preferably B8-26 clones described herein.
Antibodies or binding fragments thereof that can also bind TcdB include antibodies or binding fragments thereof that have variable heavy and variable light chain regions that are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, preferably about 98%, more preferably about 99% or most preferably about 100% identical to the variable heavy and light chain regions of B2-31, B5-40, B70-2, B6-30, B9-30, B59-3, B60-2, B56-6, preferably B8-26, B59-3 and/or B9-30.
In one embodiment, the antibody or antigen-binding fragment thereof comprises a variable heavy chain region comprising an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the variable heavy chain region amino acid sequence of A3-25 as set forth in SEQ ID NO. 49.
In one embodiment, the antibody or antigen-binding fragment thereof comprises a variable heavy chain region comprising an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the variable heavy chain region amino acid sequence of A3-25 as set forth in SEQ ID NO: 60.
In one embodiment, the antibody or antigen-binding fragment thereof comprises a variable heavy chain region comprising an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the variable heavy chain region amino acid sequence of A3-25 as set forth in SEQ ID NO: 71.
In another embodiment, the antibody or antigen-binding fragment thereof comprises a variable light chain region comprising an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the variable light chain region amino acid sequence of A3-25 as set forth in SEQ ID NO: 55.
In another embodiment, the antibody or antigen-binding fragment thereof comprises a variable light chain region comprising an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the variable light chain region amino acid sequence of A3-25 as set forth in SEQ ID NO: 66.
In another embodiment, the antibody or antigen-binding fragment thereof comprises a variable light chain region comprising an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the variable light chain region amino acid sequence of A3-25 as set forth in SEQ ID NO. 77.
The amino acid sequence of the variable heavy chain of the neutralizing antibody (B8-26mAb) for Clostridium difficile TcdB is shown in SEQ ID NO: 49. See Table 25-a.
The amino acid sequence of the variable light chain of the neutralizing antibody (B8-26mAb) of Clostridium difficile TcdB is shown in SEQ ID NO: 55. See Table 25-b.
In one embodiment, the antibody or binding fragment thereof comprises the amino acid sequences of the heavy chain CDRs represented by SEQ ID NOs 51(CDR H1),52(CDR H2) and 53(CDR H3) and/or comprises the amino acid sequences of the light chain CDRs represented by SEQ ID NOs 57(CDR L1),58(CDRL2) and 59(CDR L3).
The amino acid sequence of the variable heavy chain of the neutralizing antibody (B59-3mAb) of Clostridium difficile TcdB is shown in SEQ ID NO: 60. See Table 26-a.
The amino acid sequence of the variable light chain of the neutralizing antibody (B59-3mAb) of Clostridium difficile TcdB is shown in SEQ ID NO: 66. See Table 26-b.
In one embodiment, the antibody or binding fragment thereof comprises the amino acid sequences of the heavy chain CDRs represented by SEQ ID NOs 62(CDR H1), 63(CDR H2) and 64(CDR H3) and/or comprises the amino acid sequences of the light chain CDRs represented by SEQ ID NOs 68(CDR L1), 69(CDRL2) and 70(CDR L3).
The amino acid sequence of the variable heavy chain of the neutralizing antibody (B9-30mAb) of Clostridium difficile TcdB is shown in SEQ ID NO: 71. See Table 27-a.
The amino acid sequence of the variable light chain of the neutralizing antibody (B9-30mAb) of Clostridium difficile TcdB is shown in SEQ ID NO: 77. See Table 27-b.
In one embodiment, the antibody or binding fragment thereof comprises the amino acid sequences of the heavy chain CDRs represented by SEQ ID NOs 73(CDR H1), 74(CDR H2) and 75(CDR H3) and/or comprises the amino acid sequences of the light chain CDRs represented by SEQ ID NOs 79(CDR L1), 80(CDRL2) and 81(CDR L3).
In one aspect, the invention relates to an antibody or binding fragment thereof that is specific for wild type Clostridium difficile TcdB from any Clostridium difficile strain, such as those described above, e.g., specific for SEQ ID NO. 2. In another aspect, the invention relates to antibodies or binding fragments thereof specific for the immunogenic compositions described above. For example, in one embodiment, the antibody or binding fragment thereof is specific for an immunogenic composition comprising
Antibodies or binding fragments thereof having variable heavy and variable light chain regions that are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, preferably about 98%, more preferably about 99% or most preferably about 100% identical to the variable heavy and light chain regions of B2-31, B5-40, B70-2, B6-30, B9-30, B59-3, B60-2, B56-6 and/or preferably B8-26 can also bind TcdB.
