The present Application claims priority from U.S. provisional Patent Application61/478,474, filed 2011 at 22/4 and U.S. provisional Patent Application61/478,899, filed 2011 at 25/4. The entire contents of the above application are incorporated herein by reference in their entirety.
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, demonstrating that triple mutant A (SEQ ID NO:4), double mutant B (SEQ ID NO:5), and triple mutant B (SEQ ID NO:6) did not glycosylate Rac1 or RhoA GTPase in an in vitro glucosylation assay using UDP-14C-glucose, whereas 10. mu.g to 1ng of wild-type toxin B was glucosylated Rac 1.
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 the EC of triple mutant toxin B (SEQ ID NO:6) and heptad 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-italicized and underlined, constant region-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). mAbA50-10, 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 are graphs representing the results of in vitro cytotoxicity assays, in which ATP levels (RLU) were plotted against increased 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 poolCrude toxinControl, and purified toxin (. diamond-solid.), control. Toxins from each strain were treated with 4xEC50Values were added to the cells. FIG. 23 shows that immunogenic compositions comprising mutant TcdA (SEQ ID NO:4) and mutant TcdB (SEQ ID NO:6), wherein the mutant toxin was inactivated by EDC, e.g., according to example 29 described herein, Table 15, induced neutralizing antibodies that exhibited neutralizing activity (compared to the respective toxin-only controls) against toxins of Clostridium difficile from at least 16 different CDC strains 2007886 (FIG. 23A), 2006017 (FIG. 23B), 2007070 (FIG. 23C), 2007302 (FIG. 23D), 2007838 (FIG. 23E), 2007886 (FIG. 23F), 2009292 (FIG. 23G), 2004013 (FIG. 23H), 2009141 (FIG. 23I), 2005022 (FIG. 23J), 2006376 (FIG. 23K).
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 1 shows the amino acid sequence of wild type Clostridium difficile 630 toxin A (TcdA).
SEQ ID NO 2 shows the amino acid sequence of wild type Clostridium difficile 630 toxin B (TcdB).
SEQ ID NO 3 shows the amino acid sequence of mutant TcdA having mutations at positions 285 and 287 compared to SEQ ID NO 1.
SEQ ID NO 4 shows the amino acid sequence of mutant TcdA having mutations at positions 285, 287 and 700 compared to SEQ ID NO 1.
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 2.
SEQ ID NO 6 shows the amino acid sequence of the mutant TcdB with mutations at positions 286, 288 and 698 compared to SEQ ID NO 2.
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 1.
SEQ ID NO 8 shows the amino acid sequence of the mutant TcdB with mutations at positions 270, 273, 286, 288, 461, 463 and 698 compared to SEQ ID NO 2.
SEQ ID NO 9 shows the DNA sequence encoding wild type Clostridium difficile 630 toxin A (TcdA).
SEQ ID NO 10 shows the DNA sequence encoding wild-type Clostridium difficile 630 toxin B (TcdB).
SEQ ID NO 11 shows the DNA sequence encoding SEQ ID NO 3.
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 16 shows the DNA sequence encoding SEQ ID NO 15.
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.
SEQ ID NO 27 shows the DNA sequence of the pathogenic locus of wild type Clostridium difficile VPI 10463.
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 SEQ ID NO 2.
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 4.
SEQ ID NO 46 shows the DNA sequence encoding SEQ ID NO 5.
SEQ ID NO 47 shows the DNA sequence encoding SEQ ID NO 6.
The nucleotide sequence of the immunostimulatory oligonucleotide ODN CpG2455 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 positions 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: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 position 1 is deleted.
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 position 1 is deleted.
SEQ ID NO:85 shows the amino acid sequence of mutant Clostridium difficile toxin B with mutations at positions 270, 273, 286, 288, 461, 463 and 698 compared to SEQ ID NO:2, wherein the methionine at position 1 is deleted.
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 2, wherein the methionine at position 1 is deleted.
SEQ ID NO:87 shows the amino acid sequence of wild type Clostridium difficile 2004013 TcdA.
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 type clostridium difficile 2007070 TcdA.
SEQ ID NO 99 shows the amino acid sequence of wild type Clostridium difficile 2007217 TcdA.
100 shows the amino acid sequence of wild type Clostridium difficile 2007302 TcdA.
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 type Clostridium difficile 2007886 TcdA.
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 type Clostridium difficile 2009141 TcdA.
109 shows the amino acid sequence of wild type C.difficile 2009292 TcdA.
110 shows the amino acid sequence of wild type Clostridium difficile 2004013 TcdB.
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 type C.difficile 2007070 TcdB.
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 type Clostridium difficile 2007302 TcdB.
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 type C.difficile 2009141 TcdB.
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.
SEQ ID NO: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 SEQ ID NO 2.
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 SEQ ID NO 2. SEQ ID NO 26 describes the wild type gene for TcdB from Clostridium difficile strain VPI10463, also disclosed as GenBank accession number X53138.1.
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 toxinotype 0. Strains of toxin types I-VII, IX, XII-XV and XVIII-XXIV produce TcdA and TcdB, regardless of variation in their toxin genes.
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 SEQ ID NO 1. Unless otherwise indicated, all amino acid positions of TcdB refer to the numbering of SEQ ID NO 2 herein.
The glucosyltransferase domain of TcdA as used herein may start at exemplary residues 1, 101 or 102 of wild type clostridium difficile TcdA such as SEQ id no:1 and may end at exemplary residues 542, 516 or 293. Any minimum residue position between residues 1 and 542 of TcdA may be combined with the maximum residue position to define the sequence of the glucosyltransferase domain, provided that the DXD motif region is comprised. For example, in one embodiment, the glucosyltransferase domain of TcdA comprises SEQ ID NO:27, which is identical to residue 101-293 of SEQ ID NO:1, and which comprises the DXD motif region. In another embodiment, the glucosyltransferase domain of TcdA comprises SEQ ID NO:28, which is identical to residues 1-542 of SEQ ID NO: 1.
The glucosyltransferase domain of TcdB as used herein may start at exemplary residues 1, 101 or 102 of wild type clostridium difficile TcdB such as SEQ id no:2 and may end at exemplary residues 542, 516 or 293. Any minimum residue position between residues 1 and 543 of TcdB can be combined with the maximum residue position to define the sequence of the glucosyltransferase domain, as long as the DXD motif region is contained. For example, in one embodiment, the glucosyltransferase domain of TcdB comprises SEQ ID NO:29, which is identical to residue 101-293 of SEQ ID NO:2, and which comprises the DXD motif region. In another embodiment, the glucosyltransferase domain of TcdB comprises SEQ ID NO:30, which is identical to residues 1-543 of SEQ ID NO: 1.
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 SEQ ID NO 1, TcdA.
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 SEQ ID NO 2. Residue 544-767 of SEQ ID NO 33 and SEQ ID NO 2, TcdB are identical. In another embodiment, the cysteine protease domain of TcdB comprises residues 544-801 of SEQ ID NO 2, TcdB.
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 SEQ ID NO 4, 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: 84.
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 SEQ ID NO 6, 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 86.
