Human Antibodies To Clostridium Difficile Toxins

Abstract

The present invention provides fully human antibodies that bind to either toxin A or toxin B of Clostridium difficile, or to both toxin A and toxin B, compositions comprising the antibodies and methods of use. The antibodies of the invention are useful for neutralizing the toxins from C. difficile, thus providing a means of treating the disease and symptoms associated with a C. difficile infection, including the treatment of diarrhea, or pseudomembranous colitis caused by C. difficile. The antibodies may also prevent the severity and/or duration of the primary disease, or may prevent the number, duration, and/or the severity of recurrences, or relapses of the disease attributed to the presence of C. difficile. The antibodies of the invention may also be useful for diagnosis of an infection by C. difficile.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. application Ser. No. 13/782,444, filed Mar. 1, 2013, which claims the benefit under 35 U.S.C .sctn.119(e) of U.S. provisional application No. 61/605,914, filed Mar. 2, 2012; 61/608,255, filed Mar. 8, 2012, and 61/717,404, filed Oct. 23, 2012, all of which are herein specifically incorporated by reference in their entirety.

FIELD OF THE INVENTION

[0002] The present invention is related to human antibodies and antigen-binding fragments of human antibodies that specifically bind to toxin A and/or toxin B of Clostridium difficile, compositions comprising these antibodies and therapeutic methods of using these antibodies.

STATEMENT OF RELATED ART

[0003] Clostridium difficile (C. difficile) is a gram positive, anaerobic, spore forming bacterium, which is a major cause of hospital-acquired gastrointestinal disease in humans, resulting in symptoms ranging from mild to severe diarrhea and colitis. It is believed that treatment with broad spectrum antibiotics, such as ampicillin, amoxicillin, cephalosporins, fluoroquinolones and clindamycin, may result in disruption of normal intestinal flora, which then allows for colonization of the gut with C. difficile (Kelly and Lamont, (1998), Ann. Rev. Med. 49:375-90). Treatment of C. difficile infections may involve stopping or modifying the use of broad spectrum antibiotics and requires commencing treatment with specific anticlostridial antibiotics, such as, for example, vancomycin, metronidazole, or fidaxomicin.

[0004] The diarrhea and inflammation observed in patients suffering from a C. difficile infection is believed to be due to the production of two toxins by the bacterium, enterotoxin (toxin A) and cytotoxin (toxin B). C. difficile toxins A and B are high molecular weight glucosyltransferases that inhibit members of the Rho family of GTPases. Toxin A has a molecular weight of 308 kDa and Toxin B has a molecular weight of 270 kDa. Both toxin A and toxin B deactivate small GTPases such as Rho, Rac and Cdc42 by glucosylation of a threonine residue. Inhibition of these GTPases causes the shutdown of signal transduction cascades leading to: depolymerization of the cytoskeleton, gene transcription of certain stress-activated protein kinases, a drop in synthesis of phosphatidylinositol bisphosphate, and possibly even the loss of cell polarity. Loss of cytoskeletal structure results in cell rounding, and this loss of structure may account for the host reactions to C. difficile. Toxin B is at least 1,000 times more cytotoxic than toxin A in cell rounding assays.

[0005] C. difficile toxins A and B are 63% homologous in amino acid content and have a similar three-dimensional structure (Davies, A H, (2011), Biochem. J., 436:517-526). The C-terminal third of each toxin is made up of sequences called clostridial repetitive oligopeptides (CROPs), which are highly antigenic. The remaining N-terminal two-thirds of toxins A and B are less similar to each other with respect to sequence homology; however, it is this portion of each protein that contains the glucosyltransferase activity.

[0006] Support for the role of toxin A and/or toxin B in the onset of diarrhea and inflammation following infection with C. difficile stems from observations in animal models. For example, oral dosing with the toxins mimics the disease (Kelly and Lamont, (1998), Ann. Rev. Med. 49:375-90). Mutant strains lacking toxin A and B have reduced or altered virulence (Lyras D, O'Connor J R, Howarth P K et al., Nature 458(7242), 1176-1179 (2009); Kuehne S A, Cartman S T, Heap J T, Kelly M L, Cockayne A, Minton N P, Nature 15, 467(7316), 711-713 (2010).). Furthermore, administration of polyclonal antibodies to the toxins has been shown to protect hamsters from the disease (Gianasca et al., (1999), Infect. Immun. 66(2): 527-38). In the clinic, studies have shown that there is a correlation between the presence of anti-toxin A or anti-toxin B antibodies and protection against C. difficile associated diarrhea and disease recurrence (Warny, M. et al., (1994), Inf. Immun. 62(2): 384-389; Kyne, L. et al. (2001), Lancet 357:189-193; Leav, B. A., (2010), Vaccine 28(4):965-969). Development of anti-toxin antibody is associated with asymptomatic carriers (Kyne, L. et al. (2000), NEJM 342(6), 390-397). Furthermore, a clinical trial using a combination of C. difficile anti-toxin A and anti-toxin B antibodies in conjunction with metronidazole or vancomycin resulted in a reduction in the rate of recurrent infection with C. difficile (Lowy, I. et al., (2010), NEJM 362(3):197-205).

[0007] Monoclonal antibodies to C. difficile toxin A have been described by Wilkins, et al. in U.S. Pat. No. 4,879,218. In addition, Rothman et al. described a murine monoclonal antibody that cross-reacts with C. difficile toxins A and B. Furthermore, Coughlin et al. described a monoclonal antibody specific for C. difficile toxin B, which did not cross-react with toxin A. Other antibodies to the C. difficile toxins have been described (See, for example, U.S. Pat. No. 7,151,159; U.S. Pat. No. 7,625,559; U.S. Pat. No. 8,236,311; U.S. Pat. No. 8,257,709; US publication Nos. 2009/0087478; US2010/0233182; US2010/0233181; US2012/0288508; US2012/012160; U52011/0020356; US2012/0121607; EP1766093B1; EP1024826B1; EP1568378A1; EP2305303A2; EP2305293A2; EP2405940A1; EP2261253A2; WO2006/121422; WO2011/130650; WO2010/094970; WO2009/108652; WO2011/063346 and WO2005/058353).

BRIEF SUMMARY OF THE INVENTION

[0008] The invention provides fully human monoclonal antibodies (mAbs) and antigen-binding fragments thereof that bind specifically to either toxin A or to toxin B produced by Clostridium difficile (C. difficile), or which bind to both toxin A and toxin B of C. difficile (ie. human monoclonal antibodies that cross react with both toxin A and toxin B). Such antibodies may be useful to neutralize the toxicity associated with either toxin A or toxin B, or both, and as such, may act to lessen the severity of the primary C. difficile-associated condition or disease, or reduce the number, the duration, or the severity of disease recurrence, or ameliorate at least one symptom associated with the C. difficile-associated condition or disease. Such antibodies may be used alone or in conjunction with a second agent useful for treating a C. difficile-associated condition or disease. In certain embodiments, the antibodies specific for toxin A, toxin B, or both, may be given therapeutically in conjunction with a second agent to lessen the severity of the primary C. difficile-associated condition or disease, or to reduce the number, the duration, or the severity of disease recurrence, or ameliorate at least one symptom associated with the C. difficile-associated condition or disease. In certain embodiments, the antibodies may be used prophylactically as stand-alone therapy to protect patients who are at risk for developing a C. difficile-associated condition or disease. For example, certain patient populations may be at risk for developing a C. difficile condition or disease, including elderly patients, or patients who have chronic and/or concomitant underlying medical conditions that may pre-dispose them to a C. difficile infection. Other at-risk patient populations include patients who are hospitalized for extended periods of time and who are taking broad spectrum antibiotics that may disrupt the normal intestinal flora and which may predispose them to infection with C. difficile. More recent data suggest that patients taking proton pump inhibitors (PPIs) are at risk for developing C. difficile-associated diarrhea (Yearsley, K. et al. (2006), Aliment. Pharmacol. Ther. 24(4):613-619; Lowe, D O, et al. Clin. Infect. Dis. (2006), 43(10):1272-1276). Other patient populations at risk for developing a C. difficile infection include patients who are undergoing any type of immunosuppressive therapy, such as, but not limited to an anti-cancer drug, general radiotherapy to treat certain cancers, or a drug or drug regimen to prevent tissue or organ graft rejection following a transplant. Patients who receive a hematopoietic stem cell transplant (HSCT) may be at particularly high risk for developing a C. difficile infection because of long hospitalizations, receipt of broad-spectrum antibiotics and chemotherapy-related disruption of enteric mucosal barriers (Thibault, A. et al., ((1991), Infect. Control Hosp. Epidemiol. 12:345-8; Anand, A. et al. (1993), Clin. Infect. Dis. 17:109-13). Patients who receive a solid organ transplant may also be at risk for developing a C. difficile infection. Included in the at-risk population are patients suffering from an autoimmune disease, or patients on dialysis. More recent studies demonstrated that patients who received either an autologous or allogeneic HSCT were not only at greater risk for developing a C. difficile infection, but these patients were also at higher risk of developing gastrointestinal graft versus host disease (GI-GVHD) (Alonso, C. D., et. al. (2012), Clin Inf. Dis, 54:1053-1063). While this study clearly demonstrated that C. difficile infections were a frequent early complication following HSCT, the exact relationship or interplay between C. difficile infections (CDI) and GVHD involving the GI tract needs to be explored in greater detail. Any of these patient populations may benefit from treatment with the antibodies of the invention, when given alone or in conjunction with a second agent, such as metronidazole, vancomycin or fidaxomicin.

[0009] The antibodies of the invention can be full-length (for example, an IgG1 or IgG4 antibody) or may comprise only an antigen-binding portion (for example, a Fab, F(ab').sub.2 or scFv fragment), and may be modified to affect functionality, e.g., to eliminate residual effector functions (Reddy et al., (2000), J. Immunol. 164:1925-1933).

[0010] Accordingly, in a first aspect, the invention provides an isolated fully human monoclonal antibody or antigen-binding fragment thereof that binds to either toxin A, or to toxin B, or that binds to or cross reacts with both toxin A and toxin B of Clostridium difficile, wherein:

[0011] a) the isolated antibody or antigen-binding fragment thereof that specifically binds toxin A of Clostridium difficile comprises the three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 98, 114, 130, 146 and 162; and the three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 106, 122, 138, 154 and 170;

[0012] b) the isolated antibody or antigen-binding fragment thereof that specifically binds toxin B of Clostridium difficile comprises the HCDR1, HCDR2 and HCDR3 contained within a HCVR amino acid sequence selected from the group consisting of SEQ ID NOs: 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338 and 354; and the LCDR1, LCDR2 and LCDR3 contained within a LCVR amino acid sequence selected from the group consisting of SEQ ID NOs: 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346 and 362; and

[0013] c) the isolated antibody or antigen-binding fragment that binds to, or cross reacts with both toxin A and toxin B of Clostridium difficile comprises the HCDR1, HCDR2 and HCDR3 contained within a HCVR amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 34, 50, 66 and 82; and the LCDR1, LCDR2 and LCDR3 contained within a LCVR amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 42, 58, 74 and 90.

[0014] In one embodiment, the human monoclonal antibody that binds to/cross reacts with both toxin A and toxin B of C. difficile specifically binds to the carboxy terminal receptor binding domain (CBD) of both toxin A (CBD-A: SEQ ID NO: 375) and toxin B (CBD-B:SEQ ID NO: 376) of C. difficile.

[0015] In one embodiment, the isolated human antibody or antigen-binding fragment thereof which binds to/cross reacts with both toxin A and toxin B of C. difficile binds to toxin A and toxin B with a K.sub.D equal to or less than 10.sup.-7 M.

[0016] In one embodiment, the isolated human antibody or antigen-binding fragment thereof which binds to/cross reacts with both toxin A and toxin B of C. difficile comprises the three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within any one of the heavy chain variable region (HCVR) sequences selected from the group consisting of SEQ ID NOs: 18, 34, 50, 66 and 82; and the three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within any one of the light chain variable region (LCVR) sequences selected from the group consisting of SEQ ID NOs: 26, 42, 58, 74 and 90. Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and/or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify the boundaries of CDRs include, e.g., the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of the structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., (1997), J. Mol. Biol. 273:927-948; and Martin et al., (1989), Proc. Natl. Acad. Sci. USA 86:9268-9272. Public databases are also available for identifying CDR sequences within an antibody.

[0017] In one embodiment, the isolated human antibody or antigen-binding fragment thereof which binds to/cross reacts with both toxin A and toxin B of C. difficile comprises a HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 34, 50, 66 and 82.

[0018] In one embodiment, the isolated human antibody or antigen-binding fragment thereof which binds to/cross reacts with both toxin A and toxin B of C. difficile comprises a LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 42, 58, 74 and 90.

[0019] In one embodiment, the isolated human antibody or antigen-binding fragment thereof which binds to/cross reacts with both toxin A and toxin B of C. difficile comprises (a) a HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 34, 50, 66 and 82; and (b) a LCVR having an amino acid sequence selected from the group consisting of SEQ ID NO: 26, 42, 58, 74 and 90.

[0020] In one embodiment, the isolated human antibody or antigen-binding fragment thereof which binds to/cross reacts with both toxin A and toxin B of C. difficile comprises: [0021] (a) a HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 36, 52, 68, and 84; [0022] (b) a HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 38, 54, 70 and 86; [0023] (c) a HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 40, 56, 72 and 88; [0024] (d) a LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 44, 60, 76 and 92; [0025] (f) a LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 46, 62, 78 and 94; and [0026] (g) a LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 48, 64, 80 and 96.

[0027] In one embodiment, the human antibody or antigen binding fragment thereof that binds to/cross reacts with both toxin A and toxin B of C. difficile comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of SEQ ID NO: 20, 22 and 24, respectively and LCDR1, LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 28, 30 and 32, respectively.

[0028] In one embodiment, the human antibody or antigen binding fragment thereof that binds to/cross reacts with both toxin A and toxin B of C. difficile comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of SEQ ID NO: 36, 38 and 40, respectively and LCDR1, LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 44, 46 and 48, respectively.

[0029] In one embodiment, the isolated human antibody or antigen-binding fragment thereof which binds to/cross reacts with both toxin A and toxin B of C. difficile comprises a HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 18/26, 34/42, 50/58, 66/74 and 82/90.

[0030] In one embodiment, the isolated human antibody or antigen-binding fragment thereof which binds to/cross reacts with both toxin A and toxin B of C. difficile comprises the HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 18/26 and 34/42.