In one embodiment, the antibody or antigen-binding fragment thereof comprises a variable heavy chain region comprising an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the variable heavy chain region amino acid sequence of B8-26 (SEQ ID NO: 49).
In another embodiment, the antibody or antigen-binding fragment thereof comprises a variable light chain region comprising an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the variable light chain region amino acid sequence of B8-26 (SEQ ID NO: 55).
In another aspect, the antibody or antigen-binding fragment thereof comprises a variable heavy chain region comprising an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the variable heavy chain region amino acid sequence of B8-26 (SEQ ID NO: 49); and comprises a variable light chain region comprising an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the variable light chain region amino acid sequence of B8-26 (SEQ ID NO: 55).
In another embodiment, an antibody or binding fragment thereof having the CDRs of the variable heavy and/or variable light chains of B2-31, B5-40, B70-2, B6-30, B9-30, B59-3, B60-2, B56-6, and/or preferably B8-26 may also bind TcdB.
In one embodiment, the antibody or binding fragment thereof comprises the amino acid sequence of the heavy chain Complementarity Determining Regions (CDRs) of B8-26, and/or comprises the amino acid sequence of the light chain CDRs of B8-26.
In a preferred embodiment, the antibody or binding fragment thereof specific for Clostridium difficile toxin B specifically binds to an epitope in a region of the N-terminal of toxin B, e.g., between amino acids 1-1256 of TcdB numbered according to SEQ ID NO: 2. Examples include B2-31, B5-40, B8-26, B70-2, B6-30 and B9-30.
In an exemplary embodiment, the antibody or binding fragment thereof specific for Clostridium difficile toxin B specifically binds to an epitope in the C-terminal region of toxin B, e.g., an epitope between amino acids 1832-2710 of TcdB numbered according to SEQ ID NO: 2.
In another embodiment, the antibody or binding fragment thereof specific for C.difficile toxin B specifically binds to an epitope in the "translocation" region of C.difficile toxin B, e.g., an epitope preferably comprising residues 956-1838 of TcdB, numbered according to SEQ ID NO:2, e.g., an epitope between amino acids 659-1832 of TcdB. Examples include B59-3, B60-2 and B56-6.
Combination of antibodies
The anti-toxin antibody or binding fragment thereof can be administered in combination with other anti-clostridium difficile toxin antibodies (e.g., other monoclonal antibodies, polyclonal gamma-globulin) or antigen binding fragments thereof. Combinations that may be used include an antitoxin a antibody or binding fragment thereof and an antitoxin B antibody or antigen binding fragment thereof.
In another embodiment, the combination comprises an antitoxin a antibody or binding fragment thereof and another antitoxin a antibody or antigen binding fragment thereof. Preferably, the combination comprises a neutralizing antitoxin a monoclonal antibody or binding fragment thereof and another neutralizing antitoxin a monoclonal antibody or binding fragment thereof. Surprisingly, the inventors have found a synergistic effect of such a combination in the neutralization of toxin a cytotoxicity. For example, the combination includes a combination of at least two neutralizing antitoxin A monoclonal antibodies A3-25; a65-33; a60-22; and A80-29. More preferably, the combination comprises an A3-25 antibody and at least one neutralizing antitoxin A monoclonal antibody selected from the group consisting of A65-33; a60-22; and A80-29. Most preferably, the combination includes all four antibodies A3-25; a65-33; a60-22; and A80-29.
In additional embodiments, the combination comprises an antitoxin B antibody or binding fragment thereof and another antitoxin B antibody or antigen binding fragment thereof. Preferably, the combination comprises a neutralizing antitoxin B monoclonal antibody or binding fragment thereof and another neutralizing antitoxin B monoclonal antibody or antigen binding fragment thereof. Surprisingly, the inventors have found a synergistic effect of such a combination in the neutralization of toxin B cytotoxicity. More preferably, the combination comprises a combination of at least two neutralizing antitoxin B monoclonal antibodies B8-26; b9-30 and B59-3. Most preferably, the combination includes all three antibodies B8-26; b9-30 and B59-3.
In another embodiment, the combination comprises an antitoxin B antibody or binding fragment thereof and another antitoxin B antibody or antigen binding fragment thereof. As previously described, the present inventors have discovered that the combination of at least two neutralizing monoclonal antibodies can exhibit unexpected synergistic effects in the respective neutralization of toxin a and toxin B.