Further examples of a mutant Clostridium difficile TcdB comprise the amino acid sequence shown in SEQ ID NO 8 with the D270A, R273A, D286A, D288A, D461A, K463A and C698A mutations relative to SEQ ID NO 2, wherein the initial methionine is optionally absent. In another embodiment, the mutant Clostridium difficile toxin A comprises the amino acid sequence set forth in SEQ ID NO: 85.
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 SEQ ID NO 2 to define an exemplary mutant Clostridium difficile toxin B. For example, in one embodiment, the isolated polypeptide comprises a mutation between amino acid residues 830 and 990 of SEQ ID NO. 2. Exemplary positions of the mutation include positions 970 and 976 numbered according to SEQ ID No. 2. Preferably, the mutation between residues 830 and 990 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 E970K, E970R, E970H, E976K, E976R, E976H of SEQ ID No. 2 to define mutant clostridium difficile toxin B.
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 SEQ ID NO 6, wherein the initial methionine (at position 1) is optionally absent. In one embodiment, the initial methionine of SEQ ID NO 6 is absent. In one aspect, the invention relates to an isolated polypeptide comprising the amino acid sequence shown in SEQ ID NO 86, which is identical to SEQ ID NO 6, but in the absence of the initial methionine.
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 SEQ ID NO 8, wherein the initial methionine (at position 1) is optionally absent. In one embodiment, the isolated polypeptide comprises the amino acid sequence shown in SEQ ID NO. 85, which is identical to SEQ ID NO. 8, but absent the initial methionine.
In one aspect, the invention relates to an immunogenic composition comprising SEQ ID NO 4, wherein the initial methionine (at position 1) is optionally absent. In another aspect, the invention relates to an immunogenic composition comprising SEQ id No. 6, wherein the initial methionine (at position 1) is optionally absent. In a further aspect, the present invention relates to an immunogenic composition comprising SEQ ID NO 7, wherein the initial methionine (at position 1) is optionally absent. In another aspect, the invention relates to an immunogenic composition comprising SEQ ID NO 8, wherein the initial methionine (at position 1) is optionally absent.
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 apoptotic cell death and/or to the same but not cytotoxic agentIn a state in which one or more of the cells is normally biochemically or biologically abnormally impaired. 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.2010Oct7;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.1998Jul31;273(31): 19566-72) and preferably cytotoxicity assays as described in the examples below, where 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) Of a cell of (a) inhibits at least about 50% of a wild-type clostridium difficile toxin-induced cytopathic effect (CPE) of the cell. Other exemplary cytotoxicity assays include those described by Doern et al, JClin 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(Promega), which emits fluorescence measured as Relative Light Units (RLU). In such an assay, cell viabilityMay be proportional to the amount of ATP in the cell or the RLU value.
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), benzoylformaldehyde 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 SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 7 and/or SEQ ID NO 8 is chemically cross-linked. For example, at least one amino acid may be chemically crosslinked by a reagent comprising a carbodiimide such as EDC. Carbodiimides can form covalent bonds between free carboxyl groups (e.g., side chains from aspartic and/or glutamic acids) and amino groups (e.g., in the side chains of lysine residues) to form stable amide bonds.
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 position 1 is optionally absent, wherein said polypeptide comprises at least one amino acid side chain chemically modified with EDC and NHS. In another embodiment, the present invention relates to an isolated polypeptide having the amino acid sequence set forth in SEQ ID NO 6, wherein the methionine residue at position 1 is optionally absent, wherein said polypeptide comprises at least one amino acid side chain chemically modified with EDC and NHS. In another embodiment, the invention relates to an isolated polypeptide having an amino acid sequence as set forth in SEQ ID NO 84, 86, 83, 85, 7 or 8. The polypeptide is modified by contacting the polypeptide with EDC and NHS. See, for example, fig. 24 and 25.
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 a3-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 greater than or at 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, a toxin,800. mu.g/ml, 900. mu.g/ml, 1000. mu.g/ml or greater EC50The 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 to3mM, 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 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 the cross-linking agent50Values 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 pH5.5, 6.0, 6.5, 7.0, or 7.5, and a maximum of about pH8.5, 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 pH6.5-7.5, 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, SEQ ID NO 6 or SEQ ID NO 8. As another example, the immunogenic composition can comprise SEQ ID NO 86 or SEQ ID NO 85.
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, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46 or SEQ ID NO 47 when optimally aligned, e.g., by using the programs GAP or BESTFIT of predetermined GAP weights.
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.
Examples of sporulation genes for targeted inactivation 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,2007Sept, 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. 20110124109A1, [00137] - [0227 ].
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 to transfer 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, SEQ ID NO 12, SEQ ID NO 44 and SEQ ID NO 45.
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, SEQ ID NO 14, SEQ ID NO 46, and SEQ ID NO 47. In another embodiment, the polynucleotide encodes SEQ ID NO 83, SEQ ID NO 84, SEQ ID NO 85, or SEQ ID NO 86.
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 Stbl2TMColi competent cells (Invitrogen, Carlsbad, CA), OneStbl3TMChemically competent Escherichia coli (Invitrogen, Carlsbad, Calif.), ElectroMAXTMStbl4TME.coli competent cells (Invitrogen), and E.coli CA 434. In a preferred embodiment, the E.coli cloning host cell is not DH5 α. More preferably, the E.coli cloning host cell is MAXStbl2TME.coli competent cells.
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., IgG 1. In another preferred embodiment, the antibody is an IgE antibody.
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, Nature341:544-546,1989) 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. The binding fragment of the light chain variable region and the binding fragment of the heavy chain variable region, such as the two domains of the Fv fragment VL and VH, can be recombinantly producedAre linked by a synthetic linker that makes them a single protein chain in which the VL and VH pair to form monovalent molecules (known as single chain fv (scFv), see, e.g., Bird et al (1988) Science242: 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 SEQ ID NO 6 or SEQ ID NO 8.
In another embodiment, the antibody or binding fragment thereof is specific for an immunogenic composition comprising SEQ ID NO 6 or SEQ ID NO 8, wherein at least one amino acid of SEQ ID NO 6 or SEQ ID NO 8 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 86 or SEQ ID NO 85.
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 SEQ ID NO 6 or SEQ ID NO 8. In another embodiment, the antibody or binding fragment thereof is specific for an immunogenic composition comprising SEQ ID NO 6 or SEQ ID NO 8, wherein at least one amino acid of SEQ ID NO 6 or SEQ ID NO 8 is crosslinked with formaldehyde, EDC, NHS or a combination of EDC and NHS.
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 the A3-25 antibody and at least one neutralizing antitoxin A monoclonal antibody 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 against TcdA that may be produced include a65-33, 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 SEQ ID NO 6, wherein at least one amino acid of the mutant Clostridium difficile TcdB is chemically cross-linked, preferably methyl or 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide. Exemplary neutralizing antibodies to TcdB that may be produced include B2-31, B5-40, B70-2, B6-30, B9-30, B59-3, B60-2, B56-6, and/or B8-26.