[0031] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds to/cross reacts with both toxin A and toxin B binds to: [0032] an epitope within the carboxy terminal receptor binding domain of both toxin A and toxin B of Clostridium difficile, wherein the antibody comprises a HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 18/26 and 34/42; or [0033] an epitope outside of the carboxy terminal receptor binding domain of both toxin A and toxin B of Clostridium difficile, wherein the antibody comprises a HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 50/58, 66/74 and 82/90.

[0034] In one embodiment, the invention provides a fully human monoclonal antibody or antigen-binding fragment thereof that binds to/cross reacts with both toxin A and toxin B of C. difficile, wherein the antibody or fragment thereof exhibits one or more of the following characteristics: (i) comprises a HCVR having an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 34, 50, 66 and 82, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (ii) comprises a LCVR having an amino acid sequence selected from the group consisting of SEQ ID NO: 26, 42, 58, 74 and 90, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (iii) comprises a HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 24, 40, 56, 72 and 88, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 32, 48, 64, 80 and 96, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (iv) comprises a HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 20, 36, 52, 68 and 84, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 22, 38, 54, 70 and 86, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 28, 44, 60, 76 and 92, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 30, 46, 62, 78 and 94, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (v) binds to toxin A and to toxin B with a K.sub.D equal to or less than 10.sup.-9M.

[0035] In one embodiment, the fully human monoclonal antibody or antigen binding fragment thereof that binds to/cross reacts with both toxin A and toxin B of C. difficile comprises a HCDR1 sequence comprising the formula X.sup.1-X.sup.2-X.sup.3-X.sup.4-X.sup.5-X.sup.6-X.sup.7-X.sup.8 (SEQ ID NO: 381), wherein X.sup.1 is Gly, X.sup.2 is Phe, Val, or Ile, X.sup.3 is Thr, Ala, or Ser, X.sup.4 is Phe or Leu, X.sup.5 is Ser, Arg, or Asn, X.sup.6 is Gly, Thr, Asp, or Ser, X.sup.7 is His, or Tyr, and X.sup.8 is Gly, or Glu; a HCDR2 sequence comprising the formula X.sup.1-X.sup.2-X.sup.3-X.sup.4-X.sup.5-X.sup.6-X.sup.7-X.sup.8 (SEQ ID NO: 382), wherein X.sup.1 is Ile, X.sup.2 is Leu, Ser, or Asp, X.sup.3 is Tyr, Phe, or Ser, X.sup.4 is Asp, or Ser, X.sup.5 is Gly, X.sup.6 is Ser, Gly, Asp, or Thr, X.sup.7 is Ser, His, or Ile, and X.sup.8 is Glu, Gln, or Ile; a HCDR3 sequence comprising the formula X.sup.1-X.sup.2-X.sup.3-X.sup.4-X.sup.5-X.sup.6-X.sup.7-X.sup.8-X.sup.9-X- .sup.10-X.sup.11-X.sup.12-X.sup.13-X.sup.14-X.sup.15-X.sup.16-X.sup.17 (SEQ ID NO: 383), wherein X.sup.1 is Ala, or Val, X.sup.2 is Lys, or Arg, X.sup.3 is Gly, or Glu, X.sup.4 is Ser, or Arg, X.sup.5 is Ile, Asp, or Tyr, X.sup.6 is Leu, Ser, or Asp, X.sup.7 is Asn, Ser, Gln, or His, X.sup.8 is Arg, Tyr, or Ser, X.sup.9 is Pro, or Gly, X.sup.10 is Phe, or Tyr, X.sup.11 is Asp, Gly, or Tyr, X.sup.12 is Tyr, X.sup.13 is Phe, Leu, or absent, X.sup.14 is Gly, or absent, X.sup.15 is Met, or absent, X.sup.16 is Asp, or absent, X.sup.17 is Val, or absent; a LCDR1 sequence comprising the formula X.sup.1-X.sup.2-X.sup.3-X.sup.4-X.sup.5-X.sup.6-X.sup.7-X.sup.8-X.sup.9-X- .sup.10-X.sup.11-X.sup.12 (SEQ ID NO: 384), wherein X.sup.1 is Gln, X.sup.2 is Ser, or Glu, X.sup.3 is Ile, Val, or Thr, X.sup.4 is Leu, or Asp, X.sup.5 is Phe, Lys, or Asn, and X.sup.6 is Ser, or Trp, X.sup.7 is Ser, or absent, X.sup.8 is Asn, Asp, or absent, X.sup.9 is Asn, or absent, X.sup.10 is Lys, or absent, X.sup.11 Ile, Asn, or absent, X.sup.12 is Tyr, or absent; a LCDR2 sequence comprising the formula X.sup.1-X.sup.2-X.sup.3 (SEQ ID NO: 385), wherein X.sup.1 is Trp, Lys, or Arg, X.sup.2 is Ala or Thr, and X.sup.3 is Ser; and a LCDR3 sequence comprising the formula X.sup.1-X.sup.2-X.sup.3-X.sup.4-X.sup.5-X.sup.6-X.sup.7-X.sup.8-X.sup.9 (SEQ ID NO: 386), wherein X.sup.1 is Gln or His, X.sup.2 is Gln, or Glu, X.sup.3 is Tyr, X.sup.4 is Tyr, or Asn, X.sup.5 is Thr, or Ser, X.sup.6 is Leu, Ala, or Tyr, X.sup.7 is Pro, Phe, or Ser, X.sup.8 is Leu, Phe, or Arg and X.sup.9 is Thr, or Ala.

[0036] In one embodiment, the invention provides an isolated human monoclonal antibody or antigen-binding fragment thereof that binds specifically to toxin A of Clostridium difficile, wherein the antibody comprises the three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within any one of the HCVR amino acid sequences selected from the group consisting of SEQ ID NOs: 2, 98, 114, 130, 146 and 162; and the three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within any one of the LCVR amino acid sequences selected from the group consisting of SEQ ID NOs: 10, 106, 122, 138, 154 and 170.

[0037] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds specifically to toxin A of Clostridium difficile, comprises a HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 98, 114, 130, 146 and 162.

[0038] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds specifically to toxin A of Clostridium difficile, comprises a LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 106, 122, 138, 154 and 170.

[0039] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds specifically to toxin A of Clostridium difficile, comprises (a) a HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 98, 114, 130, 146 and 162; and (b) a LCVR having an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 106, 122, 138, 154 and 170.

[0040] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds specifically to toxin A of Clostridium difficile, comprises:

[0041] (a) a HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 100, 116, 132, 148 and 164;

[0042] (b) a HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 102, 118, 134, 150 and 166;

[0043] (c) a HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 104, 120, 136, 152 and 168;

[0044] (d) a LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 108, 124, 140, 156, and 172;

[0045] (e) a LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 110, 126, 142, 158 and 174; and

[0046] (f) a LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 112, 128, 144, 160 and 176.

[0047] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds specifically to toxin A of Clostridium difficile, comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of SEQ ID NO: 148, 150 and 152, respectively and LCDR1, LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 156, 158 and 160, respectively.

[0048] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds specifically to toxin A of Clostridium difficile, comprises a HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2/10, 98/106, 114/122, 130/138, 146/154 and 162/170.

[0049] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds specifically to toxin A of Clostridium difficile, comprises the HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 146/154.

[0050] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds specifically to toxin A of Clostridium difficile binds to: [0051] an epitope within the carboxy terminal receptor binding domain of toxin A of Clostridium difficile, wherein the antibody comprises a HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2/10, 98/106, 130/138, 146/154 and 162/170; or [0052] an epitope outside of the carboxy terminal receptor binding domain of toxin A of Clostridium difficile, wherein the antibody comprises a HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 114/122.

[0053] In one embodiment, the invention provides a fully human monoclonal antibody or antigen-binding fragment thereof that binds specifically to toxin A of C. difficile, wherein the antibody or fragment thereof exhibits one or more of the following characteristics: (i) comprises a HCVR having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 98, 114, 130, 146 and 162, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (ii) comprises a LCVR having an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 106, 122, 138, 154 and 170, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (iii) comprises a HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 104,120,136,152 and 168, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 16, 112, 128, 144, 160 and 176, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (iv) comprises a HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 100, 116, 132, 148 and 164, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 102, 118, 134, 150 and 166, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 108, 124, 140, 156 and 172, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 110, 126, 142, 158 and 174, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (v) demonstrates a K.sub.D equal to or less than 10.sup.-9; (vi) demonstrates neutralization of Toxin A (at a concentration of 32 pM) with an 1050 ranging from about 7 pM to about 65 pM in a cell viability assay.

[0054] In one embodiment, the fully human monoclonal antibody or antigen binding fragment thereof that binds specifically to toxin A of C. difficile comprises a HCDR1 sequence comprising the formula X.sup.1-X.sup.2-X.sup.3-X.sup.4-X.sup.5-X.sup.6-X.sup.7-X.sup.8 (SEQ ID NO: 387), wherein X.sup.1 is Gly, or Arg, X.sup.2 is Phe, X.sup.3 is Asn, or Thr, X.sup.4 is Phe, X.sup.5 is Gly, Ser, Asn, or Thr, X.sup.6 is Thr, Ser, Asn, or Asp, X.sup.7 is His, Tyr, or Phe and X.sup.8 is Asp, Val, Ala, or Tyr; a HCDR2 sequence comprising the formula X.sup.1-X.sup.2-X.sup.3-X.sup.4-X.sup.5-X.sup.6-X.sup.7-X.sup.8 (SEQ ID NO: 388), wherein X.sup.1 is Leu, or Ile, X.sup.2 is Thr, Gly, Ser, or Trp, X.sup.3 is Ser, Thr, Gly, or Phe, X.sup.4 is Thr, Val, Tyr, Val, Asp, or Gly, X.sup.5 is Gly, X.sup.6 is Gly, Asp, Ser, or Ala, X.sup.7 is Ser, Thr, Asn, or Ala, and X.sup.8 is Ala, Thr, Glu, Lys, or absent; a HCDR3 sequence comprising the formula X.sup.1-X.sup.2-X.sup.3-X.sup.4-X.sup.5-X.sup.6-X.sup.7-X.sup.8-X.sup.9-X- .sup.10-X.sup.11-X.sup.12-X.sup.13-X.sup.14-X.sup.15-X.sup.16-X.sup.17-X.s- up.18-X.sup.19-X.sup.20-X.sup.21-X.sup.22-X.sup.23-X.sup.24 (SEQ ID NO: 389), wherein X.sup.1 is Ala, X.sup.2 is Lys, or Arg, X.sup.3 is Thr, Asp, or Ser, X.sup.4 is Phe, Arg, His, Ala, or Leu, X.sup.5 is Asn, Gly, or Lys, X.sup.6 is Trp, Gly, Asp, or Ile, X.sup.7 is Asn, Ala, or Phe, X.sup.8 is Ser, Asn, Tyr, Gly, or Asp, X.sup.9 is Tyr, Ile, Ala, Thr, Glu, or Leu, X.sup.10 is Phe, Tyr, Ser, Gly, or absent, X.sup.11 is Asp. Ser, Gly, or absent, X.sup.12 is Tyr, Phe, Ser, Pro, or absent, X.sup.13 is Tyr, Leu, or absent, X.sup.14 is Tyr, Phe, or absent, X.sup.15 Gly, Asn, Asp, or absent, X.sup.16 is Met, Arg, Tyr, or absent, X.sup.17 is Asp, or absent, X.sup.18 is Tyr, Val, or absent, X.sup.19 is Tyr, or absent, X.sup.20 is Tyr, or absent, X.sup.21 is Gly, or absent, X.sup.22 is Met, or absent, X.sup.23 is Asp, or absent, X.sup.24 is Val, or absent; a LCDR1 sequence comprising the formula X.sup.1-X.sup.2-X.sup.3-X.sup.4-X.sup.5-X.sup.6-X.sup.7 (SEQ ID NO: 390), wherein X.sup.1 is Gln, X.sup.2 is Ser, Asp, or Thr, X.sup.3 is Ile, or Val, X.sup.4 is Ser, X.sup.5 is Thr, Asn, or Ser, X.sup.6 is Tyr, Trp, Phe, or Ser and X.sup.7 is Tyr, or absent; a LCDR2 sequence comprising the formula X.sup.1-X.sup.2-X.sup.3 (SEQ ID NO: 391), wherein X.sup.1 is Gly, Ala, Lys, or Thr, X.sup.2 is Ala, Thr, or Val and X.sup.3 is Ser; and a LCDR3 sequence comprising the formula X.sup.1-X.sup.2-X.sup.3-X.sup.4-X.sup.5-X.sup.6-X.sup.7-X.sup.8-X.sup.9-X- .sup.10 (SEQ ID NO: 392), wherein X.sup.1 is Gln or absent, X.sup.2 is Gln, Lys, or absent, X.sup.3 is Tyr, Asn, or absent, X.sup.4 is Gly, Asn, Thr, Tyr, His, or absent, X.sup.5 is Asn, Ser, or absent, X.sup.6 is Ser, Ala, Tyr, Asp, Trp, or absent, X.sup.7 is Leu, Pro, Ser, or absent, X.sup.8 is Tyr, Phe, Arg, Pro, or absent, X.sup.9 is Thr, Tyr, or absent, and X.sup.10 is Thr.

[0055] In one embodiment, the invention provides an isolated human monoclonal antibody or antigen-binding fragment thereof that binds specifically to toxin B of Clostridium difficile, wherein the antibody comprises the three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within any one of the HCVR amino acid sequences selected from the group consisting of SEQ ID NOs: 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338 and 354; and the three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within any one of the LCVR amino acid sequences selected from the group consisting of SEQ ID NOs: 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346 and 362.

[0056] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds specifically to toxin B of Clostridium difficile comprises a HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338 and 354.

[0057] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds specifically to toxin B of Clostridium difficile comprises a LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346 and 362.

[0058] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds specifically to toxin B of Clostridium difficile comprises (a) a HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338 and 354; and (b) a LCVR having an amino acid sequence selected from the group consisting of SEQ ID NO: 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346 and 362.