In another embodiment, the agents of the invention may be formulated as a mixture, or chemically or genetically linked using techniques known in the art, thereby resulting in covalently linked antibodies (or covalently linked antibody fragments) with the binding properties of both antitoxin a and antitoxin B. The formulation of the combination may be directed by determining one or more parameters of the substance, either alone or in combination with another substance, such as affinity, avidity, or biological potency.
Such combination therapy is preferably additive and/or synergistic in its therapeutic activity, e.g., in the inhibition, prevention (e.g., prevention of relapse), and/or treatment of a clostridium difficile-associated disease or condition. Administration of such combination therapy can reduce the dose of therapeutic substance (e.g., antibody or antibody fragment mixture, or cross-linked or genetically fused bispecific antibody or antibody fragment) needed to achieve the desired effect.
It is understood that any of the compositions of the invention, e.g., antitoxin a and/or antitoxin B antibodies or antigen binding fragments thereof, may be combined in different ratios or amounts for therapeutic effect. For example, antitoxin a and antitoxin B antibodies, or binding fragments thereof, respectively, may be present in the composition in a ratio ranging from 0.1:10 to 10:0.1(a: B). In another embodiment, the antitoxin a and antitoxin B antibodies, or binding fragments thereof, respectively, may be present in the composition in a ratio ranging from 0.1:10 to 10:0.1(B: a).
In another aspect, the invention relates to a method of generating neutralizing antibodies against clostridium difficile TcdA. The method comprises administering to a mammal an immunogenic composition as described above and recovering the antibodies from the mammal. In a preferred embodiment, the immunogenic composition comprises a mutant clostridium difficile TcdA having SEQ ID No. 4, wherein at least one amino acid of said mutant clostridium difficile TcdA is chemically cross-linked, preferably methyl or 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide. Exemplary neutralizing antibodies to TcdA that can be generated include a 65-33; a60-22; a80-29 and/or A3-25.
In another aspect, the invention relates to a method of generating neutralizing antibodies against the clostridium difficile TcdB. The method comprises administering to a mammal an immunogenic composition as described above and recovering the antibodies from the mammal. In a preferred embodiment, the immunogenic composition comprises a mutant Clostridium difficile TcdB having
Preparation
The compositions of the invention (e.g., compositions comprising the mutant clostridium difficile toxins, immunogenic compositions, antibodies, and/or antibody binding fragments thereof described herein) can take a variety of forms. Such forms include, for example, semi-solid and solid dosage forms, suppositories, liquid forms such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes, and/or dried forms such as, for example, lyophilized powder forms, lyophilized forms, spray dried forms, and/or foam-dried forms (foam-dried forms). For suppositories, binders and carriers include, for example, polyolefin glycols or triglycerides; such suppositories may be formed from mixtures containing the compositions of the present invention. In exemplary embodiments, the compositions are in the form of solutions or suspensions in liquid carriers suitable for injection. In further exemplary embodiments, the composition is emulsified or encapsulated in liposomes or microparticles, such as polylactic acid compounds, polyglycolide, or copolymers.
In a preferred embodiment, the composition is lyophilized and reconstituted immediately prior to use.
In one aspect, the invention relates to a pharmaceutical composition comprising any of the compositions described herein (such as, for example, a composition comprising a mutant clostridium difficile toxin, an immunogenic composition, an antibody and/or an antibody binding fragment thereof, described herein) formulated with a pharmaceutically acceptable carrier. "pharmaceutically acceptable carrier" includes any solvent, dispersion medium, stabilizer, diluent, and/or buffer that is physiologically suitable.
Exemplary stabilizers include carbohydrates, such as sorbitol, mannitol, starch, dextran, sucrose, trehalose, lactose, and/or glucose; inert proteins, such as albumin and/or casein; and/or other large, slowly metabolized macromolecules such as polysaccharides, e.g., chitosan, polylactic acid, polyglycolic acid, and co-polymers (e.g., latex functionalized SEPHAROSE)TMAgarose, cellulose, etc.), amino acids, polyamines, amino acid copolymers, and lipid aggregates (e.g., oil droplets or liposomes). In addition, these carriers can be used as immunostimulating substances (i.e., adjuvants).
Preferably, the composition comprises trehalose. Preferred amounts (% by weight) of trehalose are from a minimum of about 1%, 2%, 3%, or 4% to a maximum of about 10%, 9%, 8%, 7%, 6%, or 5%. Any minimum value may be combined with any maximum value to define a suitable range. In one embodiment, the composition comprises about 3% to 6% trehalose, most preferably 4.5% trehalose, for example, in a dose of every 0.5 mL.