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, the binding agent and carrier include, for example, polyolefin glycols or triglycerides, and 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 such as chitosan, polylactic acid, polyglycolic acid, and co-polymers (e.g., latex functionalized sephalase)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 (e.g., calcium phosphate, sodium phosphate), acetate (e.g., sodium acetate), succinate (e.g., sodium succinate), glycine, histidine, carbonate, 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, the 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 ester surfactants (e.g., ) Such as Polysorbate20 and/or Polysorbate80, polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethylene glycol monododecyl ether (Brij30), Triton X100, or t-octylphenyl polyvinyl chlorohydrol, and sorbitan esters (commonly known as SPANs), such as sorbitan trioleate (SPAN85) and monolaurate, and combinations thereof. Preferred surfactants include Polysorbate80 (polyoxyethylene sorbitan monooleate).
Preferred amounts (wt%) of Polysorbate80 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 from about 0.005% to 0.015% Polysorbate80, 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 GB2220211(GSK)), and aluminum hydroxide gel 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 CpGODN 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 C T C G T3' (SEQ ID NO:48) wherein. The CpG ODN of this sequence is known as CpG2455, which is described in WO 2010/067262. In a preferred embodiment, CpG2455 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) andan adjuvant. Thus, the composition of the invention may be provided in the form of: ISCOMs, ISCOMs containing CTB, liposomes or encapsulated in compounds such as acrylates or poly (DL-lactide-co-glycoside) to form microspheres of suitable size for adsorption. Generally, the term "ISCOM" refers to an immunogenic complex formed between glycosides, such as triterpenoid saponins (particularly Quil A), And an antigen comprising a hydrophobic region. In a preferred embodiment, the adjuvant is an ISCOMATRIX adjuvant.
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 a biological treatment, a probiotic treatment, an anal implant, an immunotherapy (such as intravenous immunoglobulin), and/or a standard of care accepted 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 may include physiological, biochemical, histological, and/or behavioral symptoms such as, for example, diarrhea, colitis, spastic colitis, fever, fecal leukocytosis, and inflammation on colonic biopsy, pseudomembranous colitis, hypoalbuminemia, generalized edema, leukocytosis, sepsis, abdominal pain, asymptomatic carrier, and/or complications and intermediate pathological phenotypes that occur during the development of the infection, combinations thereof, and the like. Thus, for example, administration of an effective amount of a composition described herein can treat, prevent, reduce the risk of, reduce the occurrence of, reduce the severity of, and/or delay the onset of diarrhea, abdominal pain, cramps, fever, inflammation on colonic biopsy, hypoalbuminemia, systemic edema, leukocytosis, sepsis, and/or asymptomatic carriers, among others (as compared to a mammal not administered the composition).
Risk factors for Clostridium difficile infection may include, for example, 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 normal colonic microflora, such as clindamycin, cephalosporins, metronidazole, vancomycin, fluoroquinolones (including levofloxacin, moxifloxacin, gatifloxacin, and ciprofloxacin), linezolid, etc.), ongoing or immediate discontinuation of metronidazole or vancomycin, ongoing or immediate contact with health care facilities (e.g., hospitals, long term care facilities, etc.) and care workers, ongoing or immediate treatment with proton pump inhibitors, H2 antagonists, and/or methotrexate, or combinations thereof, ongoing or immediate treatment with gastrointestinal disease or risk, such as inflammatory bowel disease, once, Gastrointestinal surgery or treatment of the gastrointestinal tract is being or is immediately performed on the mammal, Clostridium difficile infection and/or CDAD has been or is repeating, e.g., patients with one or more Clostridium difficile infections and/or CDAD, and people 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 individuals who have exhibited Clostridium difficile infection, Clostridium difficile-associated disease, syndrome, condition, symptom, and/or complication thereof, individuals who are exhibiting Clostridium difficile infection, Clostridium difficile-associated disease, syndrome, condition, symptom, and/or complication thereof, and individuals who are at risk of Clostridium difficile infection, Clostridium difficile-associated disease, syndrome, condition, symptom, and/or complication thereof.
Examples of individuals exhibiting symptoms of a Clostridium difficile infection include individuals who exhibit or are exhibiting the symptoms described above, individuals who have or are suffering from a Clostridium difficile infection and/or a Clostridium difficile-associated disease (CDAD), and individuals who have a Clostridium difficile infection and/or a repeat of CDAD.
Examples of patients at risk of clostridium difficile infection include individuals at risk of or undergoing planned antimicrobial application, individuals at risk of or undergoing prescription metronidazole or vancomycin, individuals at risk of or undergoing plan contact with a care facility (e.g., a hospital, a long term care facility, etc.) or a care worker, and/or individuals at risk of or undergoing treatment with proton pump inhibitors, H2 antagonists, and/or methotrexate, or combinations thereof, individuals who have or are undergoing gastrointestinal diseases such as inflammatory bowel disease, individuals who have or are undergoing gastrointestinal surgery or gastrointestinal treatment, and individuals who have or are undergoing recurrence of clostridium difficile infection and/or CDAD, for example, patients with one or more Clostridium difficile infections and/or CDAD, individuals 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 a composition of the invention can, for example, treat, prevent, reduce the risk of, reduce the occurrence of, reduce the severity of, and/or delay the onset of diarrhea, abdominal pain, spasticity, fever, inflammation on colonic biopsy, hypoalbuminemia, systemic edema, leukocytosis, sepsis, and/or asymptomatic carriers (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 recurrence of diarrhea, reduce the severity and/or recurrence of abdominal pain, cramps, fever, inflammation on colonic biopsy, hypoalbuminemia, systemic edema, leukocytosis, sepsis, and/or asymptomatic carriers, etc., as compared to a mammal not administered the composition. 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.
Example 45 identification of mutant toxin drug substances
The primary structure of the triple mutant toxin A is shown in SEQ ID NO. 4. NH at position 1 of SEQ ID NO 42The terminal Met residue is derived from the start codon of SEQ ID NO:12 and is deleted in the isolated protein (see, e.g., SEQ ID NO: 84). Thus, in examples 12 to 45, "SEQ ID NO: 4" refers to SEQ ID NO:4 in which the initiating methionine (at position 1) is deleted. Both the purified triple mutant toxin A (SEQ ID NO:4) (drug substance intermediate-Lot L44993-132) and the EDC/NHS treated triple mutant toxin A (SEQ ID NO:4) ("mutant toxin A drug substance" -Lot L44898-012) exhibited a single NH starting with SLISKEELIKLAYSI (positions 2-16 of SEQ ID NO:4)2-a terminal sequence.
The primary structure of the triple mutant toxin B is shown in SEQ ID NO 6. NH at position 1 of SEQ ID NO 62The terminal Met residue is derived from the start codon of SEQ ID NO:12 and is deleted in the isolated protein (see, e.g., SEQ ID NO: 86). Thus, in example 12 to example 45, "SEQ ID NO: 6" refers to SEQ ID NO:6 in which the initiating methionine (at position 1) is deleted. Both purified triple mutant toxin B (SEQ ID NO:6) (drug substance intermediate-Lot 010) and EDC/NHS treated triple mutant toxin B (SEQ ID NO:6) ("mutant toxin B drug substance" -Lot L44906-153) were shown to exhibit SLVNRKQLEKMANVR (positions 2-16 of SEQ ID NO:6) starting with a single NH2-a terminal sequence.