[0059] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds specifically to toxin B of Clostridium difficile comprises

[0060] (a) a HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 180, 196, 212, 228, 244, 260, 276, 292, 308, 324, 340 and 356;

[0061] (b) a HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 182, 198, 214, 230, 246, 262, 278, 294, 310, 326, 342 and 358;

[0062] (c) a HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 184, 200, 216, 232, 248, 264, 280, 296, 312, 328, 344 and 360;

[0063] (d) a LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 188, 204, 220, 236, 252, 268, 284, 300, 316, 332, 348 and 364;

[0064] (e) a LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 190, 206, 222, 238, 254, 270, 286, 302, 318, 334, 350 and 366; and

[0065] (f) a LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 192, 208, 224, 240, 256, 272, 288, 304, 320, 336, 352 and 368.

[0066] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds specifically to toxin B of Clostridium difficile, comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of SEQ ID NO: 276, 278 and 280, respectively and LCDR1, LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 284, 286 and 288, respectively.

[0067] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds specifically to toxin B of Clostridium difficile comprises a HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 178/186, 194/202, 210/218, 226/234, 242/250, 258/266, 274/282, 290/298, 306/314, 322/330, 338/346 and 354/362.

[0068] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds specifically to toxin B of Clostridium difficile comprises the HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 274/282.

[0069] In one embodiment, the isolated human antibody or antigen-binding fragment thereof that binds specifically to toxin B of Clostridium difficile binds to:

[0070] an epitope within the carboxy terminal receptor binding domain of toxin B of Clostridium difficile, wherein the antibody comprises a HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 178/186; or

[0071] an epitope outside of the carboxy terminal receptor binding domain of toxin B of Clostridium difficile, wherein the antibody comprises a HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 194/202, 210/218, 226/234, 242/250, 258/266, 274/282 and 290/298.

[0072] In one embodiment, the invention provides a fully human monoclonal antibody or antigen-binding fragment thereof that binds specifically to toxin B of C. difficile, wherein the antibody or fragment thereof exhibits one or more of the following characteristics: (i) comprises a HCVR having an amino acid sequence selected from the group consisting of SEQ ID NO: 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338 and 354, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (ii) comprises a LCVR having an amino acid sequence selected from the group consisting of SEQ ID NO:186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346 and 362, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (iii) comprises a HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NO:184, 200, 216, 232, 248, 264, 280, 296, 312, 328, 344 and 360, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NO:192, 208, 224, 240, 256, 272, 288, 304, 320, 336, 352 and 368, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (iv) comprises a HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 180,196, 212, 228, 244, 260, 276, 292, 308, 324, 340 and 356, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 182, 198, 214, 230, 246, 262, 278, 294, 310, 326, 342 and 358, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 188, 204, 220, 236, 252, 268, 284, 300, 316, 332, 348 and 364, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 190, 206, 222, 238, 254, 270, 286, 302, 318, 334, 350 and 366, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (v) demonstrates a K.sub.D equal to or less than 10.sup.-9M; (vi) demonstrates neutralization of Toxin B (at a concentration of 0.03 pM) with an 1050 ranging from about 25 pM to about 320 pM in a cell viability assay.

[0073] In one embodiment, the fully human monoclonal antibody or antigen binding fragment thereof that binds specifically to toxin B of C. difficile comprises a HCDR1 sequence comprising the formula X.sup.1-X.sup.2-X.sup.3-X.sup.4-X.sup.5-X.sup.6-X.sup.7-X.sup.8-X.sup.9-X- .sup.10 (SEQ ID NO: 393), wherein X.sup.1 is Gly, X.sup.2 is Phe, Asp, or Tyr, X.sup.3 is Thr, Asn, Ser, or Val, X.sup.4 is Phe, or Val, X.sup.5 is Ser, Arg, Lys, Glu, or Thr, X.sup.6 is Ser, Ile, Asp, or Arg, X.sup.7 is Phe, Tyr, or Asn, X.sup.8 is Gly, Ala, Ser, or Tyr; X.sup.9 is Ala, or absent and X.sup.10 is Ala or absent; a HCDR2 sequence comprising the formula X.sup.1-X.sup.2-X.sup.3-X.sup.4-X.sup.5-X.sup.6-X.sup.7-X.sup.8-X- .sup.9 (SEQ ID NO: 394), wherein X.sup.1 is Ile, or Thr, X.sup.2 is Ser, Gly, Tyr, or Asn, X.sup.3 is Thr, Gly, Tyr, Trp, Pro, or Ser, X.sup.4 is Asp, Ser, Asn, Arg, Lys, or Asp, X.sup.5 is Gly, Ser, or Thr, X.sup.6 is Ser, Asp, Gly, Lys, or Asn, X.sup.7 is Lys, Arg, Asn, Ser, Trp, or Gly, X.sup.8 is Lys, Thr, Ile, or Tyr, X.sup.9 is His, or absent; a HCDR3 sequence comprising the formula X.sup.1-X.sup.2-X.sup.3-X.sup.4-X.sup.5-X.sup.6-X.sup.7-X.sup.8-X.sup.9-X- .sup.10-X.sup.11-X.sup.12-X.sup.13-X.sup.14-X.sup.15-X.sup.16 (SEQ ID NO: 395), wherein X.sup.1 is Ala, or Val, X.sup.2 is Arg, Lys, Thr, or Ser, X.sup.3 is Val, Gly, Asp, Arg, or Tyr, X.sup.4 is Gly, Trp, Arg, Lys, or Asn, X.sup.5 is Glu, Tyr, Arg, Ser, or Trp, X.sup.6 is Leu, Tyr, Ser, Pro, or Asn, X.sup.7 Leu, Asp, Tyr, Ser, or Asp, X.sup.8 is Asn, Ser, Phe, Lys, Arg, Asp, or Gly, X.sup.9 is Tyr, Gly, Phe, Asp, Trp, or Val, X.sup.10 is Ser, Tyr, Asn, Asp, or absent, X.sup.11 is Tyr, Leu, Val, Gly, or absent, X.sup.12 is Tyr, Leu, Phe, Val, or absent, X.sup.13 is Asn, Gly, Asp, Phe, or absent, X.sup.14 is Tyr, Met, Asp, or absent, X.sup.15 Asp, Tyr, or absent, and X.sup.16 is Val, or absent; a LCDR1 sequence comprising the formula X.sup.1-X.sup.2-X.sup.3-X.sup.4-X.sup.5-X.sup.6-X.sup.7 (SEQ ID NO: 396), wherein X.sup.1 is Gln, Leu, or Arg, X.sup.2 is Gly, Asp, or Ser, X.sup.3 is Ile, or Val, X.sup.4 is Arg, Ser, Gly, or Tyr, X.sup.5 is Ser, or Asn, X.sup.6 is Trp, His, Asn, Phe, Ser, or Asp, and X.sup.7 is Tyr, or absent; a LCDR2 sequence comprising the formula X.sup.1-X.sup.2-X.sup.3 (SEQ ID NO: 397), wherein X.sup.1 is Ala, Ser, Asp, or Gly, X.sup.2 is Ala, or Thr, and X.sup.3 is Ser; and a LCDR3 sequence comprising the formula X.sup.1-X.sup.2-X.sup.3-X.sup.4-X.sup.5-X.sup.6-X.sup.7-X.sup.8-X- .sup.9 (SEQ ID NO: 398), wherein X.sup.1 is Gln, His, or Leu, X.sup.2 is Gln, X.sup.3 is Ala, Tyr, Arg, Asp, His, or Val, X.sup.4 is Tyr, Gly, Asn, Ser, Ile, or Lys, X.sup.5 is Ser, Leu, Pro, Ile, Asn, Thr, or Gly, X.sup.6 is Phe, Tyr, Trp, or Ser, X.sup.7 is Pro, X.sup.8 is Leu, Pro, Phe, Val, or Tyr and X.sup.9 is Thr.

[0074] In one embodiment, the invention provides an isolated antibody or antigen-binding fragment thereof that competes for specific binding to C. difficile toxin A and/or toxin B with an antibody or antigen-binding fragment comprising the complementarity determining regions (CDRs) of a heavy chain variable region (HCVR), wherein the HCVR has an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338 and 354; and the CDRs of a light chain variable region (LCVR), wherein the LCVR has an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346 and 362.

[0075] In a related embodiment, the invention provides an isolated antibody or antigen-binding fragment thereof that competes for specific binding to C. difficile toxin A and/or toxin B with an antibody or antigen-binding fragment comprising the heavy and light chain CDRs contained within heavy and light chain sequence pairs selected from the group consisting of SEQ ID NOs: 18/26, 34/42, 146/154 and 274/282.

[0076] In one embodiment, the invention provides an isolated antibody or antigen-binding fragment thereof that binds the same epitope on C. difficile toxin A and/or toxin B as an antibody or antigen-binding fragment comprising the CDRs of a heavy chain variable region (HCVR), wherein the HCVR has an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338 and 354; and the CDRs of a light chain variable region (LCVR), wherein the LCVR has an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346 and 362.

[0077] In a related embodiment, the invention provides an isolated antibody or antigen-binding fragment thereof that binds the same epitope on C. difficile toxin A and/or toxin B as an antibody or antigen-binding fragment comprising the heavy and light chain CDRs contained within heavy and light chain sequence pairs selected from the group consisting of SEQ ID NOs: 18/26, 34/42, 146/154, 274/282.

[0078] In certain embodiments of the invention, the antibodies may interact with, or bind to, amino acid residues 468-863 of the carboxy terminal receptor binding domain of toxin A produced by Clostridium difficile, the sequence of which is shown in SEQ ID NO: 375. This region corresponds to amino acid residues ranging from residues 2315-2710 of SEQ ID NO: 378 (full length toxin A). In certain embodiments of the invention, the antibodies may interact with, or bind to, an epitope in the carboxy terminal receptor binding domain of toxin A produced by Clostridium difficile, wherein the epitope is selected from the group consisting of residues 468-488 of SEQ ID NO: 375, residues 510-530 of SEQ ID NO: 375, residues 602-610 of SEQ ID NO: 375, residues 644-703 of SEQ ID NO: 375, residues 724-794 of SEQ ID NO: 375, residues 799-814 of SEQ ID NO: 375 and residues 858-863 of SEQ ID NO: 375. These residues correspond to the amino acid sequences found in the full length toxin A sequence having SEQ ID NO: 378, with the particular regions identified as residues 2315-2335 of SEQ ID NO: 378, residues 2357-2377 of SEQ ID NO: 378, residues 2449-2457 of SEQ ID NO: 378, residues 2491-2550 of SEQ ID NO: 378, residues 2571-2641 of SEQ ID NO: 378, residues 2646-2661 of SEQ ID NO: 378 and residues 2705-2710 of SEQ ID NO: 378. In one embodiment, the antibody that binds to or interacts with an epitope in the carboxy terminal receptor binding domain of toxin A produced by Clostridium difficile, selected from the group consisting of residues 468-488 of SEQ ID NO: 375, residues 510-530 of SEQ ID NO: 375, residues 602-610 of SEQ ID NO: 375, residues 644-703 of SEQ ID NO: 375, residues 724-794 of SEQ ID NO: 375, residues 799-814 of SEQ ID NO: 375 and residues 858-863 of SEQ ID NO: 375 comprises the HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 146/154. In one embodiment, the antibody that binds to or interacts with an epitope in the carboxy terminal receptor binding domain of toxin A produced by Clostridium difficile, selected from the group consisting of residues 468-488 of SEQ ID NO: 375, residues 510-530 of SEQ ID NO: 375, residues 602-610 of SEQ ID NO: 375, residues 644-703 of SEQ ID NO: 375, residues 724-794 of SEQ ID NO: 375, residues 799-814 of SEQ ID NO: 375 and residues 858-863 of SEQ ID NO: 375 is combined with a second antibody that binds specifically to toxin B of Clostridium difficile in a pharmaceutical composition. In one embodiment, this second antibody that interacts with or binds to toxin B of Clostridium difficile comprises the HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 274/282.

[0079] In a second aspect, the invention provides nucleic acid molecules encoding anti-toxin A and/or anti-toxin B antibodies or fragments thereof. Recombinant expression vectors carrying the nucleic acids of the invention, and host cells into which such vectors have been introduced, are also encompassed by the invention, as are methods of producing the antibodies by culturing the host cells under conditions permitting production of the antibodies, and recovering the antibodies produced.

[0080] In one embodiment, the invention provides an antibody or fragment thereof comprising a HCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, 17, 33, 49, 65, 81, 97, 113, 129, 145, 161, 177, 193, 209, 225, 241, 257, 273, 289, 305, 321, 337 and 353 or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereof.

[0081] In one embodiment, the HCVR is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 17, 33, 145 and 273.

[0082] In one embodiment, the antibody or fragment thereof further comprises a LCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9, 25, 41, 57, 73, 89, 105, 121, 137, 153, 169, 185, 201, 217, 233, 249, 265, 281, 297, 313, 329, 345 and 361, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereof.

[0083] In one embodiment, the LCVR is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 25, 41, 153 and 281.

[0084] In one embodiment, the invention also provides an antibody or antigen-binding fragment of an antibody comprising a HCDR3 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 7, 23, 39, 55, 71, 87, 103, 119, 135, 151, 167, 183, 199, 215, 231, 247, 263, 279, 295, 311, 327, 343 and 359 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a LCDR3 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 15, 31, 47, 63, 79, 95, 111, 127, 143, 159, 175, 191, 207, 223, 239, 255, 271, 287, 303, 319, 335, 351 and 367, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0085] In one embodiment, the invention provides an antibody or fragment thereof further comprising a HCDR1 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, 19, 35, 51, 67, 83, 99, 115, 131, 147, 163, 179, 195, 211, 227, 243, 259, 275, 291, 307, 323, 339 and 355, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a HCDR2 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 5, 21, 37, 53, 69, 85, 101, 117, 133, 149, 165, 181, 197, 213, 229, 245, 261, 277, 293, 309, 325, 341 and 357, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a LCDR1 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 11, 27, 43, 59, 75, 91, 107, 123, 139, 155, 171, 187, 203, 219, 235, 251, 267, 283, 299, 315, 331, 347 and 363, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a LCDR2 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 13, 29, 45, 61, 77, 93, 109, 125, 141, 157, 173, 189, 205, 221, 237, 253, 269, 285, 301, 317, 333, 349 and 365, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0086] In a third aspect, the invention features a human antibody or antigen-binding fragment specific for toxin A and/or toxin B of C. difficile comprising a HCVR encoded by nucleotide sequence segments derived from V.sub.H, D.sub.H and J.sub.H germline sequences, and a LCVR encoded by nucleotide sequence segments derived from V.sub.K and J.sub.K germline sequences, with combinations as shown in Table 2.