Examples of suitable diluents include distilled water, saline, physiological phosphate buffered saline, glycerol, alcohols (e.g., ethanol), ringer's solution, dextrose solution, hanks' balanced salt solution, and/or lyophilized excipients.
Exemplary buffering agents include phosphate salts (e.g., calcium phosphate, sodium phosphate); acetates (such as sodium acetate); succinate salts (such as sodium succinate); glycine; (ii) histidine; carbonate, Tris (Tris hydroxymethyl aminomethane), and/or bicarbonate (e.g., ammonium bicarbonate) buffers. Preferably, the composition comprises Tris buffer. Preferred amounts of Tris buffer include from a minimum of about 1mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM to a maximum of about 100mM, 50mM, 20mM, 19mM, 18mM, 17mM, 16mM, 15mM, 14mM, 13mM, 12mM, or 11 mM. Any minimum value may be combined with any maximum value to define a suitable range. In one embodiment, the composition comprises between about 8mM and 12mM Tris buffer, most preferably 10mM Tris buffer, for example, in a dose of every 0.5 mL.
In another preferred embodiment, the composition comprises histidine buffer. Preferred amounts of histidine buffer include from a minimum of about 1mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM to a maximum of about 100mM, 50mM, 20mM, 19mM, 18mM, 17mM, 16mM, 15mM, 14mM, 13mM, 12mM, or 11 mM. Any minimum value may be combined with any maximum value to define a suitable range. In one embodiment, the composition comprises about 8mM to 12mM histidine buffer, most preferably 10mM histidine buffer, for example, in a dose of every 0.5 mL.
In a further preferred embodiment, the composition comprises a phosphate buffer. Preferred amounts of phosphate buffer include from a minimum of about 1mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM to a maximum of about 100mM, 50mM, 20mM, 19mM, 18mM, 17mM, 16mM, 15mM, 14mM, 13mM, 12mM, or 11 mM. Any minimum value may be combined with any maximum value to define a suitable range. In one embodiment, the composition comprises about 8mM to 12mM phosphate buffer, most preferably 10mM phosphate buffer, for example, in a dose of every 0.5 mL.
The pH of the buffer will typically be selected to stabilize the active material selected, and can be determined by one skilled in the art by known methods. Preferably, the pH of the buffer will be within the physiological pH range. Thus, a preferred pH range is from about 3 to about 8; more preferably, from about 6.0 to about 8.0; more preferably, from about 6.5 to about 7.5; and most preferably, from about 7.0 to about 7.2.
In some embodiments, the pharmaceutical composition may include a surfactant. Any surfactant, whether amphoteric, nonionic, cationic or anionic, is suitable. Exemplary surfactants include polyoxyethylene sorbitan esters surfactants(for example,) Such as Polysorbate (Polysorbate)20 and/or Polysorbate 80; polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethylene glycol monododecyl ether (Brij 30); triton X100, or t-octylphenoxypolyethoxyethanol; and sorbitan esters (commonly known as SPAN), such as sorbitan trioleate (SPAN 85) and sorbitan monolaurate, and combinations thereof. Preferred surfactants include polysorbate 80 (polyoxyethylene sorbitan monooleate).
Preferred amounts (% by weight) of polysorbate 80 include from a minimum of about 0.001%, 0.005%, or 0.01%, to a maximum of about 0.010%, 0.015%, 0.025%, or 1.0%. Any minimum value may be combined with any maximum value to define a suitable range. In one embodiment, the composition comprises about 0.005% to 0.015% polysorbate 80, most preferably 0.01% polysorbate 80.
In an exemplary embodiment, the immunogenic composition includes trehalose and phospate 80. In further exemplary embodiments, the immunogenic composition comprises Tris buffer and polysorbate 80. In further exemplary embodiments, the immunogenic composition comprises histidine buffer and polysorbate 80. In further exemplary embodiments, the immunogenic composition comprises phosphate buffer and polysorbate 80.
In an exemplary embodiment, the immunogenic composition comprises trehalose, Tris buffer and polysorbate 80. In further exemplary embodiments, the immunogenic composition comprises trehalose, histidine buffer and polysorbate 80. In further exemplary embodiments, the immunogenic composition comprises trehalose, phosphate buffer and polysorbate 80.
The compositions described herein may also include ingredients of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, and/or mineral oil. Examples include glycols such as propylene glycol or polyethylene glycol.