Circular Dichroism (CD) spectroscopy was used to assess the secondary and tertiary structures of triple mutant A (SEQ ID NO:4) and mutant toxin A drug substances. CD spectra were also used to assess the secondary and tertiary structures of triple mutant B (SEQ ID NO:6) and mutant toxin B drug substances. CD spectroscopy was also used to assess the potential effect of pH on structure. The effect of EDC treatment on triple mutant toxin a was analyzed by comparing CD data from mutant toxin a drug substance with data from triple mutant toxin a. The effect of EDC treatment on triple mutant toxin B (SEQ ID NO:6) was analyzed by comparing CD data from mutant toxin B drug substance to data from triple mutant toxin B.
far-UV CD data for mutant toxin a drug substances were obtained at various phs. The spectra recorded at ph5.0-7.0 indicate a high proportion of alpha-helices in secondary structure, indicating that the polypeptide backbone of the protein adopts a well-defined conformation in which alpha-helices predominate.
near-UV CD spectra of mutant toxin a drug substances were also obtained. The strong negative ellipticity between 260-300nm indicates that the aromatic side chains are in a unique rigid environment, i.e., that the mutant toxin A drug substance has a tertiary structure. Indeed, the characteristic features derived from individual types of aromatic side chains can be distinguished in the spectrum by shoulders at 290nm and a maximum negative peak at 283nm due to absorption of polarized light by the ordered tryptophan side chain, the negative peak at 276nm coming from the tyrosine side chain, and small shoulders at 262 and 268nm indicating phenylalanine residues involved in three-dimensional contacts. The far-and near-UV results provide evidence that the mutant toxin a drug substance remains in an intact folded structure at physiological pH. Almost identical far-and near-UV CD spectra observed at pH5.0-7.0 indicate that no detectable structural changes occur in this pH range. CD data could not be collected at pH3.0 and 4.0 because the protein was not soluble at such pH points. In the comparative far-and near-UV CD spectra of mutant toxin a drug substance versus triple mutant toxin a, the spectra of both proteins were essentially identical under all experimental conditions studied, indicating that EDC treatment had no detectable effect on the secondary and tertiary structures of triple mutant toxin a. This finding is consistent with gel filtration and analytical ultracentrifugation results that show no detectable change in both stokes radius and sedimentation/friction coefficient, respectively.
The mutant toxin a drug substance (as well as the triple mutant toxin a) contains 25 tryptophan residues, which are distributed throughout the primary sequence and can be used as convenient internal fluorescent probes. Fluorescence emission spectra of the mutant toxin A drug substance between 300-400nm as a function of temperature were obtained. The mutant toxin a drug substance exhibited a characteristic tryptophan fluorescence emission spectrum upon excitation at 280nm at 6.8 ℃. The fluorescence emission maximum was observed at 335nm, indicating that the tryptophan residue is in a non-polar environment, typical of the protein interior environment and not a polar aqueous environment. Fluorescence emission spectroscopy results, along with the CD experimental results given in this report, confirm that the mutant toxin a drug substance remains in an intact folded structure.
The fluorescence of the external probe 8-anilino-1-naphthalenesulfonic Acid (ANS) was used to characterize possible conformational changes in mutant toxin a drug substance and triple mutant toxin a upon pH change. From the results, it can be seen that there was no substantial increase in ANS fluorescence density when either the mutant toxin a drug substance or the triple mutant toxin a was titrated with the probe at ph7.0, indicating that no hydrophobic surface was exposed on the protein under these conditions. Changing the pH to 2.6 causes a drastic change in the ANS fluorescence quantum field as the probe concentration increases until the fluorescence quantum field reaches apparent saturation. This increase in ANS fluorescence quantum field indicates that at low pH (2.6), both the mutant toxin a drug substance and the triple mutant toxin a undergo a pH-induced conformational change exposing a hydrophobic surface. Such conformational changes indicate that EDC-induced modification and inactivation of triple mutant toxin a does not limit the conformational plasticity of the mutant toxin a Drug Substance (DS).
The effect of EDC treatment on the hydrodynamic properties of triple mutant toxin a was evaluated on a G4000SWXL column using size exclusion chromatography. Mutant toxin a drug substance and triple mutant toxin a were injected onto G4000SWXL columns (equilibrated at ph7.0, 6.0, and 5.0). The data indicate that no difference in stokes radius between mutant toxin a drug substance and triple mutant toxin a could be detected using size exclusion chromatography. Thus, EDC treatment did not dramatically affect the hydrodynamic properties of triple mutant toxin a, and thus, correspondingly, did not drastically affect its overall molecular shape.
Triple mutant toxin a and mutant toxin a drug substances were further analyzed using the multi-angle laser light scattering (MALLS) technique. Treatment of triple mutant toxin a with EDC resulted in the production of a heterogeneous mixture, which consisted of various multimeric and monomeric species. This heterogeneity reflects the introduction of a large number of EDC-induced intermolecular and intramolecular covalent bonds (between carboxyl groups and primary amines of proteins).
The data obtained provide the physical and chemical characteristics of the triple mutant toxin a and mutant toxin a drug substance (EDC treated triple mutant toxin a) and describe key features in their primary, secondary and tertiary structures. The data generated demonstrate that EDC-treated triple mutant toxin a results in covalent modification of its polypeptide chains, but does not affect the secondary and tertiary structure of the protein. Treatment with EDC results in intramolecular and intermolecular crosslinking. The biochemical and biophysical parameters obtained for the mutant toxin a drug substance (as well as the triple mutant toxin a) are given in table 60.
far-UV CD data for mutant toxin B drug substances were obtained at various phs. The spectra recorded at ph5.0-7.0 indicate a high proportion of alpha-helices in secondary structure, indicating that the polypeptide backbone of the protein adopts a well-defined conformation in which alpha-helices predominate.
near-UV CD spectra of the mutant toxin B drug substance were also obtained. The strong negative ellipticity between 260-300nm indicates that the aromatic side chains are in a unique rigid environment, i.e., that the mutant toxin B drug substance has a tertiary structure. Indeed, the characteristic features derived from individual types of aromatic side chains can be distinguished in the spectrum by shoulders at 290nm and a maximum negative peak at 283nm due to absorption of polarized light by the ordered tryptophan side chain, the negative peak at 276nm coming from the tyrosine side chain, and small shoulders at 262 and 268nm indicating phenylalanine residues involved in three-dimensional contacts. The far-and near-UV results provide evidence that the mutant toxin B drug substance remains in an intact folded structure at physiological pH. Very similar far-and near-UV CD spectra observed at pH5.0-7.0 indicate that no detectable structural changes occur in this pH range. CD data could not be collected at pH3.0 and 4.0 because the protein was not soluble at such pH points.
The spectra of both proteins were very similar between ph5.0-7.0 in the comparative far-and near-UVCD spectra of the mutant toxin B drug substance and the triple mutant toxin B, indicating that EDC treatment had no detectable effect on the secondary and tertiary structure of the protein.