[0087] The invention encompasses antibodies having a modified glycosylation pattern. In some applications, modification to remove undesirable glycosylation sites may be useful, or e.g., removal of a fucose moiety to increase antibody dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, modification of galactosylation can be made in order to modify complement dependent cytotoxicity (CDC).

[0088] In a fourth aspect, the invention provides a pharmaceutical composition comprising at least one isolated fully human monoclonal antibody or antigen-binding fragment thereof that binds to either toxin A or toxin B of C. difficile, or that binds to both toxin A and toxin B of C. difficile and a pharmaceutically acceptable carrier or diluent. In one embodiment, the invention provides a pharmaceutical composition comprising an isolated fully human monoclonal antibody or antigen-binding fragment thereof that binds specifically to only toxin A of C. difficile and a pharmaceutically acceptable carrier or diluent. In one embodiment, the invention provides a pharmaceutical composition comprising an isolated fully human monoclonal antibody or antigen-binding fragment thereof that binds specifically to only toxin B of C. difficile and a pharmaceutically acceptable carrier or diluent. In one embodiment, the invention provides a pharmaceutical composition comprising two fully human monoclonal antibodies or antigen-binding fragments thereof, one that binds specifically to toxin A and one that binds specifically to toxin B of C. difficile and a pharmaceutically acceptable carrier or diluent. In one embodiment, the invention provides a pharmaceutical composition comprising one dual binding fully human monoclonal antibody (an antibody that binds to both toxin A and toxin B) and a pharmaceutically acceptable carrier or diluent. In one embodiment, the invention provides a pharmaceutical composition comprising two dual binding fully human monoclonal antibodies (an antibody that binds to both toxin A and toxin B) and a pharmaceutically acceptable carrier or diluent. The dual antibodies used in the pharmaceutical composition may recognize and/or bind to the same epitope on toxin A or toxin B, or may recognize and/or bind to different epitopes on toxin A or toxin B. It is to be understood that any combination of antibodies as described herein may be used in a pharmaceutical composition to achieve the desired results in the patient population in need of such therapy. For example, two antibodies that recognize and/or bind only toxin A may be used in a composition. Alternatively, two antibodies that recognize and/or bind only toxin B may be used in a composition. In one embodiment, one antibody that recognizes/binds to only toxin A or toxin B may be combined with a dual binding antibody in a composition. In one embodiment, one antibody that recognizes/binds to only toxin A may be combined with one antibody that recognizes/binds to only toxin B and this combination may be used in a composition.

[0089] In one embodiment, the pharmaceutical composition comprises a fully human monoclonal antibody that binds to the carboxy terminal receptor binding domain of both toxin A and toxin B of C. difficile having any one or more of the characteristics described herein. The antibody that binds to the carboxy terminal receptor binding domain of both toxin A and toxin B of C. difficile binds toxin A and toxin B with a K.sub.D equal to or less than 10.sup.-7M.

[0090] In one embodiment, the composition comprises an antibody that binds both toxin A and toxin B of C. difficile and has a HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 18/26, 34/42, 50/58, 66/74 and 82/90.

[0091] In one embodiment, the composition comprises an antibody that binds both toxin A and toxin B of C. difficile and has a HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 18/26 and 34/42.

[0092] In one embodiment, the pharmaceutical composition comprises at least one antibody that binds a Clostridium difficile toxin, wherein the antibody is selected from the group consisting of:

[0093] a) an isolated antibody or antigen-binding fragment thereof that specifically binds toxin A of Clostridium difficile, wherein the antibody comprises the three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) contained within any one of the heavy chain variable region (HCVR) amino acid sequences selected from the group consisting of SEQ ID NOs: 2, 98, 114, 130, 146 and 162; and the three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) contained within any one of the light chain variable region (LCVR) amino acid sequences selected from the group consisting of SEQ ID NOs: 10, 106, 122, 138, 154 and 170;

[0094] b) an isolated antibody or antigen-binding fragment thereof that specifically binds toxin B of Clostridium difficile, wherein the antibody comprises the three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within any one of the HCVR amino acid sequences selected from the group consisting of SEQ ID NOs: 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338 and 354; and the three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within any one of the LCVR amino acid sequences selected from the group consisting of SEQ ID NOs: 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346 and 362; and

[0095] c) an isolated antibody or antigen-binding fragment that binds to/cross reacts with both toxin A and toxin B of Clostridium difficile, wherein the antibody comprises the three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within any one of the HCVR amino acid sequences selected from the group consisting of SEQ ID NOs: 18, 34, 50, 66 and 82; and the three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within any one of the LCVR amino acid sequences selected from the group consisting of SEQ ID NOs: 26, 42, 58, 74 and 90.

[0096] In one embodiment, the pharmaceutical composition comprises an isolated first fully human monoclonal antibody or antigen-binding fragment thereof that specifically binds toxin A of Clostridium difficile, as described herein, and an isolated second fully human monoclonal antibody or antigen-binding fragment thereof that specifically binds toxin B of Clostridium difficile, as described herein, and a pharmaceutically acceptable carrier or diluent.

[0097] In one embodiment, the composition comprises at least one antibody, or an antigen-binding fragment thereof that binds specifically to toxin A of Clostridium difficile and at least one antibody, or an antigen-binding fragment thereof that binds specifically to toxin B of Clostridium difficile, wherein:

[0098] a) the antibody or antigen-binding fragment thereof that binds specifically to toxin A comprises the three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) contained within any one of the heavy chain variable region (HCVR) amino acid sequences selected from the group consisting of SEQ ID NOs: 2, 98, 114, 130, 146 and 162; and the three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) contained within any one of the light chain variable region (LCVR) amino acid sequences selected from the group consisting of SEQ ID NOs: 10, 106, 122, 138, 154 and 170; and wherein

[0099] b) the antibody or antigen-binding fragment thereof that binds specifically to toxin B comprises the three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within any one of the HCVR amino acid sequences selected from the group consisting of SEQ ID NOs: 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338 and 354; and the three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within any one of the LCVR amino acid sequences selected from the group consisting of SEQ ID NOs: 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346 and 362.

[0100] In one embodiment, the pharmaceutical composition comprises:

[0101] a) an isolated first fully human monoclonal antibody, or antigen-binding fragment thereof that specifically binds toxin A of Clostridium difficile, which comprises a HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 98, 114, 130, 146 and 162; and a LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 106, 122, 138, 154 and 170; and

[0102] b) an isolated second fully human monoclonal antibody, or antigen-binding fragment thereof that specifically binds toxin B of Clostridium difficile, which comprises a HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338 and 354; and a LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346 and 362.

[0103] In one embodiment, the pharmaceutical composition comprises an isolated first fully human monoclonal antibody or antigen-binding fragment thereof that specifically binds toxin A of C. difficile, which comprises a HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2/10, 98/106, 114/122, 130/138, 146/154 and 162/170; and an isolated second fully human monoclonal antibody or antigen-binding fragment thereof that specifically binds toxin B of C. difficile, which comprises a HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 178/186, 194/202, 210/218, 226/234, 242/250, 258/266, 274/282, 290/298, 306/314, 322/330, 338/346 and 354/362.

[0104] In another embodiment, the pharmaceutical composition comprises an isolated first fully human monoclonal antibody or antigen-binding fragment thereof that specifically binds toxin A of C. difficile, which comprises a HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 146/154; and an isolated second fully human antibody or antigen-binding fragment thereof that specifically binds toxin B of C. difficile, which comprises a HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 274/282.

[0105] In another related embodiment, the pharmaceutical composition comprises:

[0106] a) an isolated first human antibody, or antigen-binding fragment thereof that specifically binds toxin A of Clostridium difficile, comprising a HCDR1 having the amino acid sequence of SEQ ID NO: 148, a HCDR2 having the amino acid sequence of SEQ ID NO: 150, a HCDR3 having the amino acid sequence of SEQ ID NO: 152, a LCDR1 having the amino acid sequence of SEQ ID NO: 156, a LCDR2 having the amino acid sequence of SEQ ID NO: 158, a LCDR3 having the amino acid sequence of SEQ ID NO: 160;

[0107] b) an isolated second human antibody, or antigen-binding fragment thereof that specifically binds toxin B of Clostridium difficile, comprising a HCDR1 having the amino acid sequence of SEQ ID NO: 276, a HCDR2 having the amino acid sequence of SEQ ID NO: 278, a HCDR3 having the amino acid sequence of SEQ ID NO: 280, a LCDR1 having the amino acid sequence of SEQ ID NO: 284, a LCDR2 having the amino acid sequence of SEQ ID NO: 286, a LCDR3 having the amino acid sequence of SEQ ID NO: 288; and

[0108] c) a pharmaceutically acceptable carrier or diluent.

[0109] In one embodiment, the antibodies of the invention, or compositions containing one or more antibodies of the invention may be used to neutralize either toxin A, or toxin B, or both toxin A and B from any strain of Clostridium difficile.

[0110] In one embodiment, the antibodies of the invention, or compositions containing one or more antibodies of the invention may be used to neutralize toxins A and/or B from a hypervirulent strain of Clostridium difficile.

[0111] In one embodiment, the antibodies of the invention, or compositions containing one or more antibodies of the invention may be used to neutralize toxins A and/or B from a BI/NAP1/027 strain.

[0112] In one embodiment, the antibodies of the invention, or compositions containing one or more antibodies of the invention, may be used to neutralize toxins A and/or B from a BI/NAP1/027 strain, wherein the BI/NAP1/027 strain is selected from the group consisting of VA5, VA17, 6336 and 6443.

[0113] In one embodiment, the antibody composition comprising a first antibody that binds specifically to toxin A, may be administered alone as a separate composition and the antibody composition comprising the second antibody that binds specifically to toxin B may also be administered as a separate composition. Each composition may be prepared for delivery to the patient in separate syringes, or delivery devices, or vials. When formulated separately as two compositions, both compositions may be delivered separately, with one antibody composition being given immediately prior to the other antibody composition. Alternatively, the two antibody compositions may be mixed together shortly before administration and given concurrently.

[0114] In one embodiment, the invention features a composition, which is a combination of an antibody or antigen-binding fragment of an antibody of the invention, and a second therapeutic agent.

[0115] The second therapeutic agent may be a small molecule drug, a protein/polypeptide, an antibody, a nucleic acid molecule, such as an anti-sense molecule, or a siRNA. The second therapeutic agent may be synthetic or naturally derived.

[0116] The second therapeutic agent may be any agent that is advantageously combined with the antibody or fragment thereof of the invention, for example, a probiotic, an antibiotic, a toxoid, a vaccine specific for C. difficile, or a second different antibody against C. difficile toxin A and/or toxin B.

[0117] In certain embodiments, the second therapeutic agent may be an agent that helps to counteract or reduce any possible side effect(s) associated with the antibody or antigen-binding fragment of an antibody of the invention, if such side effect(s) should occur.

[0118] It will also be appreciated that the antibodies and pharmaceutically acceptable compositions of the present invention can be employed in combination therapies, that is, the antibodies and pharmaceutically acceptable compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. The particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder (for example, an antibody may be administered concurrently with another agent used to treat the same disorder), or they may achieve different effects (e.g., control of any adverse effects). As used herein, additional therapeutic agents that are normally administered to treat or prevent a particular disease, or condition, are appropriate for the disease, or condition, being treated.

[0119] When multiple therapeutics are co-administered, dosages may be adjusted accordingly, as is recognized in the pertinent art.

[0120] A fifth aspect of the invention provides a method for treating a patient suffering from a Clostridium difficile-associated condition or disease, or for treating at least one symptom or complication associated with the condition or disease, or for preventing the development of a Clostridium difficile-associated condition or disease in a patient at risk thereof, the method comprising administering to the patient an effective amount of an antibody or an antigen-binding fragment thereof that binds to C. difficile toxin A and/or toxin B; or a pharmaceutical composition comprising an effective amount of an antibody or an antigen-binding fragment thereof that binds to Clostridium difficile toxin A and/or toxin B, such that the Clostridium difficile-associated condition or disease is either prevented, or lessened in severity and/or duration, or at least one symptom or complication associated with the condition or disease is prevented, or ameliorated, or that the frequency and/or duration of, or the severity of recurrences, or relapses with Clostridium difficile is reduced.

[0121] In one embodiment, the invention provides for use of one or more antibodies of the invention, or pharmaceutical compositions comprising one or more antibodies of the invention in the manufacture of a medicament for use in treating a patient suffering from a Clostridium difficile-associated condition or disease, or for treating at least one symptom or complication associated with the condition or disease, or for preventing the development of a Clostridium difficile-associated condition or disease in a patient at risk thereof, wherein the Clostridium difficile-associated condition or disease is either prevented, or lessened in severity and/or duration, or at least one symptom or complication associated with the condition or disease is prevented, or ameliorated, or that the frequency and/or duration of, or the severity of recurrences, or relapses with Clostridium difficile is reduced. The at least one symptom or complication associated with the Clostridium difficile-associated condition or disease may be selected from the group consisting of anorexia, abdominal pain, abdominal bloating, diarrhea with or without bleeding, dehydration, malnutrition, pseudomembranous colitis, complete or segmental colonic resection, fever and systemic infection (sepsis), death, relapse of the Clostridium difficile condition or disease, and rejection of a transplanted tissue or organ.

[0122] In one embodiment, the patient to be treated with the antibodies of the invention, or with the pharmaceutical compositions comprising one or more antibodies of the invention are infected with a hypervirulent isolate of Clostridium difficile, such as one belonging to the BI/NAP1/027 group, or may be at risk for developing an infection with a hypervirulent strain, as described herein.

[0123] In a related embodiment, the antibodies of the invention, or a pharmaceutical composition containing one or more antibodies of the invention may be used to neutralize the toxins produced by a hypervirulent strain of Clostridium difficile, such as but not limited to any of those belonging to the BI/NAP1/027 group of strains. Included in these hypervirulent strains are clinical isolates noted herein as VA5, VA17, 6336 and 6443, described herein in Example 10.