In some embodiments, the pharmaceutical composition further comprises formaldehyde. For example, in a preferred embodiment, a pharmaceutical composition further comprising formaldehyde has an immunogenic composition, wherein the mutant clostridium difficile toxin of the immunogenic composition has been contacted with a chemical cross-linker comprising formaldehyde. The amount of formaldehyde present in The pharmaceutical composition of The amount of formaldehyde present in can vary from a minimum of about 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.010%, 0.013%, or 0.015%, to a maximum of about 0.020%, 0.019%, 0.018%, 0.017% 0.016%, 0.015%, 0.014%, 0.013%, 0.012% 0.011%, or 0.010%. Any minimum value may be combined with any maximum value to define a suitable range. In one embodiment, the pharmaceutical composition comprises about 0.010% formaldehyde.
In some further embodiments, the pharmaceutical compositions described herein do not include formaldehyde. For example, in a preferred embodiment, the pharmaceutical composition that does not include formaldehyde has an immunogenic composition in which at least one amino acid of the mutant clostridium difficile toxin is chemically cross-linked with a substance that includes EDC. More preferably, in such embodiments, the mutant clostridium difficile toxin has not been contacted with a chemical crosslinker comprising formaldehyde. As another exemplary embodiment, the pharmaceutical composition in lyophilized form does not include formaldehyde.
In another embodiment, the compositions described herein may include an adjuvant as described below. Preferred adjuvants boost the intrinsic immune response to an immunogen without causing a conformational change in the immunogen that can affect the qualitative form of the immune response.
Exemplary adjuvants include 3 De-O-acylated monophosphoryl lipid A (MPL)TM) (see GB 2220211 (GSK)); aluminum hydroxide gels such as AlhydrogelTM(Brenntag Biosector, Denmark); aluminium salts (e.g. aluminium hydroxide, aluminium phosphate, aluminium sulphate), which may be used in the presence or absence of an immunostimulant such as MPL or 3-DMP, QS-21,polymeric or monomeric amino acids such as polyglutamic acid or polylysine.
Further exemplary adjuvants are immunostimulatory oligonucleotides such as CpG oligonucleotides (see, e.g., WO1998/040100, WO2010/067262), or saponins and immunostimulatory oligonucleotides such as CpG oligonucleotides (see, e.g., WO 00/062800). In a preferred embodiment, the adjuvant is a CpG oligonucleotide, most preferably a CpG oligodeoxynucleotide (CpG odn). Preferred CpG ODN are B classes that preferentially activate B cells. For the purposes of the present invention, CpG ODN have the amino acid sequence 5 'T C G T G T C G T3' (SEQ ID NO:48) wherein. The CpG ODN of this sequence is known as CpG 2455, which is described in WO 2010/067262. In a preferred embodiment, CpG 2455 is used with an aluminium hydroxide salt such as aluminium gel (Alhydrogel).
Another exemplary class of adjuvants includes saponin adjuvants, such as StimulonTM(QS-21, which is a triterpene glycoside or saponin, Aquila, Framingham, Mass.) or particles produced therefrom such as ISCOMs (immune stimulating complexes) and
Other exemplary adjuvants include RC-529, GM-CSF, and Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA).
Another class of exemplary adjuvants are glycolipid analogs including N-glycosylamides, N-glycosylureas, and N-glycosylcarbamates, each of which is substituted with an amino acid in the sugar residue.
Optionally, the pharmaceutical composition comprises two or more different adjuvants. Preferred adjuvant combinations include, for example, any adjuvant combination comprising at least two adjuvants selected from the group consisting of alum, MPL, QS-21, ISCOMATRIX, CpG, and alumina gel. Exemplary adjuvant combinations include combinations of CpG and alumina gel.
Alternatively, in one embodiment, the composition is administered to the mammal in the absence of an adjuvant.
The compositions described herein may be administered by any route of administration, such as, for example, parenteral, topical, intravenous, mucosal, oral, subcutaneous, intraarterial, intracranial, intradural, intraperitoneal, intranasal, intramuscular, intradermal, infusion, rectal, and/or transdermal routes for prophylactic and/or therapeutic applications. In a preferred embodiment, the route of administration of the composition is parenteral, more preferably intramuscular. Typical intramuscular administration is performed at the arm or leg muscles.
The compositions described herein can be administered in combination with other therapies (at least partially effective in the prevention and/or treatment of clostridium difficile infection). For example, the compositions of the present invention may be administered prior to, concurrently with, or subsequent to the treatment with a biological agent; probiotic treatment; implanting the anus; immunotherapy (e.g., intravenous immunoglobulin); and/or accepted standard of care for Clostridium Difficile Associated Disease (CDAD) antibiotic treatment, such as metronidazole and/or vancomycin.