Triple mutant toxin B contains 16 tryptophan residues, which are distributed throughout the primary sequence and can be used as a convenient internal fluorescent probe. Fluorescence emission spectra of the mutant toxin B drug substance between 300-400nm as a function of temperature were obtained. The mutant toxin B drug substance exhibited a characteristic tryptophan fluorescence emission spectrum upon excitation at 7 ℃ at 280 nm. The fluorescence emission maximum was observed at 335nm, indicating that the tryptophan residue is in a non-polar environment, typical of the protein interior environment and not a polar aqueous environment. This result, together with the results of the CD experiment (see above), confirms that the mutant toxin B drug substance remains in an intact folded structure.
The fluorescence of the external probe 8-anilino-1-naphthalenesulfonic Acid (ANS) was used to characterize possible conformational changes in mutant toxin B drug substance and triple mutant toxin B upon pH change. From the results, it can be seen that there was no substantial increase in ANS fluorescence density when either the mutant toxin B drug substance or the triple mutant toxin B was titrated with the probe at ph7.0, indicating that no hydrophobic surface was exposed on the protein under these conditions. Changing the pH to 2.6 resulted in a dramatic change in the ANS fluorescence quantum field (in the presence of mutant toxin B drug substance) with increasing probe concentration until the fluorescence quantum field reached apparent saturation. This increase in ANS fluorescence quantum field indicates that at low pH (2.6), the mutant toxin B drug substance undergoes a pH-induced conformational change exposing a hydrophobic surface. Such conformational changes indicate that EDC-induced modification and inactivation of triple mutant toxin B does not limit the conformational plasticity of the mutant toxin B Drug Substance (DS).
The effect of EDC treatment on the hydrodynamic properties of triple mutant toxin B was evaluated on a G4000SWXL column using size exclusion chromatography. Mutant toxin B drug substance and triple mutant toxin B were injected onto G4000SWXL columns (equilibrated at ph7.0, 6.0, and 5.0). The data indicate that no difference in stokes radius between the mutant toxin B drug substance and the triple mutant toxin B could be detected using size exclusion chromatography, and therefore EDC treatment did not dramatically affect the hydrodynamic properties of the protein, and thus, the overall molecular shape, accordingly.
Triple mutant toxin B and mutant toxin B drug substance were further analyzed using multi-angle laser light scattering (MALLS) technology. Treatment of triple mutant toxin B with EDC resulted in the production of a more heterogeneous mixture, consisting of various multimeric and monomeric species. This heterogeneity reflects the introduction of a large number of EDC-induced intermolecular and intramolecular covalent bonds (between carboxyl groups and primary amines of proteins).
The data obtained provide the physical and chemical characteristics of the triple mutant toxin B and mutant toxin B drug substance (EDC treated triple mutant toxin B) and describe key features in their primary, secondary and tertiary structures. The data generated demonstrate that EDC-treated triple mutant toxin B results in covalent modification of its polypeptide chain, but does not affect the secondary and tertiary structure of the protein. Treatment with EDC results in intramolecular and intermolecular crosslinking. The main biochemical and biophysical parameters obtained for the mutant toxin B drug substance (as well as the triple mutant toxin B) are given in table 61.
Aspects of the invention
The following items describe further embodiments of the invention:
C1. an isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO 4, wherein the methionine residue at position 1 is optionally absent, and wherein said polypeptide comprises at least one amino acid side chain chemically modified with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) (EDC) and N-hydroxysuccinimide (NHS).
C2. An isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO 6, wherein the methionine residue at position 1 is optionally absent, and wherein said polypeptide comprises an amino acid side chain chemically modified with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) (EDC) and N-hydroxysuccinimide (NHS).
C3. The isolated polypeptide of clauses C1 or C2, wherein at least one aspartic acid residue side chain of the polypeptide or at least one glutamic acid residue side chain of the polypeptide is chemically modified with glycine.
C4. The isolated polypeptide of any one of entries C1-C3, wherein the polypeptide comprises:
a) at least one cross-link between an aspartic acid residue side chain of said polypeptide and a lysine residue side chain of said 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.
C5. The isolated polypeptide of any one of entries C1-C4, wherein the polypeptide comprises a β -alanine moiety attached to at least one lysine residue side chain of the polypeptide.
C6. The isolated polypeptide of clause C4, wherein the 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.
C7. An isolated polypeptide comprising the amino acid sequence set forth in SEQ ID No. 4, wherein the methionine residue at position 1 is optionally absent, and wherein at least one lysine residue of said polypeptide is side-chain linked to a β -alanine moiety.
C8. An isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO 6, wherein the methionine residue at position 1 is optionally absent, and wherein at least one lysine residue of said polypeptide is side-chain linked to a β -alanine moiety.
C9. The isolated polypeptide of clauses C7 or C8, wherein the side chain of the second lysine residue of the polypeptide is linked to the side chain of an aspartic acid residue or to the side chain of a glutamic acid residue.
C10. The isolated polypeptide of any one of entries C7-C9, wherein the side chain of an aspartic acid residue or the side chain of a glutamic acid residue of the polypeptide is attached to a glycine moiety.
C11. The isolated polypeptide of any of items C1-C10, wherein the polypeptide has an EC of at least about 100 μ g/ml50。
C12. An immunogenic composition comprising an isolated polypeptide having an amino acid sequence shown in SEQ ID No. 4, wherein the methionine residue at position 1 is optionally absent, and an isolated polypeptide having an amino acid sequence shown in SEQ ID No. 6, wherein the methionine residue at position 1 is optionally absent, and wherein said polypeptide has at least one amino acid side chain chemically modified with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) (EDC) and N-hydroxysuccinimide (NHS).
C13. The immunogenic composition of item C12, wherein the polypeptide comprises at least one of any of:
a) at least one β -alanine moiety attached to a lysine side chain of said polypeptide;
b) at least one cross-link between the side chain of an aspartic acid residue and the side chain of a lysine residue of said polypeptide, and
c) at least one crosslink between a glutamic acid residue side chain and a lysine residue side chain of the polypeptide.
C14. The immunogenic composition of item C12, wherein the polypeptide has an EC of at least about 100 μ g/ml50。
C15. An immunogenic composition comprising an isolated polypeptide having an amino acid sequence shown in SEQ ID NO. 4, wherein the methionine residue at position 1 is optionally absent, and an isolated polypeptide having an amino acid sequence shown in SEQ ID NO. 6, wherein the methionine residue at position 1 is optionally absent, and
a) Wherein at least one lysine residue of SEQ ID NO 4 is side-chain linked to a beta-alanine moiety, and
b) wherein at least one lysine residue of SEQ ID NO 6 is side-chain linked to a beta-alanine moiety.
C16. The immunogenic composition of item C15, wherein the second lysine residue side chain of SEQ ID NO:4 is 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.
C17. The immunogenic composition of any of items C12-C16, wherein the side chain of an aspartic acid residue or the side chain of a glutamic acid residue of the polypeptide is linked to a glycine moiety, wherein the polypeptide has the amino acid sequence set forth in SEQ ID No. 4, wherein the methionine residue at position 1 is optionally absent.
C18. The immunogenic composition of any one of items C12-C16, wherein the side chain of an aspartic acid residue or the side chain of a glutamic acid residue of the polypeptide is linked to a glycine moiety, wherein the polypeptide has the amino acid sequence shown in SEQ ID No. 6, wherein the methionine residue at position 1 is optionally absent.