[0124] In one embodiment, the patient at risk of developing a Clostridium difficile-associated condition or disease, who may benefit from treatment with the antibodies of the invention, or with a composition comprising one or more antibodies of the invention, may be selected from the group consisting of an elderly (.gtoreq.65 years old) patient, a patient who is immunocompromised due to underlying illness or due to administration of immunosuppressive therapeutics, a patient who has some underlying medical condition that may pre-dispose them to acquiring a Clostridium difficile infection, a patient hospitalized for an extended period of time (one week or more), a patient who has been treated for an extended period of time (.gtoreq.14 days) with broad spectrum antibiotics, a cancer patient, a transplant patient, and a patient on therapy with agents such as but not limited to a proton pump inhibitor, or histamine H2 receptor inhibitor that are used for treatment of gastrointestinal diseases or conditions to reduce or treat gastric acidity, gastroesophageal reflux disease (GERD), stomach and small intestine ulcers, or heartburn.

[0125] In one embodiment, the patient at risk of developing a Clostridium difficile-associated condition or disease is a cancer patient. In a related embodiment, the cancer patient is undergoing treatment with an anti-cancer drug, or undergoing radiotherapy to treat a cancer.

[0126] In one embodiment, the patient at risk of developing a Clostridium difficile-associated condition or disease is a transplant patient. In a related embodiment, the transplant patient is a patient receiving a hematopoietic stem cell transplant, or a solid tissue or organ transplant. In certain embodiments, the transplant patient is being treated with an immunosuppressive drug, or any transplant rejection drug, or is a patient who is undergoing treatment with a drug regimen to prevent tissue or organ graft rejection following the transplant.

[0127] In one embodiment, the antibody is administered therapeutically (administered after the infection has been established and given throughout the course of the infection) to a patient suffering from a Clostridium difficile-associated condition or disease, or suffering from at least one symptom or complication associated with the condition or disease. In one embodiment, the antibody is administered prophylactically (administered prior to development of the infection) to a patient at risk for developing a Clostridium difficile-associated condition or disease, or at risk for developing at least one symptom or complication associated with the Clostridium difficile condition or disease. For example, such "patients at risk for developing a Clostridium difficile infection" include the elderly (65 years of age or older), or patients who may be immunocompromised due to illness or due to administration of immunosuppressive therapeutics, or patients who have some underlying medical condition that may pre-dispose them to acquiring a Clostridium difficile infection, or patients hospitalized for long periods of time (generally one week or longer), or patients who have been treated for a long period of time with broad spectrum antibiotics (generally 14 days or longer), or patients on therapy with proton pump inhibitors for treatment of gastrointestinal diseases or conditions. Other patients at risk for developing a Clostridium difficile infection are those patients that are in need of a tissue or organ transplant, who would be undergoing treatment with immunosuppressive drugs to prevent tissue or organ rejection. This patient population includes individuals in need of either an autologous or allogeneic hematopoietic stem cell transplant. The long hospitalization required for these patients, in addition to receipt of high doses of antibiotic therapy to prevent other types of infections may pre-dispose these patients to acquiring a primary C. difficile infection. Alternatively, if a patient in need of such a transplant already suffers from a C. difficile infection, or has exhibited symptoms of a C. difficile infection, that patient may be prone to a recurrence, or exacerbation of such infection when placed on high dose antibiotic therapy, then followed by immunosuppressive therapy to prevent graft rejection. Furthermore, these transplant patients may be at risk not only for acquiring a C. difficile infection, but also may be at risk for rejection of the transplant due to GI related graft versus host disease (GI-GVHD), which appears to be enhanced in transplant patients suffering from infection with C. difficile (See Alonso, C. D. et al., (2012), Clin. Infect. Dis. 54, 1053-1063. The relationship between C. difficile infection and GVHD involving the GI tract is unclear at this time, but it is appears that this patient population would benefit from therapy with the anti-toxin A and/or anti-toxin B antibodies of the invention. While it is envisioned that this patient population may be treated therapeutically (after the start of the infection), it is also contemplated that these patients would benefit from prophylactic (prior to infection) administration of any of the antibodies of the invention. The patients who are candidates for treatment with the antibodies of the invention may be administered the compositions comprising one or more antibodies by any route of delivery suitable for administration, including but not limited to intravenous injection, or subcutaneous injection.

[0128] In one embodiment, the pharmaceutical composition comprising the antibodies of the invention is administered to the patient in combination with one or more therapeutic agents useful for treating a C. difficile infection.

[0129] In one embodiment, the one or more therapeutic agents may be selected from the group consisting of a toxoid, a probiotic, a C. difficile vaccine (e.g., inactivated toxins A and B, such as, but not limited to ACAM-CDIFF.TM.), an antibiotic (e.g. metronidazole, vancomycin or fidaxomicin), another different antibody to C. difficile toxin A and/or B, and any other palliative therapy useful for reducing the severity of the C. difficile disease or for reducing the frequency of recurrence of the C. difficile disease or for ameliorating at least one symptom associated with a C. difficile-associated condition or disease.

[0130] In another embodiment, the one symptom or complication associated with the C. difficile-associated condition or disease is selected from the group consisting of diarrhea, pseudomembranous colitis, relapse/recurrence of the Clostridium difficile condition or disease, and rejection of a transplanted tissue or organ.

[0131] Other embodiments will become apparent from a review of the ensuing detailed description.

BRIEF DESCRIPTION OF THE FIGURES

[0132] FIG. 1 shows the domain structures of Toxin A and Toxin B from Clostridium difficile (See Davies A H, et al., Biochem. J. (2011), 436:517-526).

[0133] FIG. 2 is a graph showing results of hamster relapse assays as the percentage of hamsters surviving clindamycin and vancomycin treatment following C. difficile challenge and the effect of treatment with anti-toxin A and anti-toxin B mAbs. All antibodies were given subcutaneously once a day on days 3-6. Positive control antibodies are comparator antibodies, anti-Toxin A (control I) and anti-Toxin B (control II). Vancomycin was given as an oral dose at 10 mg/kg on days 1-3 to all animals. (.cndot. with dotted line: PBS control; .DELTA. with dotted line: Negative isotype control at 10 mg/kg; .quadrature. with solid line: Control I/Control II at 5 mg/kg each (5/5): .diamond-solid. with solid line: H1H3330P/H1H3347P at 5 mg/kg each (5/5)).

[0134] FIG. 3 is a graph showing results of hamster relapse assays as the percentage of hamsters surviving clindamycin and vancomycin treatment following C. difficile challenge and the effect of anti-toxin A and anti-toxin B mAbs. All antibodies were given subcutaneously once on day 3. Positive control antibodies are comparator antibodies, anti-Toxin A (control I) and anti-Toxin B (control II). Vancomycin was given as an oral dose at 10 mg/kg on days 1-3 to all animals. (.DELTA. with dotted line: Negative isotype control at 10 mg/kg; .quadrature. with solid line: Control I/Control II at 5 mg/kg each (5/5); .diamond-solid. with solid line: H1H3330P/H1H3347P at 5 mg/kg each (5/5); .smallcircle. with solid line: Control I/Control II at 2 mg/kg each (2/2); .quadrature. with solid line: H1H3330P/H1H3347P at 2 mg/kg each (2/2)).

[0135] FIG. 4 is a graph showing survival results in an acute model of C. difficile infection in hamsters. Results are shown as the percentage of hamsters surviving C. difficile challenge (day 0) following clindamycin treatment (day -1). All antibodies were given subcutaneously on each of 4 days from day -3 to day 0. Antibodies were administered at 50 mg/kg each (50/50), 16.6 mg/kg each (16.6/16.6), 5.5 mg/kg each (5.5/5.5) and 1.85 mg/kg each (1.85/1.85). (.gradient. with solid line: Uninfected; .cndot. with dotted line: PBS control; .DELTA. with dotted line: Negative isotype control at 100 mg/kg; .diamond-solid. with solid line: H1H3330P/H1H3347P at 50 mg/kg each (50/50); .smallcircle. with solid line: H1H3330P/H1H3347P at 16.6 mg/kg each (16.6/16.6); with solid line: H1H3330P/H1H3347P at 5.5 mg/kg each (5.5/5.5); with a solid line: H1H3330P/H1H3347P at 1.85 mg/kg each (1.85/1.85).

[0136] FIG. 5 is a graph showing survival results in an acute model of C. difficile infection in hamsters. Results are shown as the percentage of hamsters surviving C. difficile challenge (day 0) following clindamycin treatment (day -1). All antibodies were given subcutaneously on each of 4 days from day -3 to day 0. Antibodies were administered at 20 mg/kg each (20/20), or at 5 mg/kg each (5/5). (.gradient. with solid line: Uninfected; .quadrature. with dotted line: PBS control; .DELTA. with dotted line: Negative isotype control at 40 mg/kg; .quadrature. with solid line: Control I/Control II at 20 mg/kg each (20/20); with solid line: Control I/Control II at 5 mg/kg each (5/5); .diamond-solid. with solid line: H1H3330P/H1H3347P at 20 mg/kg each (20/20); .diamond. with solid line: H1H3330P/H1H3347P at 5 mg/kg each (5/5)).

DETAILED DESCRIPTION

[0137] Before the present methods are described, it is to be understood that this invention is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0138] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference in their entirety.

DEFINITIONS

[0139] The term "toxin A" (also referred to as "tcdA") refers to the toxin A protein produced by Clostridium difficile (also referred to herein as "C. difficile"). The amino acid sequence of "toxin A' is provided in GenBank as accession number CAA63564 and is also referred to herein as SEQ ID NO: 378. Toxin A is encoded by the nucleic acid provided herein as SEQ ID NO: 377, and is also found in GenBank as accession number AM180355.

[0140] The term "toxin B" (also referred to as "tcdB") refers to the toxin B protein produced by Clostridium difficile. The amino acid sequence of "toxin B' is provided in GenBank as accession number CAJ67492 and is also referred to herein as SEQ ID NO: 380. Toxin B is encoded by the nucleic acid provided herein as SEQ ID NO: 379, and is also found in GenBank as accession number AM180355.

[0141] The "carboxy terminal receptor binding domain of toxin A and toxin B of Clostridium difficile" refers to the portion of toxin A and toxin B from C. difficile that is responsible for binding to the target cell, thus allowing for subsequent receptor mediated endocytosis. As described herein, the amino acid sequence of the carboxy terminal receptor binding domain of toxin A is shown in SEQ ID NO: 375. The amino acid sequence of the carboxy terminal receptor binding domain of toxin B is shown in SEQ ID NO: 376. The various domains of toxin A and toxin B from C. difficile are illustrated in FIG. 1 and further described in Davies et al. (Davies, A H, et al., Biochem. J. (2011), 436:517-526).

[0142] The "BI/NAP1/027" designation for Clostridium difficile refers to a highly virulent group of isolates of Clostridium difficile that has been associated with an increase in morbidity and mortality throughout Europe and North America (Loo, V G, et al., (2005), N Engl J Med, 353:2442-9; McDonald, L C et al. (2006), Emerg Infect Dis, 12:409-15; McDonald, L C, et al., (2005), N Engl J Med, 353:2433-41; Redelings, M D, et al., (2007), Emerg Infect Dis 13:1417-9). The "BI/NAP1/027" designation further refers to North American pulsed-field type I (NAP1), ribotype 027, and group BI by restriction endonuclease analysis. It was originally identified in the 1980s, but was not originally identified as being resistant to the newer fluoroquinolone agents and was not epidemic prior to 2000 (Warny, M. et al., (2005), Lancet 366:1079-84; Kelly, C P, et al., N Engl J Med 359:1932-40). The "BI/NAP1/027" strain of Clostridium difficile is also characterized by increased toxin A and toxin B production, by the presence of an additional toxin (binary toxin), and increased resistance to fluoroquinolones (McDonald, L C, et al., (2005), N Engl J Med, 353:2433-41; Warny, M. et al., (2005), Lancet 366:1079-84).

[0143] The term "antibody", as used herein, is intended to refer to immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds (i.e., "full antibody molecules"), as well as multimers thereof (e.g. IgM) or antigen-binding fragments thereof. Each heavy chain is comprised of a heavy chain variable region ("HCVR" or "V.sub.H") and a heavy chain constant region (comprised of domains C.sub.H1, C.sub.H2 and C.sub.H3). Each light chain is comprised of a light chain variable region ("LCVR or "V.sub.L") and a light chain constant region (C.sub.L). The V.sub.H and V.sub.L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each V.sub.H and V.sub.L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the invention, the FRs of the antibody (or antigen binding fragment thereof) may be identical to the human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0144] Substitution of one or more CDR residues or omission of one or more CDRs is also possible. Antibodies have been described in the scientific literature in which one or two CDRs can be dispensed with for binding. Padlan et al. (1995 FASEB J. 9:133-139) analyzed the contact regions between antibodies and their antigens, based on published crystal structures, and concluded that only about one fifth to one third of CDR residues actually contact the antigen. Padlan also found many antibodies in which one or two CDRs had no amino acids in contact with an antigen (see also, Vajdos et al. 2002 J Mol Biol 320:415-428).

[0145] CDR residues not contacting antigen can be identified based on previous studies (for example residues H60-H65 in CDRH2 are often not required), from regions of Kabat CDRs lying outside Chothia CDRs, by molecular modeling and/or empirically. If a CDR or residue(s) thereof is omitted, it is usually substituted with an amino acid occupying the corresponding position in another human antibody sequence or a consensus of such sequences. Positions for substitution within CDRs and amino acids to substitute can also be selected empirically. Empirical substitutions can be conservative or non-conservative substitutions.

[0146] The fully human anti-toxin A and/or anti-toxin B monoclonal antibodies disclosed herein may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. The present invention includes antibodies, and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as "germline mutations"). A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and/or CDR residues within the V.sub.H and/or V.sub.L domains are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and/or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies of the present invention may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antibodies and antigen-binding fragments that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present invention.

[0147] The present invention also includes fully human anti-toxin A and/or anti-toxin B monoclonal antibodies comprising variants of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present invention includes anti-toxin A and anti-toxin B antibodies having HCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein.

[0148] The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human mAbs of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term "human antibody", as used herein, is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., mouse), have been grafted onto human FR sequences.

[0149] The term "specifically binds," or "binds specifically to", or the like, means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiologic conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1.times.10.sup.-6 M or less (e.g., a smaller K.sub.D denotes a tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. As described herein, antibodies have been identified by surface plasmon resonance, e.g., BIACORE.TM., which bind specifically to either toxin A, or specifically to toxin B from C. difficile, while others have been identified that bind specifically to the carboxy terminal receptor binding domain of both toxin A and B. Moreover, multi-specific antibodies that bind to toxin A or toxin B and one or more additional antigens or a bi-specific that binds to two different regions of toxin A or toxin B are nonetheless considered antibodies that "specifically bind", as used herein.