The compositions of the invention involving toxin a and toxin B may be administered to a mammal in any combination. For example, an immunogenic composition comprising a mutant clostridium difficile TcdA may be administered before, simultaneously with, or after an immunogenic composition comprising a mutant clostridium difficile TcdB. In turn, the immunogenic composition comprising the mutant clostridium difficile TcdB may be administered before, simultaneously with, or after the immunogenic composition comprising the mutant clostridium difficile TcdA.
In another embodiment, the immunogenic composition comprising an anti-toxin a antibody or binding fragment thereof can be administered prior to, simultaneously with, or subsequent to the immunogenic composition comprising an anti-toxin B antibody or binding fragment thereof. In turn, the immunogenic composition comprising the antitoxin B antibody or binding fragment thereof may be administered prior to, simultaneously with, or subsequent to the immunogenic composition comprising the antitoxin a antibody or binding fragment thereof.
In further embodiments, the compositions of the present invention may be administered prior to, simultaneously with, or subsequent to a pharmaceutically acceptable carrier. For example, the adjuvant may be administered prior to, simultaneously with, or after the composition comprising the mutant clostridium difficile toxin. Thus, the composition of the invention and the pharmaceutically acceptable carrier may be packaged in the same bottle or they may be packaged in different bottles and mixed prior to use. The compositions may be formulated for single dose administration and/or multiple dose administration.
Method for the protection and/or treatment of clostridium difficile infection in mammals
In one aspect, the invention relates to a method of inducing an immune response in a mammal against a clostridium difficile toxin. The method comprises administering to the mammal an effective amount of a composition described herein. For example, the method can comprise administering an effective amount to generate an immune response in the mammal against each clostridium difficile toxin.
In exemplary embodiments, the invention relates to a method of inducing an immune response against clostridium difficile TcdA in a mammal. The method comprises administering to a mammal an effective amount of an immunogenic composition comprising a mutant clostridium difficile TcdA. In further exemplary embodiments, the invention relates to a method of inducing an immune response against clostridium difficile TcdB in a mammal. The method comprises administering to the mammal an effective amount of an immunogenic composition comprising a mutant clostridium difficile TcdB.
In further embodiments, the method comprises administering to the mammal an effective amount of an immunogenic composition comprising a mutant clostridium difficile TcdA and an effective amount of an immunogenic composition comprising a mutant clostridium difficile TcdB. In further aspects, the compositions described herein can be used to treat, prevent, reduce the risk of, reduce the occurrence, reduce the severity of, and/or delay the onset of a clostridium difficile infection, Clostridium Difficile Associated Disease (CDAD), syndrome, condition, symptom, and/or complication thereof in a mammal compared to a mammal not administered the composition. The method comprises administering to the mammal an effective amount of the composition.
Based on the severity of the infection, three clinical syndromes caused by c. The most severe form is pseudomembranous colitis (PMC), which is characterized by violent diarrhea, abdominal pain, signs of systemic disease, and distinctive endoscopic appearance of the colon.
Antibiotic Associated Colitis (AAC) is also characterized by boluses, abdominal pain and tenderness, systemic signs (e.g., fever), and leukocytosis. Intestinal damage in AAC is less severe than in PMC, the characteristic endoscopic appearance of the colon in PMC is absent, and mortality is low.
Finally, antibiotic-associated diarrhea (AAD, also known as clostridium difficile-associated diarrhea (CDAD) is a relatively mild syndrome characterized by mild to moderate diarrhea, lack of coliform inflammation (characterized by, for example, abdominal pain and tenderness), and signs of systemic infection (e.g., fever).
These three different devices typically occur at increasing frequencies one after the other. That is, PMC usually occurs less frequently than AAC, while AAD is usually the most common clinical manifestation of clostridium difficile disease.
A common complication of c difficile infection is recurrent or relapsing disease, occurring in up to 20% of all patients recovering from c difficile disease. Relapse is clinically characterized by AAD, AAC, or PMC. Patients who relapse once are more likely to relapse again.
As used herein, conditions of clostridium difficile infection include, for example, mild to moderate, and severe clostridium difficile infections. The condition of clostridium difficile infection may vary depending on the manifestation of the infection and/or the severity of the symptoms.