C19. The immunogenic composition of any one of items C12-C18, wherein the polypeptide has an EC of at least about 100 μ g/ml50。
C20. An immunogenic composition comprising an isolated polypeptide having an amino acid sequence set forth in SEQ ID No. 84 and an isolated polypeptide having an 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 said polypeptide and a lysine residue side chain of said 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 beta-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.
C21. An immunogenic composition comprising a mutant clostridium difficile toxin a comprising a glucosyltransferase domain having at least one mutation and a cysteine protease domain having at least one mutation, relative to a corresponding wild-type clostridium difficile toxin a.
C22. The composition of item C21, wherein the mutation is a non-conservative amino acid substitution.
C23. The composition of item C22, wherein the substitution comprises an alanine substitution.
C24. The composition of any of entries C21-C23, wherein the wild-type clostridium difficile toxin a comprises a sequence having at least 95% identity to SEQ id No. 1.
C25. The composition of item C24, wherein the wild-type clostridium difficile toxin a comprises a sequence having at least 98% identity to SEQ ID NO: 1.
C26. The composition of item C25, wherein the wild-type Clostridium difficile toxin A comprises SEQ ID NO 1.
C27. The composition of any one of entries C21-C26, wherein the glucosyltransferase domain comprises at least two mutations.
C28. The composition of item C27, wherein the at least two mutations are present at amino acid positions 101, 269, 272, 285, 287, 269, 272, 460, 462, 541, or 542, according to the numbering of SEQ ID NO: 1.
C29. The composition of any one of entries C21-C26, wherein the glucosyltransferase domain comprises SEQ ID NO: 29.
C30. The composition of item C29, wherein the glucosyltransferase domain comprises at least two non-conservative mutations present at amino acid position 101, 269, 272, 285, 287, 269, 272, 460, 462, 541, or 542 of SEQ ID NO:29, or any combination thereof.
C31. The composition of any one of items C21-C26, wherein the cysteine protease domain comprises a mutation present at position 700, 589, 655, 543, or any combination thereof, according to the numbering of SEQ ID NO: 1.
C32. The composition of any one of entries C21-C26, wherein the cysteine protease domain comprises SEQ ID NO: 32.
C33. The composition of item C32, wherein the cysteine protease domain comprises a non-conservative mutation that is present at position 1, 47, 113, 158 of SEQ ID NO:32, or any combination thereof.
C34. The composition of item C21, wherein the mutant Clostridium difficile toxin A comprises SEQ ID NO 4.
C35. The composition of item C21, wherein the mutant Clostridium difficile toxin A comprises SEQ ID NO: 84.
C36. The composition of item C21, wherein the mutant Clostridium difficile toxin A comprises SEQ ID NO 7.
C37. The composition of item C21, wherein the mutant Clostridium difficile toxin A comprises SEQ ID NO 83.
C38. The composition of any of entries C21-C33, wherein at least one amino acid of the mutant clostridium difficile toxin a is chemically cross-linked.
C39. The composition of clause C38, wherein the amino acid is chemically crosslinked by formaldehyde.
C40. The composition of clause C38, wherein the amino acid is chemically crosslinked by 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide.
C41. The composition of clauses C38 or C40, wherein the amino acids are chemically crosslinked by N-hydroxysuccinimide.
C42. The composition of any one of items C21-C41, wherein the composition is recognized by an antitoxin a neutralizing antibody or binding fragment thereof.
C43. An immunogenic composition comprising a mutant clostridium difficile toxin a comprising, relative to a corresponding wild-type clostridium difficile toxin a: a glucosyltransferase domain comprising SEQ ID No. 29 with amino acid substitutions at positions 285 and 287; and a cysteine protease domain comprising SEQ ID NO:32 with an amino acid substitution at position 158, wherein at least one amino acid of the mutant Clostridium difficile toxin A is chemically cross-linked.
C44. 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 chemically cross-linked.
C45. The composition of clauses C43 or C44, wherein the at least one amino acid is crosslinked by formaldehyde.
C46. The composition of clauses C43 or C44, wherein the at least one amino acid is crosslinked by 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide.
C47. The composition of entry C43, C44, or C46, wherein the at least one amino acid is crosslinked by N-hydroxysuccinimide.
C48. The composition of clauses C43 or C44, wherein the composition is recognized by an antitoxin a neutralizing antibody or binding fragment thereof.
C49. An immunogenic composition comprising SEQ ID NO 4.
C50. An immunogenic composition comprising SEQ ID NO 84.
C51. An immunogenic composition comprising SEQ ID NO 7.
C52. An immunogenic composition comprising SEQ ID NO 83.
C53. The composition of any of items C49-C52, wherein at least one amino acid is chemically crosslinked.
C54. The composition of any one of entries C21-C51, wherein the composition exhibits reduced cytotoxicity relative to corresponding wild-type clostridium difficile toxin a.
C55. An isolated polypeptide comprising SEQ ID NO 84.
C56. An isolated polypeptide comprising SEQ ID NO 86.
C57. 83 comprising SEQ ID NO.
C58. An isolated polypeptide comprising SEQ ID NO 85.
C59. An immunogenic composition comprising a mutant clostridium difficile toxin B comprising a glucosyltransferase domain having at least one mutation and a cysteine protease domain having at least one mutation, relative to a corresponding wild-type clostridium difficile toxin B.
C60. The composition of item C59, wherein the mutation is a non-conservative amino acid substitution.
C61. The composition of item C60, wherein the substitution comprises an alanine substitution.
C62. The composition of any of entries C59-C61, wherein the wild-type clostridium difficile toxin B comprises a sequence having at least 95% identity to SEQ id No. 2.
C63. The composition of item C62, wherein the wild-type clostridium difficile toxin B comprises a sequence having at least 98% identity to SEQ ID NO: 2.
C64. The composition of item C63, wherein the wild-type Clostridium difficile toxin B comprises SEQ ID NO 2.
C65. The composition of any one of entries C59-C64, wherein the glucosyltransferase domain comprises at least two mutations.
C66. The composition of entry C65, wherein the at least two mutations are present at amino acid positions 102, 286, 288, 270, 273, 384, 461, 463, 520, or 543 according to the numbering of SEQ ID NO: 2.
C67. The composition of any one of entries C59-C64, wherein the glucosyltransferase domain comprises SEQ ID NO: 31.
C68. The composition of clause C67, wherein the glucosyltransferase domain comprises at least two non-conservative mutations present at amino acid positions 102, 286, 288, 270, 273, 384, 461, 463, 520, or 543 of SEQ ID No. 31.
C69. The composition of any one of entries C59-C64, wherein the cysteine protease domain comprises a mutation at position 698, 653, 587, 544, or any combination thereof, according to the numbering of SEQ ID No. 2.
C70. The composition of any one of entries C59-C64, wherein the cysteine protease domain comprises SEQ ID NO 33.
C71. The composition of item C70, wherein the cysteine protease domain comprises a non-conservative mutation that is present at position 1, 44, 110, 155 of SEQ ID NO:33, or any combination thereof.