[0150] The term "high affinity" antibody refers to those mAbs having a binding affinity to toxin A or toxin B, expressed as K.sub.D, of at least 10.sup.-8 M; preferably 10.sup.-9 M; more preferably 10.sup.-19M, even more preferably 10.sup.-11 M, even more preferably 10.sup.-12 M, as measured by surface plasmon resonance, e.g., BIACORE.TM. or solution-affinity ELISA.

[0151] By the term "slow off rate", "Koff" or "kd" is meant an antibody that dissociates from toxin A or toxin B, or both, with a rate constant of 1.times.10.sup.-3 s.sup.-1 or less, preferably 1.times.10.sup.-4 s.sup.-1 or less, as determined by surface plasmon resonance, e.g., BIACORE.TM..

[0152] The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. The terms "antigen-binding portion" of an antibody, or "antibody fragment", as used herein, refers to one or more fragments of an antibody that retain the ability to bind to toxin A or toxin B or both.

[0153] The specific embodiments, antibody or antibody fragments of the invention may be conjugated to a therapeutic moiety ("immunoconjugate"), such as an antibiotic, a second anti-toxin A or B antibody, or a C. difficile vaccine, or a toxoid, or any other therapeutic moiety useful for treating a disease or condition caused by C. difficile.

[0154] An "isolated antibody", as used herein, is intended to refer to an antibody that is substantially free of other antibodies (Abs) having different antigenic specificities (e.g., an isolated antibody that specifically binds toxin A or toxin B, or a fragment thereof, is substantially free of Abs that specifically bind antigens other than toxin A or toxin B.

[0155] A "blocking antibody" or a "neutralizing antibody", as used herein (or an "antibody that neutralizes toxin A and/or toxin B activity"), is intended to refer to an antibody whose binding to toxin A and/or toxin B results in inhibition of at least one biological activity of toxin A and/or toxin B. For example, an antibody of the invention may aid in preventing the primary disease caused by C. difficile. Alternatively, an antibody of the invention may demonstrate the ability to prevent a recurrence or relapse of the disease caused by C. difficile, or at least one symptom caused by C. difficile infection, including diarrhea or psudomembranous colitis. This inhibition of the biological activity of toxin A and/or toxin B can be assessed by measuring one or more indicators of toxin A and/or toxin B biological activity by one or more of several standard in vitro assays (such as a neutralization assay, as described herein) or in vivo assays known in the art (for example, animal models to look at protection from challenge with C. difficile following administration of one or more of the antibodies described herein).

[0156] The term "surface plasmon resonance", as used herein, refers to an optical phenomenon that allows for the analysis of real-time biomolecular interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIACORE.TM. system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).

[0157] The term "K.sub.D", as used herein, is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

[0158] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. The term "epitope" also refers to a site on an antigen to which B and/or T cells respond. It also refers to a region of an antigen that is bound by an antibody. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of the structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes may also be conformational, that is, composed of non-linear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics, and/or specific charge characteristics.

[0159] The term "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or GAP, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0160] As applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 90% sequence identity, even more preferably at least 95%, 98% or 99% sequence identity. Preferably, residue positions, which are not identical, differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, which is herein incorporated by reference. Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate and glutamate, and 7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443 45, herein incorporated by reference. A "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

[0161] Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG software contains programs such as GAP and BESTFIT which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA with default or recommended parameters; a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the invention to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215: 403 410 and (1997) Nucleic Acids Res. 25:3389 402, each of which is herein incorporated by reference.

[0162] In specific embodiments, the antibody or antibody fragment for use in the method of the invention may be mono-specific, bi-specific, or multi-specific. Multi-specific antibodies may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for epitopes of more than one target polypeptide. An exemplary bi-specific antibody format that can be used in the context of the present invention involves the use of a first immunoglobulin (Ig) C.sub.H3 domain and a second Ig C.sub.H3 domain, wherein the first and second Ig C.sub.H3 domains differ from one another by at least one amino acid, and wherein at least one amino acid difference reduces binding of the bi-specific antibody to Protein A as compared to a bi-specific antibody lacking the amino acid difference. In one embodiment, the first Ig C.sub.H3 domain binds Protein A and the second Ig C.sub.H3 domain contains a mutation that reduces or abolishes Protein A binding such as an H95R modification (by IMGT exon numbering; H435R by EU numbering). The second C.sub.H3 may further comprise an Y96F modification (by IMGT; Y436F by EU). Further modifications that may be found within the second C.sub.H3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1 mAbs; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU) in the case of IgG2 mAbs; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU) in the case of IgG4 mAbs. Variations on the bi-specific antibody format described above are contemplated within the scope of the present invention.

[0163] By the phrase "therapeutically effective amount" is meant an amount that produces the desired effect for which it is administered. The exact amount will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

General Description

[0164] Clostridium difficile is a gram-positive, spore-forming, toxin producing bacterium, which is a leading cause of nosocomial antibiotic-associated diarrhea and colitis in humans (Bartlett, J. G. et al. (1978), N. Engl. J. Med. 298:531-534; Kyne, L., et al. (2001), Clin. N. Am. 30:753-777). The perturbation of the colonic environment resulting from administration of broad-spectrum antibiotics leads to colonization of the gut by the bacterium (Johnson, S. C. et al. (1990), Lancet 336:97-100). A large percentage of this patient population that becomes colonized with C. difficile develops diarrhea, which in certain instances leads to pseudomembranous colitis, which is believed to be due to the production of two exotoxins by C. difficile, toxin A and toxin B. Treatment consists of the discontinuation of the offending antibiotic, or alterations in the dosing of the offending antibiotic, or no change in the offending antibiotic, followed by the administration of metronidazole, vancomycin, or fidaxomicin. While this treatment regimen is usually successful, many patients relapse when therapy is discontinued (Fekety, R., (1997), Am. J. Gastroenterology, 92:739-750). Furthermore, in many instances, the C. difficile bacterium becomes resistant to the therapy used, thus leading to treatment failures and in some instances increased mortality rates (Dworczynski, A. et al. (1991), Cytobios. 65:149-153; Fekety, R. et al. (1993), JAMA, 269:71-75). Accordingly, there is a need for more effective therapies to combat this disease and/or to prevent the recurrence of this disease in patients colonized with C. difficile. In addition, there is a need to treat patients who are at risk for developing a C. difficile infection by prophylactic administration of an effective agent. Included in this at risk patient population are the elderly, in particular, patients 65 years of age and older, although patients younger than 65 may be at greater risk depending on the presence of any underlying disease that may predispose them to infection with C. difficile. Patients that have been infected with C. difficile previously may be at greater risk of recurrences. Other patients at risk include patients who are pre-disposed to a C. difficile infection because of an underlying medical condition, or patients who are hospitalized for long periods of time (at least one week or longer) and/or, who are on long term treatment (.gtoreq.14 days) with broad spectrum antibiotics, as well as patients who are on proton pump inhibitors to treat gastroesophageal reflux disease (GERD), stomach and small intestine ulcers and inflammation of the esophagus. These agents include dexlansoprazole, esomeprazole, lansoprazole, omeprazole, pantoprazole sodium, or rabeprazole sodium. Other agents that are under study for placing a patient at risk for developing a C. difficile infection include histamine-H2 receptor blockers, such as cimetidine, famotidine, nizatidine and ranitidine. Other studies noted an age-specific incidence of C. difficile-associated diarrhea, more specifically, an increase in patients after the age of 50 years, and an increase in mortality rate in patients after the age of 60 (Loo, V G, et al., (2005), N Engl J Med 353:2442-9). This study was in fact, consistent with an earlier study that showed an age-related increase in the incidence of positive assays for C. difficile toxin (Karlstrom, O. et al. (1998), Clin Infect Dis 26:141-5).

[0165] To address the need for more effective therapies against C. difficile, many studies have been conducted to determine if anti-toxin A and/or B antibodies, when used alone, or as adjunct therapy, could be used as a means of treating this disease, or at least as a means of preventing the recurrence of the diarrhea or colitis associated with C. difficile infection. (Corthier, et al. (1991), Infect. Immun. 59(3):1192-1195; Kink, J. A. and Willilams, J. A., (1998), Infect. Immun. 66(5):2018-2025; Lowy, I. et al. (2010), N. Engl. J. Med. 362(3):197-205; Babcock, G. J., et al.; (2006), Infection and Immunity, 74(11):6339-6347). More particularly, animal models of infection with C. difficile have been used to study the effect of antibodies to toxin A and/or toxin B from C. difficile on primary infection, as well as on relapse rates in vivo (Corthier, G. et al. (1991), Infect. Immun. 59(3):1192-1195; Kink, J. A. et al. (1998), Infect. Immun. 66(5):2018-2025; Babcock, G. J. et al. (2006), 74(11):6339-6347). The results in animal models of C. difficile showed significant protection, thus prompting further clinical trials using anti-toxin A and anti-toxin B antibodies in human patients with the disease (Lowy, I., et al., (2010), N. Engl. J. Med. 362(3):197-205).

[0166] The antibodies described herein demonstrate specific binding to toxin A and/or to toxin B of C. difficile and may be useful for treating patients suffering from infection with C. difficile. The use of such antibodies may be an effective means of treating patients suffering from a primary infection with C. difficile, or they may be used to prevent a relapse or recurrence of the disease and the accompanying symptoms associated with the disease, or may be used to lessen the severity of the diarrhea or colitis associated with a primary infection or with the recurrence of the infection. They may be used alone or as adjunct therapy with other therapeutic moieties or modalities known in the art for treating C. difficile infections, such as, but not limited to, antibiotic therapy, for example, with metronidazole, vancomycin, or fidaxomicin. They may be used in conjunction with a C. difficile vaccine, or with use of a toxoid, or with a second or third different antibody specific for toxin A and/or B.

[0167] In certain embodiments of the invention, combinations of the antibodies of the invention may be used to treat an infection caused by a hypervirulent strain of C. difficile. The most notable hypervirulent epidemic isolate group to date is one referred to as "BI/NAP1/027". This has been associated with outbreaks of C. difficile infections throughout Europe and North America. Isolates that fall into this designation are characterized by increased toxin A and toxin B production, by the presence of an additional toxin (binary toxin) and by an increased resistance to fluoroquinolones (McDonald, L C, et al., (2005), N Engl J Med 353:2433-41; Warny, M E, et al., (2005), Lancet 366:1079-84). This group of isolates may also be referred to as the North American pulsed-field type 1 (NAP1), ribotype 027, group BI strains. This group of strains contains an 18 base pair tcdC gene deletion and the binary toxin, which it produces is encoded by cdtA and cdtB genes. It has been reported that this group produces toxin A and toxin B in quantities 16 and 23 times, respectively, greater than control strains (Warny, M E, et al., (2005), Lancet 366:1079-84). Since the antibodies of the present invention have been shown to neutralize the toxin produced by four different clinically isolated C. difficile BI/NAP1/027 strains (VA5, VA17, 6336 and 6443), it is envisioned that compositions comprising the antibodies of the present invention may be administered therapeutically to patients suffering from an infection with the above-noted hypervirulent strains of C. difficile, or may be administered prophylactically to patients who are at risk for developing an infection with the hypervirulent strains noted herein, as well as with any other clinically relevant hypervirulent strains. The means by which to identify these strains are known to those skilled in the art, and these methods may include pulsed-field gel electrophoresis (PFGE) of C. difficile isolates (See for example, Fawley, W N, et al., (2002), J. Clin Microbiol 40:3546-7), PCR analyses for binary toxin genes and partial deletions of the tcdC gene (See, for example, Gongalves, C. et al. (2004), J Clin Microbiol 42:1933-9; and Cohen, S H et al., (2000), J Infect Dis 181:659-63), and restriction-endonuclease analyses (See, for example, Clabots, C R, et al., (1993), J Clin Microbiol 31:1870-5).

[0168] In certain embodiments, the antibodies of the invention are obtained from mice immunized with a primary immunogen, such as a native, inactivated, toxin A (See GenBank accession number CAA63564 (SEQ ID NO: 378)), or toxin B (See GenBank accession number CAJ67492 (SEQ ID NO: 380)) from C. difficile, or with a recombinant, but inactivated form of the toxins, or toxin fragments, followed by immunization with a secondary immunogen, or with an immunogenically active fragment of the native toxin. Animals may be immunized with either inactivated toxin A alone or inactivated toxin B alone, or with both inactivated toxin A and inactivated toxin B concurrently. The toxins can be inactivated prior to use as an immunogen using standard procedures for preparing toxoids, including by treatment with formaldehyde, glutaraldehyde, peroxide, or oxygen treatment (Relyveld, et al. Methods in Enzymology, 93:24, 1983, Woodrow and Levine, eds. New Generation Vaccines, Marcel Dekker, Inc., New York, 1990). Another means of inactivation is by use of UDP-dialdehyde (Genth et al., (2000), Infect. Immun. 68(3):1094-1101), which may act to preserve the native structure of the toxin compared to other inactivation methods, thereby enhancing the likelihood of eliciting antibodies that are more reactive with the native toxin.

[0169] Alternatively, mutant toxins from C. difficile, which exhibit reduced toxicity, may be produced using standard recombinant techniques and used as immunogens (See, for example, U.S. Pat. Nos. 5,085,862; 5,221,618; 5,244,657; 5,332,583; 5,358,868; and 5,433,945). Such mutants may contain deletions or point mutations in the active site of the toxin.

[0170] The immunogen may be a biologically active and/or immunogenic fragment of native toxin A or toxin B, or DNA encoding the active fragment thereof. The fragment may be derived from the N-terminal or C-terminal domain of either toxin A or toxin B. The fragment may be derived from any of the known domains of toxin A or toxin B (See FIG. 1), including the glucosylating enzymatic domain (A), the autocatalytic processing domain (C), the translocating domain (D) or the binding domain (B). In certain embodiments of the invention, the immunogen is the carboxy terminal receptor binding domain of toxin A that ranges from about amino acid residues 1832-2710 of SEQ ID NO: 378. In certain embodiments of the invention, the immunogen is the carboxy terminal receptor binding domain of toxin A that is shown in SEQ ID NO: 375. In certain embodiments of the invention, the immunogen is the carboxy terminal receptor binding domain of toxin B that ranges from about amino acid residues 1834-2366 of SEQ ID NO: 380. In certain embodiments of the invention, the immunogen is the carboxy terminal receptor binding domain of toxin B that is shown in SEQ ID NO: 376.