Symptoms of clostridium difficile infection can include physiological, biochemical, histological, and/or behavioral symptoms, such as, for example, diarrhea; colitis; spastic colitis, fever, fecal leukocytosis, and inflammation on colon biopsy; pseudomembranous colitis; hypoalbuminemia; generalized edema; leukocytosis; sepsis; abdominal pain; asymptomatic bacteria-carrying; and/or complications and intermediate pathological phenotypes that arise during the development of the infection, as well as combinations thereof and the like. Thus, for example, administration of an effective amount of a composition described herein can be effective, for example, against diarrhea; abdominal pain, cramping, fever, inflammation on colonic biopsy, hypoalbuminemia, systemic edema, leukocytosis, sepsis, and/or asymptomatic carriers, and the like, are treated, prevented, reduced in risk for, reduced in occurrence of, reduced in severity of, and/or delayed in onset (as compared to a mammal not administered the composition).
Risk factors for clostridium difficile infection may include, for example, the ongoing or immediate use of antimicrobial agents (encompassing any antimicrobial substance having an antimicrobial spectrum and/or activity against anaerobes, including, for example, vitamins that disrupt the normal colonic microflora, e.g., clindamycin, cephalosporins, metronidazole, vancomycin, fluoroquinolones (including levofloxacin, moxifloxacin, gatifloxacin, and ciprofloxacin), linezolid, and the like); the prescribed metronidazole or vancomycin is being or immediately discontinued; is or is immediately in contact with a health care facility (e.g., hospital, long term care facility, etc.) and care worker; is or is immediately about to be treated with a proton pump inhibitor, an H2 antagonist, and/or methotrexate, or a combination thereof; is suffering from or at risk of suffering from a gastrointestinal disorder, such as inflammatory bowel disease; surgery or treatment of the gastrointestinal tract of a mammal at one time, at the time, or immediately; clostridium difficile infection and/or CDAD has been or is repeating, e.g., a patient with one or more clostridium difficile infections and/or CDAD; and persons at least or over the age of about 65 years.
In the methods described herein, the mammal can be any mammal, such as, for example, a mouse, hamster, primate, and human. In a preferred embodiment, the mammal is a human. According to the present invention, the human may include an individual who has exhibited a clostridium difficile infection, a clostridium difficile-associated disease, syndrome, condition, symptom, and/or complication thereof; an individual who is exhibiting clostridium difficile infection, clostridium difficile-associated disease, syndrome, condition, symptom, and/or complication thereof; and individuals at risk for clostridium difficile infection, clostridium difficile-associated diseases, syndromes, conditions, symptoms, and/or complications thereof.
Examples of individuals who exhibit symptoms of a clostridium difficile infection include individuals who exhibit or are exhibiting the symptoms described above; an individual who has or is suffering from clostridium difficile infection and/or Clostridium Difficile Associated Disease (CDAD); and individuals with clostridium difficile infection and/or repeated CDAD.
Examples of patients at risk of clostridium difficile infection include individuals at risk of, or undergoing, a planned antimicrobial application; an individual at risk of or at risk of discontinuing the prescription of metronidazole or vancomycin; an individual at risk of, or who is contacting, a care facility or care worker who is planning to contact the care facility (e.g., hospital, long-term care facility, etc.); and/or an individual at risk of, or undergoing planned treatment with, a proton pump inhibitor, an H2 antagonist, and/or methotrexate, or a combination thereof; individuals who have had or are experiencing a gastrointestinal disorder such as inflammatory bowel disease; an individual who has undergone or is undergoing gastrointestinal surgery or gastrointestinal manipulation; and individuals who have experienced or are experiencing a clostridium difficile infection and/or a relapse in CDAD, e.g., patients with one or more clostridium difficile infections and/or CDAD; an individual aged about 65 years or older. Patients at such risk may or may not currently show symptoms of clostridium difficile infection.
In asymptomatic patients, prevention and/or treatment may begin at any age (e.g., at about 10, 20, or 30 years of age). In one embodiment, however, it is not necessary to begin treatment until the patient reaches approximately 45, 55, 65, 75, or 85 years of age. For example, the compositions described herein may be administered to an asymptomatic human of 50-85 years of age.