C72. The composition of item C59, wherein the mutant Clostridium difficile toxin B comprises SEQ ID NO 6.
C73. The composition of item C59, wherein the mutant Clostridium difficile toxin B comprises SEQ ID NO 86.
C74. The composition of item C59, wherein the mutant Clostridium difficile toxin B comprises SEQ ID NO 8.
C75. The composition of item C59, wherein the mutant Clostridium difficile toxin B comprises SEQ ID NO: 85.
C76. The composition of any of entries C59-C71, wherein at least one amino acid of the mutant clostridium difficile toxin B is chemically cross-linked.
C77. The composition of entry C76, wherein the amino acid is chemically crosslinked by formaldehyde.
C78. The composition of clause C76, wherein the amino acid is chemically crosslinked by 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide.
C79. The composition of clauses C76 or C78, wherein the at least one amino acid is chemically crosslinked by N-hydroxysuccinimide.
C80. The composition of any one of items C59-C79, wherein the composition is recognized by an antitoxin B neutralizing antibody or binding fragment thereof.
C81. An immunogenic composition comprising a mutant clostridium difficile toxin B comprising, relative to a corresponding wild-type clostridium difficile toxin B: a glucosyltransferase domain comprising SEQ ID No. 31 with amino acid substitutions at positions 286 and 288; and a cysteine protease domain comprising SEQ ID NO 33 having an amino acid substitution at position 155; wherein at least one amino acid of the mutant clostridium difficile toxin B is chemically cross-linked.
C82. An immunogenic composition comprising SEQ ID NO 6 or SEQ ID NO 8 wherein at least one amino acid of SEQ ID NO 6 or SEQ ID NO 8 is chemically cross-linked.
C83. The composition of clauses C81 or C82, wherein the at least one amino acid is crosslinked by formaldehyde.
C84. The composition of clauses C81 or C82, wherein the at least one amino acid is crosslinked by 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide.
C85. The composition of entry C81, C82, or C84, wherein the at least one amino acid is crosslinked by N-hydroxysuccinimide.
C86. The composition of clauses C81 or C82, wherein the composition is recognized by an antitoxin B neutralizing antibody or binding fragment thereof.
C87. An immunogenic composition comprising SEQ ID NO 6.
C88. An immunogenic composition comprising SEQ ID NO 86.
C89. An immunogenic composition comprising SEQ ID NO 8.
C90. An immunogenic composition comprising SEQ ID NO 85.
C91. The composition of any one of entries C59-C89, wherein the composition exhibits reduced cytotoxicity relative to corresponding wild-type clostridium difficile toxin B.
C92. Immunogenic compositions comprising SEQ ID NO. 4 and immunogenic compositions comprising SEQ ID NO. 6, wherein at least one amino acid of each of SEQ ID NO. 4 and 6 is chemically cross-linked.
C93. Immunogenic compositions comprising SEQ ID NO 84 and immunogenic compositions comprising SEQ ID NO 86, wherein at least one amino acid of each of SEQ ID NO 84 and 86 is chemically cross-linked.
C94. The composition of clauses C92 or C93, wherein the at least one amino acid is crosslinked by formaldehyde.
C95. The composition of clauses C92 or C93, wherein the at least one amino acid is crosslinked by 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide.
C96. The composition of entry C92, C93, or C95, wherein the at least one amino acid is crosslinked by N-hydroxysuccinimide.
C97. The recombinant cell, or progeny thereof, comprises SEQ ID NO 11, 12, 13, 14, 44, 45, 46, or 47.
C98. The recombinant cell, or progeny thereof, comprises a nucleic acid sequence encoding SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 7, or SEQ ID NO 8.
C99. A recombinant cell, or progeny thereof, comprising a nucleic acid sequence encoding SEQ ID NO: 84.
C100. The recombinant cell, or progeny thereof, comprises a nucleic acid sequence encoding SEQ ID NO 86.
C101. A recombinant cell, or progeny thereof, comprising a nucleic acid sequence encoding SEQ ID NO 83.
C102. A recombinant cell, or progeny thereof, comprising a nucleic acid sequence encoding SEQ ID NO 85.
C103. The recombinant cell of clauses C97 or C98, wherein the cell is derived from a gram-positive bacterial cell.
C104. The recombinant cell of entry C97, C98, or C99, wherein the cell is derived from a clostridium difficile cell.
C105. The recombinant cell of any one of items C97-C104, wherein the cell lacks an endogenous polynucleotide encoding a toxin.
C106. The cell of item C104 or C105, wherein the cell is derived from a clostridium difficile cell selected from the group consisting of: clostridium difficile 1351, clostridium difficile 3232, clostridium difficile 7322, clostridium difficile 5036, clostridium difficile 4811 and clostridium difficile VPI 11186.
C107. The cell of item C106, wherein the cell is a Clostridium difficile VPI11186 cell.
C108. The cell of item C106 or C107, wherein the sporulation gene of the clostridium difficile cell is inactivated.
C109. The cell of entry C108, wherein the sporulation gene comprises the spo0A gene or the spolie gene.
C110. A method of producing a mutant clostridium difficile toxin, comprising:
culturing the recombinant cell or progeny thereof under suitable conditions to express a polynucleotide encoding a mutant clostridium difficile toxin, wherein the cell comprises the polynucleotide encoding the 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.
C111. The method of item C110, wherein the cell lacks an endogenous polynucleotide encoding a toxin.
C112. The method of item C110, wherein said recombinant cell or progeny thereof comprises a cell of any of items C97-C111.
C113. The method of item C110, further comprising isolating the mutant Clostridium difficile toxin.
C114. The method of clause C113, further comprising contacting the isolated mutant Clostridium difficile toxin with formaldehyde.
C115. The method of clause C114, wherein the contacting occurs for up to 14 days.
C116. The method of clause C115, wherein the contacting occurs for up to 48 hours.
C117. The method of clause C114, wherein the contacting occurs at about 25 ℃.
C118. The method of clause C113, further comprising contacting the isolated mutant Clostridium difficile toxin with ethyl-3- (3-dimethylaminopropyl) carbodiimide.
C119. The method of clause C118, wherein the contacting occurs for up to 24 hours.
C120. The method of clause C120, wherein the contacting occurs for up to 4 hours.
C121. The method of clause C118, wherein the contacting occurs at about 25 ℃.
C122. The method of clause C118, further comprising contacting the isolated mutant Clostridium difficile toxin with N-hydroxysuccinimide.
C123. An immunogenic composition produced by the method of any one of items C110-C122.
C124. A method of generating neutralizing antibodies against clostridium difficile toxin a, comprising: administering to a mammal an immunogenic composition comprising SEQ ID No. 4, wherein the methionine residue at position 1 is optionally absent, wherein at least one amino acid of SEQ ID No. 4 is crosslinked by formaldehyde, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, or a combination of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, and recovering the antibody from the mammal.
C125. A method of generating neutralizing antibodies against clostridium difficile toxin a, comprising: administering to a mammal an immunogenic composition comprising SEQ ID NO:84, wherein at least one amino acid of SEQ ID NO:84 is crosslinked by formaldehyde, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, or a combination of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, and recovering the antibody from the mammal.