[0171] The full-length amino acid sequence of toxin A from C. difficile is shown as SEQ ID NO: 378.

[0172] The full-length amino acid sequence of toxin B from C. difficile is shown as SEQ ID NO: 380.

[0173] In certain embodiments, antibodies that bind specifically to C. difficile toxin A or toxin B may be prepared using fragments of the above-noted regions, or peptides that extend beyond the designated regions by about 5 to about 20 amino acid residues from either, or both, the N or C terminal ends of the regions described herein. In certain embodiments, any combination of the above-noted regions or fragments thereof may be used in the preparation of toxin A or toxin B specific antibodies. In certain embodiments, any one or more of the above-noted regions of toxin A or toxin B, or fragments thereof may be used for preparing monospecific, bispecific, or multispecific antibodies.

Antigen-Binding Fragments of Antibodies

[0174] Unless specifically indicated otherwise, the term "antibody," as used herein, shall be understood to encompass antibody molecules comprising two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., "full antibody molecules") as well as antigen-binding fragments thereof. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. The terms "antigen-binding portion" of an antibody, or "antibody fragment", as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to either toxin A and/or toxin B of C. difficile. An antibody fragment may include a Fab fragment, a F(ab').sub.2 fragment, a Fv fragment, a dAb fragment, a fragment containing a CDR, or an isolated CDR. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and (optionally) constant domains. Such DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

[0175] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigen-binding fragment," as used herein.

[0176] An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR, which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a V.sub.H domain associated with a V.sub.L domain, the V.sub.H and V.sub.L domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain V.sub.H-V.sub.H, V.sub.H-V.sub.L or V.sub.L-V.sub.L dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric V.sub.H or V.sub.L domain.

[0177] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present invention include: (i) V.sub.H-C.sub.H1; (ii) V.sub.H-C.sub.H2; (iii) V.sub.H-C.sub.H3; (iv) V.sub.H-C.sub.H1-C.sub.H2; (v) V.sub.H-C.sub.H1-C.sub.H2-C.sub.H3, (vi) V.sub.H-C.sub.H2-C.sub.H3; V.sub.H-C.sub.L; V.sub.L-C.sub.H1; (ix) C.sub.H2, (x) V.sub.L-C.sub.H3; (xi) V.sub.L-C.sub.H1-C.sub.H2; (xii) V.sub.L-C.sub.H1-C.sub.H2-C.sub.H3; (xiii) --C.sub.H2-C.sub.H3; and (xiv) V.sub.L-C.sub.L. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody of the present invention may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric V.sub.H or V.sub.L domain (e.g., by disulfide bond(s)).

[0178] As with full antibody molecules, antigen-binding fragments may be mono-specific or multi-specific (e.g., bi-specific). A multi-specific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multi-specific antibody format, including the exemplary bi-specific antibody formats disclosed herein, may be adapted for use in the context of an antigen-binding fragment of an antibody of the present invention using routine techniques available in the art.

Preparation of Human Antibodies

[0179] Methods for generating human antibodies in transgenic mice are known in the art. Any such known methods can be used in the context of the present invention to make human antibodies that specifically bind to toxin A and/or toxin B of C. difficile.

[0180] Using VELOCIMMUNE.RTM. technology (see, for example, U.S. Pat. No. 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE.RTM.) or any other known method for generating monoclonal antibodies, high affinity chimeric antibodies to toxin A and/or toxin B of C. difficile are initially isolated having a human variable region and a mouse constant region. The VELOCIMMUNE.RTM. technology involves generation of a transgenic mouse having a genome comprising human heavy and light chain variable regions operably linked to endogenous mouse constant region loci such that the mouse produces an antibody comprising a human variable region and a mouse constant region in response to antigenic stimulation. The DNA encoding the variable regions of the heavy and light chains of the antibody are isolated and operably linked to DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in a cell capable of expressing the fully human antibody.

[0181] Generally, a VELOCIMMUNE.RTM. mouse is challenged with the antigen of interest, and lymphatic cells (such as B-cells) are recovered from the mice that express antibodies. The lymphatic cells may be fused with a myeloma cell line to prepare immortal hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the variable regions of the heavy chain and light chain may be isolated and linked to desirable isotypic constant regions of the heavy chain and light chain. Such an antibody protein may be produced in a cell, such as a CHO cell. Alternatively, DNA encoding the antigen-specific chimeric antibodies or the variable domains of the light and heavy chains may be isolated directly from antigen-specific lymphocytes.

[0182] Initially, high affinity chimeric antibodies are isolated having a human variable region and a mouse constant region. As in the experimental section below, the antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with a desired human constant region to generate the fully human antibody of the invention, for example wild-type or modified IgG1 or IgG4. While the constant region selected may vary according to specific use, high affinity antigen-binding and target specificity characteristics reside in the variable region.

[0183] In general, the antibodies of the instant invention possess very high affinities, typically possessing K.sub.D of from about 10.sup.-12 through about 10.sup.-9 M, when measured by binding to antigen either immobilized on solid phase or in solution phase. The mouse constant regions are replaced with desired human constant regions to generate the fully human antibodies of the invention. While the constant region selected may vary according to specific use, high affinity antigen-binding and target specificity characteristics reside in the variable region.

Bioequivalents

[0184] The anti-toxin A and anti-toxin B antibodies and antibody fragments of the present invention encompass proteins having amino acid sequences that vary from those of the described antibodies, but that retain the ability to bind toxin A or toxin B. Such variant antibodies and antibody fragments comprise one or more additions, deletions, or substitutions of amino acids when compared to parent sequence, but exhibit biological activity that is essentially equivalent to that of the described antibodies. Likewise, the antibody-encoding DNA sequences of the present invention encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to the disclosed sequence, but that encode an antibody or antibody fragment that is essentially bioequivalent to an antibody or antibody fragment of the invention.

[0185] Two antigen-binding proteins, or antibodies, are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered at the same molar dose under similar experimental conditions, either single does or multiple dose. Some antibodies will be considered equivalents or pharmaceutical alternatives if they are equivalent in the extent of their absorption but not in their rate of absorption and yet may be considered bioequivalent because such differences in the rate of absorption are intentional and are reflected in the labeling, are not essential to the attainment of effective body drug concentrations on, e.g., chronic use, and are considered medically insignificant for the particular drug product studied.

[0186] In one embodiment, two antigen-binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency.

[0187] In one embodiment, two antigen-binding proteins are bioequivalent if a patient can be switched one or more times between the reference product and the biological product without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity, or diminished effectiveness, as compared to continued therapy without such switching.

[0188] In one embodiment, two antigen-binding proteins are bioequivalent if they both act by a common mechanism or mechanisms of action for the condition or conditions of use, to the extent that such mechanisms are known.

[0189] Bioequivalence may be demonstrated by in vivo and/or in vitro methods. Bioequivalence measures include, e.g., (a) an in vivo test in humans or other mammals, in which the concentration of the antibody or its metabolites is measured in blood, plasma, serum, or other biological fluid as a function of time; (b) an in vitro test that has been correlated with and is reasonably predictive of human in vivo bioavailability data; (c) an in vivo test in humans or other mammals in which the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) in a well-controlled clinical trial that establishes safety, efficacy, or bioavailability or bioequivalence of an antibody.

[0190] Bioequivalent variants of the antibodies of the invention may be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences not needed for biological activity. For example, cysteine residues not essential for biological activity can be deleted or replaced with other amino acids to prevent formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation. In other contexts, bioequivalent antibodies may include antibody variants comprising amino acid changes, which modify the glycosylation characteristics of the antibodies, e.g., mutations that eliminate or remove glycosylation.

Biological Characteristics of the Antibodies

[0191] In general, the antibodies of the present invention may function by binding to either toxin A or to toxin B of C. difficile, or to both toxin A and toxin B of C. difficile (cross-reactive antibodies), or to a fragment of either A or B.

[0192] In certain embodiments, the antibodies of the present invention may bind to an epitope located in at least the C-terminal receptor binding domain of toxin A and/or toxin B of C. difficile. In one embodiment, the antibodies may bind to the C-terminal region of toxin A, ranging from amino acid residue 1832-2710 of the carboxy terminal receptor binding domain of toxin A, which spans amino acid residues 1832-2710 of SEQ ID NO: 378. In certain embodiments of the invention, the antibodies may bind the carboxy terminal receptor binding domain of toxin A that is shown in SEQ ID NO: 375. In certain embodiments of the invention, the antibodies may interact with, or bind to, amino acid residues 468-863 of the carboxy terminal receptor binding domain of toxin A produced by Clostridium difficile, the sequence of which is shown in SEQ ID NO: 375. In certain embodiments of the invention, the antibodies may interact with, or bind to, an epitope in the carboxy terminal receptor binding domain of toxin A produced by Clostridium difficile, wherein the epitope is selected from the group consisting of residues 468-488 of SEQ ID NO: 375, residues 510-530 of SEQ ID NO: 375, residues 602-610 of SEQ ID NO: 375, residues 644-703 of SEQ ID NO: 375, residues 724-794 of SEQ ID NO: 375, residues 799-814 of SEQ ID NO: 375 and residues 858-863 of SEQ ID NO: 375. In one embodiment, the antibody that binds to or interacts with an epitope in the carboxy terminal receptor binding domain of toxin A produced by Clostridium difficile, selected from the group consisting of residues 468-488 of SEQ ID NO: 375, residues 510-530 of SEQ ID NO: 375, residues 602-610 of SEQ ID NO: 375, residues 644-703 of SEQ ID NO: 375, residues 724-794 of SEQ ID NO: 375, residues 799-814 of SEQ ID NO: 375 and residues 858-863 of SEQ ID NO: 375 comprises the HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 146/154. In one embodiment, the antibody that binds to or interacts with an epitope in the carboxy terminal receptor binding domain of toxin A produced by Clostridium difficile, selected from the group consisting of residues 468-488 of SEQ ID NO: 375, residues 510-530 of SEQ ID NO: 375, residues 602-610 of SEQ ID NO: 375, residues 644-703 of SEQ ID NO: 375, residues 724-794 of SEQ ID NO: 375, residues 799-814 of SEQ ID NO: 375 and residues 858-863 of SEQ ID NO: 375 is combined with a second antibody that binds specifically to toxin B of Clostridium difficile in a pharmaceutical composition. In one embodiment, this second antibody that interacts with or binds to toxin B of Clostridium difficile comprises the HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 274/282.

[0193] In certain embodiments of the invention, the antibodies may bind to the carboxy terminal receptor binding domain of toxin B that ranges from about amino acid residues 1834-2366 of SEQ ID NO: 380. In certain embodiments of the invention, the antibodies may bind to the carboxy terminal receptor binding domain of toxin B that is shown in SEQ ID NO: 376.

[0194] In certain embodiments, the antibodies of the present invention may function by blocking or inhibiting the toxicity associated with toxin A of C. difficile by binding to any other region or fragment of the full length native toxin A protein, the amino acid sequence of which is shown in SEQ ID NO: 378, which is encoded by the nucleic acid sequence shown in SEQ ID NO: 377.

[0195] In certain embodiments, the antibodies of the present invention may function by blocking or inhibiting the toxicity associated with toxin B of C. difficile by binding to any other region or fragment of the full length native toxin B protein, the amino acid sequence of which is shown in SEQ ID NO: 380, which is encoded by the nucleic acid sequence shown in SEQ ID NO: 379.

[0196] In certain embodiments, the antibodies of the present invention may be bi-specific antibodies. The bi-specific antibodies of the invention may bind one epitope in toxin A and may also bind one epitope in toxin B. In certain embodiments, the bi-specific antibodies of the invention may bind two different epitopes in toxin A. In certain embodiments, the bi-specific antibodies of the invention may bind two different epitopes in toxin B. In certain embodiments, the bi-specific antibodies of the invention may bind to two different sites within the same domain on either one of toxin A or toxin B, or may bind to the same domain on both toxin A and toxin B.

[0197] In one embodiment, the invention provides a fully human monoclonal antibody or antigen-binding fragment thereof that binds to the carboxy terminal receptor binding domain of both toxin A and toxin B of C. difficile, wherein the antibody or fragment thereof exhibits one or more of the following characteristics: (i) comprises a HCVR having an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 34, 50, 66 and 82, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (ii) comprises a LCVR having an amino acid sequence selected from the group consisting of SEQ ID NO: 26, 42, 58, 74 and 90, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (iii) comprises a HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 24, 40, 56, 72 and 88, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 32, 48, 64, 80 and 96, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (iv) comprises a HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 20, 36, 52, 68 and 84, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 22, 38, 54, 70 and 86, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 28, 44, 60, 76 and 92, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 30, 46, 62, 78 and 94, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (v) binds to toxin A and toxin B with a K.sub.D equal to or less than 10.sup.-9M.

[0198] In one embodiment, the invention provides a fully human monoclonal antibody or antigen-binding fragment thereof that binds specifically to toxin A (but not to toxin B) of C. difficile, wherein the antibody or fragment thereof exhibits one or more of the following characteristics: (i) comprises a HCVR having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 98,114,130,146 and 162, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (ii) comprises a LCVR having an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 106, 122, 138, 154 and 170, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (iii) comprises a HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 104,120,136,152 and 168, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 16, 112, 128, 144, 160 and 176, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (iv) comprises a HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 100, 116, 132, 148 and 164, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 102, 118, 134, 150 and 166, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 108, 124, 140, 156 and 172, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 110, 126, 142, 158 and 174, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (v) demonstrates a K.sub.D equal to or less than 10.sup.-9M; (vi) demonstrates neutralization of Toxin A (at a concentration of 32 pM) with an 1050 ranging from about 7 pM to about 65 pM in a cell viability assay.

[0199] In one embodiment, the invention provides a fully human monoclonal antibody or antigen-binding fragment thereof that binds specifically to toxin B (but not to toxin A) of C. difficile, wherein the antibody or fragment thereof exhibits one or more of the following characteristics: (i) comprises a HCVR having an amino acid sequence selected from the group consisting of SEQ ID NO: 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338 and 354, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (ii) comprises a LCVR having an amino acid sequence selected from the group consisting of SEQ ID NO:186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346 and 362, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (iii) comprises a HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NO:184, 200, 216, 232, 248, 264, 280, 296, 312, 328, 344 and 360, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NO:192, 208, 224, 240, 256, 272, 288, 304, 320, 336, 352 and 368, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (iv) comprises a HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 180,196, 212, 228, 244, 260, 276, 292, 308, 324, 340 and 356, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 182, 198, 214, 230, 246, 262, 278, 294, 310, 326, 342 and 358, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 188, 204, 220, 236, 252, 268, 284, 300, 316, 332, 348 and 364, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 190, 206, 222, 238, 254, 270, 286, 302, 318, 334, 350 and 366, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (v) demonstrates a K.sub.D equal to or less than 10.sup.-9M; (vi) demonstrates neutralization of Toxin B (at a concentration of 0.03 pM) with an 1050 ranging from about 25 pM to about 320 pM in a cell viability assay.