In one embodiment, a method of treating, preventing, reducing the risk of, reducing the occurrence of, reducing the severity of, and/or delaying the onset of a clostridium difficile infection, clostridium difficile-associated disease, syndrome, condition, symptom, and/or complication thereof in a mammal comprises administering to a mammal in need thereof, a mammal at risk of a clostridium difficile infection, and/or a mammal suspected of having a clostridium difficile infection an effective amount of a composition described herein. An effective amount includes, for example, an amount sufficient to treat, prevent, reduce the risk of, reduce the occurrence of, reduce the severity of, and/or delay the onset of a clostridium difficile infection, clostridium difficile-associated disease, syndrome, condition, symptom, and/or complication thereof (as compared to a mammal not administered the composition). Administration of an effective amount of the composition of the invention can, for example, be effective against diarrhea; abdominal pain, cramping, fever, inflammation on colonic biopsy, hypoalbuminemia, systemic edema, leukocytosis, sepsis, and/or asymptomatic carriers, and the like, are treated, prevented, reduced in risk for, reduced in occurrence of, reduced in severity of, and/or delayed in onset (as compared to a mammal not administered the composition). In a preferred embodiment, the method comprises administering to a mammal in need thereof, a mammal at risk of a clostridium difficile infection, and/or a mammal suspected of having a clostridium difficile infection an effective amount of an immunogenic composition described herein.
In additional embodiments, a method of treating, preventing, reducing the risk of, reducing the occurrence of, reducing the severity of, and/or delaying the onset of a clostridium difficile infection, clostridium difficile-associated disease, syndrome, condition, symptom, and/or complication thereof in a mammal comprises administering to a mammal suspected of having or experiencing a clostridium difficile infection an effective amount of a composition described herein. An effective amount includes, for example, an amount sufficient to treat, prevent, reduce the risk of, reduce the occurrence of, reduce the severity of, and/or delay the onset of a clostridium difficile infection, clostridium difficile-associated disease, syndrome, condition, symptom, and/or complication thereof (as compared to a mammal not administered the composition).
Administration of an effective amount of the composition can improve at least one sign or symptom of clostridium difficile infection in a subject, such as those described below. Administration of an effective amount of a composition described herein can, for example, reduce the severity and/or reduce the recurrence of diarrhea as compared to a mammal not administered the composition; reducing the severity and/or recurrence of abdominal pain, cramping, fever, inflammation on colonic biopsy, hypoalbuminemia, systemic edema, leukocytosis, sepsis, and/or asymptomatic carriers, and the like. Optionally, the presence of symptoms, signs and/or risk factors of the infection is determined prior to treatment. In a preferred embodiment, the method comprises administering to a mammal suspected of having or experiencing a clostridium difficile infection an effective amount of an antibody and/or binding fragment thereof described herein.
Thus, an effective amount of a composition refers to an amount sufficient to achieve a desired effect (e.g., a prophylactic and/or therapeutic effect) in the methods of the invention. For example, the amount of immunogen used for administration can vary from a minimum of about 1 μ g, 5 μ g, 25 μ g, 50 μ g, 75 μ g, 100 μ g, 200 μ g, 500 μ g, or 1mg to a maximum of about 2mg, 1mg, 500 μ g, 200 μ g per injection. Any minimum value may be combined with any maximum value to define a suitable range. Typically about 10, 20, 50 or 100 μ g of each immunogen is used for each human injection.
The amount of the composition of the invention administered to a subject may depend on the severity of the infection and/or characteristics of the individual, such as overall health, age, sex, weight, and tolerance to drugs. It may also depend on the extent, severity, and type of the disease. The effective amount may also vary depending on factors such as the route of administration, the target site, the physiological state of the patient, the age of the patient, whether the patient is a human or an animal, other therapies administered, and whether the treatment is prophylactic or therapeutic. One skilled in the art will be able to determine appropriate amounts based on these and other factors.
An effective amount can include an effective dose or multiple effective doses (such as, for example, 2, 3, 4 doses or more) for use in the methods herein. Effective dosages may need to be escalated to optimize safety and efficacy.
A combination of an amount and frequency of administration sufficient to achieve prophylactic and/or therapeutic use is defined as a prophylactically effective or therapeutically effective regimen. In prophylactic and/or therapeutic regimens, the composition is typically administered in more than one dose until a sufficient immune response is achieved. Typically, the immune response is monitored and repeated doses are administered as the immune response diminishes.
The composition may be administered in multiple doses over a period of time. Treatment can be monitored by measuring antibody, or activated T-cell or B-cell responses, against time, against a therapeutic substance (e.g., an immunogenic composition comprising a mutant clostridium difficile toxin). If the response is reduced, a booster dose is indicated.
Examples
- 上一篇：一种医用注射器针头装配设备
- 下一篇：植物氮吸收相关蛋白NAR及其编码基因与应用