C126. A method of generating neutralizing antibodies against clostridium difficile toxin B, comprising: administering to a mammal an immunogenic composition comprising SEQ ID No. 6, wherein the methionine residue at position 1 is optionally absent, wherein at least one amino acid of SEQ ID No. 6 is crosslinked by formaldehyde, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, or a combination of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, and recovering the antibody from the mammal.
C127. A method of generating neutralizing antibodies against clostridium difficile toxin a, comprising: administering to a mammal an immunogenic composition comprising SEQ ID NO 86, wherein at least one amino acid of SEQ ID NO 86 is crosslinked by formaldehyde, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, or a combination of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, and recovering the antibody from the mammal.
C128. An antibody or antibody binding fragment thereof specific for an immunogenic composition comprising SEQ ID No. 4 wherein the methionine residue at position 1 is optionally absent, or SEQ ID No. 7 wherein the methionine residue at position 1 is optionally absent.
C129. The antibody or antibody-binding fragment thereof of item C128, wherein the at least one amino acid in SEQ ID NO:4 where the methionine residue at position 1 is optionally absent, or the at least one amino acid in SEQ ID NO:7 where the methionine residue at position 1 is optionally absent, is crosslinked by formaldehyde, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, or a combination of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide.
C130. An antibody or antibody binding fragment thereof comprising: the amino acid sequences of the heavy chain Complementarity Determining Regions (CDRs) shown in SEQ ID NO:41(CDR H1), SEQ ID NO:42(CDR H2) and SEQ ID NO:43(CDR H3), and the amino acid sequences of the light chain CDRs shown in SEQ ID NO:38(CDR L1), SEQ ID NO:39(CDRL2) and SEQ ID NO:40(CDR L3).
C131. An antibody or antibody-binding fragment thereof of item C128, C129, or C130, wherein the antibody or antibody-binding fragment thereof comprises: a heavy chain comprising the amino acid sequence set forth in SEQ ID NO 37; and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 36.
C132. A composition comprising two or more antibodies or antibody binding fragments thereof, wherein said antibodies or antibody binding fragments thereof are selected from any antibody or antibody binding fragment thereof of any one of items C128-C131.
C133. An antibody or antibody binding fragment thereof specific for an immunogenic composition comprising: 6, or 8, wherein the methionine residue at position 1 is optionally absent.
C134. The antibody or antibody-binding fragment thereof of item C133, wherein the at least one amino acid in SEQ ID NO:6 where the methionine residue at position 1 is optionally absent, or the at least one amino acid in SEQ ID NO:8 where the methionine residue at position 1 is optionally absent, is crosslinked by formaldehyde, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, or a combination of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide.
C135. An antibody or antibody binding fragment thereof comprising: the amino acid sequences of the heavy chain Complementarity Determining Regions (CDRs) shown in SEQ ID NO:51(CDR H1), SEQ ID NO:52(CDR H2) and SEQ ID NO:53(CDR H3), and the amino acid sequences of the light chain CDRs shown in SEQ ID NO:57(CDR L1), SEQ ID NO:58(CDR L2) and SEQ ID NO:59(CDR L3).
C136. An antibody or antibody binding fragment thereof comprising: the amino acid sequences of the heavy chain Complementarity Determining Regions (CDRs) shown in SEQ ID NO:61(CDR H1), SEQ ID NO:62(CDR H2) and SEQ ID NO:63(CDR H3), and the amino acid sequences of the light chain CDRs shown in SEQ ID NO:68(CDR L1), SEQ ID NO:69(CDR L2) and SEQ ID NO:70(CDR L3).
C137. An antibody or antibody binding fragment thereof comprising: the amino acid sequences of the heavy chain Complementarity Determining Regions (CDRs) shown in SEQ ID NO:73(CDR H1), SEQ ID NO:74(CDR H2) and SEQ ID NO:75(CDR H3), and the amino acid sequences of the light chain CDRs shown in SEQ ID NO:79(CDR L1), SEQ ID NO:80(CDR L2) and SEQ ID NO:81(CDR L3).
C138. A composition comprising two or more antibodies or antibody binding fragments thereof, wherein said antibodies or antibody binding fragments thereof are selected from any one of items C133-C137.
C139. A method of treating clostridium difficile infection in a mammal, comprising: administering to the mammal an immunogenic composition comprising SEQ ID NO. 4 in which the methionine residue at position 1 is optionally absent, and an immunogenic composition comprising SEQ ID NO. 6 in which the methionine residue at position 1 is optionally absent, wherein at least one amino acid of each of SEQ ID NO. 4 and 6 is cross-linked by formaldehyde.
C140. A method of treating clostridium difficile infection in a mammal, comprising: administering to the mammal an immunogenic composition comprising SEQ ID NO 4 wherein the methionine residue at position 1 is optionally absent, and an immunogenic composition comprising SEQ ID NO 6 wherein the methionine residue at position 1 is optionally absent, wherein at least one amino acid of each of SEQ ID NO 4 and SEQ ID NO 6 is crosslinked by 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, or a combination of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide.
C141. A method of treating a clostridium difficile infection in a mammal, comprising administering to the mammal: an immunogenic composition comprising SEQ ID NO 84 and an immunogenic composition comprising SEQ ID NO 86, wherein at least one amino acid of each of SEQ ID NO 84 and SEQ ID NO 86 is crosslinked by 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide.
C142. A method of inducing an immune response to clostridium difficile in a mammal, comprising administering to the mammal: an immunogenic composition comprising SEQ ID NO 4 wherein the methionine residue at position 1 is optionally absent; and an immunogenic composition comprising SEQ ID NO 6, wherein the methionine residue at position 1 is optionally absent; wherein at least one amino acid of each of SEQ ID NO. 4 and SEQ ID NO. 6 is crosslinked by formaldehyde.
C143. A method of inducing an immune response to clostridium difficile in a mammal, comprising administering to the mammal: an immunogenic composition comprising SEQ ID NO 4 wherein the methionine residue at position 1 is optionally absent; and an immunogenic composition comprising SEQ ID NO 6, wherein the methionine residue at position 1 is optionally absent; wherein each of SEQ ID NO 4 and SEQ ID NO 6 has at least one amino acid crosslinked by 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, or a combination of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide.
C144. A method of inducing an immune response to clostridium difficile in a mammal, comprising administering to the mammal: an immunogenic composition comprising SEQ ID NO 84 and an immunogenic composition comprising SEQ ID NO 86, wherein at least one amino acid of each of SEQ ID NO 84 and SEQ ID NO 86 is crosslinked by 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide.
C145. The method of any one of items C139-C144, wherein the mammal is a mammal in need thereof.
C146. The method of any one of items C139-C144, wherein the mammal has a recurrent Clostridium difficile infection.
C147. The method of any of clauses C139-C144, wherein the composition is administered parenterally.
C148. The method of any one of items C139-C144, wherein the composition further comprises an adjuvant.
C149. The method of clause C148, wherein the adjuvant comprises aluminum.
C150. The method of item C148, wherein the adjuvant comprises aluminum hydroxide gel and CpG oligonucleotide.
C151. The method of item C148, wherein said adjuvant comprises