[0200] Certain anti-toxin A or anti-toxin B antibodies of the present invention are able to bind to and neutralize the toxicity of either toxin A, or toxin B, or both, of C. difficile, as determined by in vitro or in vivo assays. The ability of the antibodies of the invention to bind to and neutralize the activity of the toxins may be measured using any standard method known to those skilled in the art, including binding assays, or neutralization of toxicity (protection from cell death) assays, as described herein.

[0201] Non-limiting, exemplary in vitro assays for measuring binding activity are illustrated in Examples 4, 5 and 6, herein. In Examples 4 and 5, the binding affinities and kinetic constants of human anti-toxin A or anti-toxin B antibodies were determined by surface plasmon resonance and the measurements were conducted on a T200 Biacore instrument. In Example 6, the binding studies were conducted using size exclusion chromatography. In Example 7, a neutralization bioassay was developed in Vero cells to detect cell viability after treatment with toxin A or B and antibodies to either toxin A or to toxin B.

[0202] The present invention also includes anti-toxin A or B antibodies and antigen binding fragments thereof which bind to at least one biologically active fragment of any of the following proteins, or peptides: SEQ ID NO: 378 (full length toxin A), residue numbers 1832-2710 of SEQ ID NO: 378 (C-terminal domain of toxin A); SEQ ID NO: 380 (full length toxin B), residues 1834-2366 of SEQ ID NO: 380; SEQ ID NO: 375 (carboxy terminal receptor binding domain of toxin A); or SEQ ID NO: 376. The present invention also provides for antibodies that interact with or bind to an epitope within the carboxy terminal receptor binding domain of toxin A produced by Clostridium difficile, or an antigen binding fragment thereof, wherein the epitope is contained within residues ranging from about residue 468 to about 863 of SEQ ID NO: 375. In one embodiment, the epitope for an antibody that binds toxin A is selected from the group consisting of residues 468-488 of SEQ ID NO: 375, residues 510-530 of SEQ ID NO: 375, residues 602-610 of SEQ ID NO: 375, residues 644-703 of SEQ ID NO: 375, residues 724-794 of SEQ ID NO: 375, residues 799-814 of SEQ ID NO: 375 and residues 858-863 of SEQ ID NO: 375. Any of the toxin A or toxin B peptides described herein, or fragments thereof, may be used to generate anti-toxin A or anti-toxin B antibodies.

[0203] The peptides may be modified to include addition or substitution of certain residues for tagging or for purposes of conjugation to carrier molecules, such as, KLH. For example, a cysteine may be added at either the N terminal or C terminal end of a peptide, or a linker sequence may be added to prepare the peptide for conjugation to, for example, KLH for immunization. The antibodies specific for toxin A or toxin B may contain no additional labels or moieties, or they may contain an N-terminal or C-terminal label or moiety. In one embodiment, the label or moiety is biotin. In a binding assay, the location of a label (if any) may determine the orientation of the peptide relative to the surface upon which the peptide is bound. For example, if a surface is coated with avidin, a peptide containing an N-terminal biotin will be oriented such that the C-terminal portion of the peptide will be distal to the surface.

Epitope Mapping and Related Technologies

[0204] Various techniques known to persons of ordinary skill in the art can be used to determine whether an antibody "interacts with one or more amino acids" within a polypeptide or protein. Exemplary techniques include, for example, a routine cross-blocking assay such as that described Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., N.Y.) can be performed. Other methods include alanine scanning mutational analysis, peptide blot analysis (Reineke (2004) Methods Mol Biol 248:443-63), peptide cleavage analysis crystallographic studies and NMR analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer (2000) Protein Science 9: 487-496). Another method that can be used to identify the amino acids within a polypeptide with which an antibody interacts is hydrogen/deuterium exchange detected by mass spectrometry. In general terms, the hydrogen/deuterium exchange method involves deuterium-labeling the protein of interest, followed by binding the antibody to the deuterium-labeled protein. Next, the protein/antibody complex is transferred to water and exchangeable protons within amino acids that are protected by the antibody complex undergo deuterium-to-hydrogen back-exchange at a slower rate than exchangeable protons within amino acids that are not part of the interface. As a result, amino acids that form part of the protein/antibody interface may retain deuterium and therefore exhibit relatively higher mass compared to amino acids not included in the interface. After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing the deuterium-labeled residues that correspond to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0205] The term "epitope" refers to a site on an antigen to which B and/or T cells respond. B-cell epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.

[0206] Modification-Assisted Profiling (MAP), also known as Antigen Structure-based Antibody Profiling (ASAP) is a method that categorizes large numbers of monoclonal antibodies (mAbs) directed against the same antigen according to the similarities of the binding profile of each antibody to chemically or enzymatically modified antigen surfaces (US 2004/0101920, herein specifically incorporated by reference in its entirety). Each category may reflect a unique epitope either distinctly different from or partially overlapping with epitope represented by another category. This technology allows rapid filtering of genetically identical antibodies, such that characterization can be focused on genetically distinct antibodies. When applied to hybridoma screening, MAP may facilitate identification of rare hybridoma clones that produce mAbs having the desired characteristics. MAP may be used to sort the antibodies of the invention into groups of antibodies binding different epitopes.

[0207] In certain embodiments, the anti-toxin A or anti-toxin B antibody or antigen-binding fragments thereof binds an epitope within any one of the regions exemplified in FIG. 1, or in SEQ ID NOS: 378, or 380, or at least one of the carboxy terminal receptor binding domains of toxin A or toxin B, or to a fragment thereof, wherein the carboxy terminal receptor binding domain of toxin A is shown in SEQ ID NO: 375, and wherein the carboxy terminal receptor binding domain of toxin B is shown as SEQ ID NO: 376.

[0208] The present invention includes anti-toxin A or anti-toxin B antibodies that bind to the same epitope as any of the specific exemplary antibodies described herein in Table 1. Likewise, the present invention also includes anti-toxin A or anti-toxin B antibodies that compete for binding to toxin A or B or a toxin A or B fragment with any of the specific exemplary antibodies described herein in Table 1.

[0209] One can easily determine whether an antibody binds to the same epitope as, or competes for binding with, a reference anti-toxin A or anti-toxin B antibody by using routine methods known in the art. For example, to determine if a test antibody binds to the same epitope as a reference anti-toxin A or anti-toxin B antibody of the invention, the reference antibody is allowed to bind to a toxin A or B protein or peptide under saturating conditions. Next, the ability of a test antibody to bind to the toxin A or B molecule is assessed. If the test antibody is able to bind to toxin A or B following saturation binding with the reference anti-toxin A or anti-toxin B antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-toxin A or anti-toxin B antibody. On the other hand, if the test antibody is not able to bind to the toxin A or B molecule following saturation binding with the reference anti-toxin A or anti-toxin B antibody, then the test antibody may bind to the same epitope as the epitope bound by the reference anti-toxin A or anti-toxin B antibody of the invention.

[0210] To determine if an antibody competes for binding with a reference anti-toxin A or anti-toxin B antibody, the above-described binding methodology is performed in two orientations: In a first orientation, the reference antibody is allowed to bind to a toxin A or B molecule under saturating conditions followed by assessment of binding of the test antibody to the toxin A or B molecule. In a second orientation, the test antibody is allowed to bind to a toxin A or B molecule under saturating conditions followed by assessment of binding of the reference antibody to the toxin A or B molecule. If, in both orientations, only the first (saturating) antibody is capable of binding to the toxin A or B molecule, then it is concluded that the test antibody and the reference antibody compete for binding to toxin A or B. As will be appreciated by a person of ordinary skill in the art, an antibody that competes for binding with a reference antibody may not necessarily bind to the identical epitope as the reference antibody, but may sterically block binding of the reference antibody by binding an overlapping or adjacent epitope.

[0211] Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) binding of the other to the antigen. That is, a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibits binding of the other by at least 50% but preferably 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990 50:1495-1502). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

[0212] Additional routine experimentation (e.g., peptide mutation and binding analyses) can then be carried out to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody or if steric blocking (or another phenomenon) is responsible for the lack of observed binding. Experiments of this sort can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry or any other quantitative or qualitative antibody-binding assay available in the art.

Immunoconjugates

[0213] The invention encompasses a human anti-toxin A or anti-toxin B monoclonal antibody conjugated to a therapeutic moiety ("immunoconjugate"), such as an agent that is capable of reducing the severity of primary infection with C. difficile, or to ameliorate at least one symptom associated with C. difficile infection, including diarrhea or colitis, or the severity thereof. Such an agent may be a second different antibody to either or both toxin A or toxin B of C. difficile, or a toxoid, or a C. difficile vaccine. The type of therapeutic moiety that may be conjugated to the anti-toxin A or anti-toxin B antibody and will take into account the condition to be treated and the desired therapeutic effect to be achieved. Alternatively, if the desired therapeutic effect is to treat the sequelae or symptoms associated with C. difficile infection, or any other condition resulting from such infection, such as, but not limited to, pseudomembranous colitis, it may be advantageous to conjugate an agent appropriate to treat the sequelae or symptoms of the condition, or to alleviate any side effects of the antibodies of the invention. Examples of suitable agents for forming immunoconjugates are known in the art, see for example, WO 05/103081.

Multi-Specific Antibodies

[0214] The antibodies of the present invention may be mono-specific, bi-specific, or multi-specific. Multi-specific antibodies may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. The antibodies of the present invention can be linked to or co-expressed with another functional molecule, e.g., another peptide or protein. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment to produce a bi-specific or a multi-specific antibody with a second binding specificity. For example, the present invention includes bi-specific antibodies wherein one arm of an immunoglobulin is specific for toxin A of C. difficile, or a fragment thereof, and the other arm of the immunoglobulin is specific for toxin B of C. difficile, or a second therapeutic target, or is conjugated to a therapeutic moiety. In certain embodiments of the invention, one arm of an immunoglobulin is specific for an epitope on the C-terminal domain of toxin A or a fragment thereof, and the other arm of the immunoglobulin is specific for an epitope on the C-terminal domain of toxin B, or a fragment thereof.

[0215] An exemplary bi-specific antibody format that can be used in the context of the present invention involves the use of a first immunoglobulin (Ig) C.sub.H3 domain and a second Ig C.sub.H3 domain, wherein the first and second Ig C.sub.H3 domains differ from one another by at least one amino acid, and wherein at least one amino acid difference reduces binding of the bi-specific antibody to Protein A as compared to a bi-specific antibody lacking the amino acid difference. In one embodiment, the first Ig C.sub.H3 domain binds Protein A and the second Ig C.sub.H3 domain contains a mutation that reduces or abolishes Protein A binding such as an H95R modification (by IMGT exon numbering; H435R by EU numbering). The second C.sub.H3 may further comprise a Y96F modification (by IMGT; Y436F by EU). Further modifications that may be found within the second C.sub.H3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1 antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU) in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU) in the case of IgG4 antibodies. Variations on the bi-specific antibody format described above are contemplated within the scope of the present invention.

Therapeutic Administration and Formulations

[0216] The invention provides therapeutic compositions comprising the anti-toxin A or anti-toxin B antibodies or antigen-binding fragments thereof of the present invention. The administration of therapeutic compositions in accordance with the invention will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN.TM.), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0217] The dose of each of the antibodies of the invention may vary depending upon the age and the size of a subject to be administered, target disease, conditions, route of administration, and the like. When the antibodies of the present invention are used for treating a C. difficile infection in a patient, or for treating one or more symptoms associated with a C. difficile infection, such as the diarrhea or colitis associated with a C. difficile infection in a patient, or for preventing a relapse of the disease, or for lessening the severity of the disease, it is advantageous to administer each of the antibodies of the present invention intravenously or subcutaneously normally at a single dose of about 0.01 to about 30 mg/kg body weight, more preferably about 0.1 to about 20 mg/kg body weight, or about 0.1 to about 15 mg/kg body weight, or about 0.02 to about 7 mg/kg body weight, about 0.03 to about 5 mg/kg body weight, or about 0.05 to about 3 mg/kg body weight, or about 1 mg/kg body weight, or about 3.0 mg/kg body weight, or about 10 mg/kg body weight, or about 20 mg/kg body weight. Multiple doses may be administered as necessary. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted. In certain embodiments, the antibodies or antigen-binding fragments thereof of the invention can be administered as an initial dose of at least about 0.1 mg to about 800 mg, about 1 to about 600 mg, about 5 to about 300 mg, or about 10 to about 150 mg, to about 100 mg, or to about 50 mg. In certain embodiments, the initial dose may be followed by administration of a second or a plurality of subsequent doses of the antibodies or antigen-binding fragments thereof in an amount that can be approximately the same or less than that of the initial dose, wherein the subsequent doses are separated by at least 1 day to 3 days; at least one week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.

[0218] Various delivery systems are known and can be used to administer the pharmaceutical composition of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.

[0219] The pharmaceutical composition can be also delivered in a vesicle, in particular a liposome (see, for example, Langer (1990) Science 249:1527-1533).

[0220] In certain situations, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used. In another embodiment, polymeric materials can be used. In yet another embodiment, a controlled release system can be placed in proximity of the composition's target, thus requiring only a fraction of the systemic dose.

[0221] The injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in an appropriate ampoule.

[0222] A pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0223] Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present invention. Examples include, but certainly are not limited to AUTOPEN.TM. (Owen Mumford, Inc., Woodstock, UK), DISETRONIC.TM. pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75/25.TM. pen, HUMALOG.TM. pen, HUMALIN 70/30.TM. pen (Eli Lilly and Co., Indianapolis, Ind.), NOVOPEN.TM. I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR.TM. (Novo Nordisk, Copenhagen, Denmark), BD.TM. pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPEN.TM., OPTIPEN PRO.TM., OPTIPEN STARLET.TM., and OPTICLIK.TM. (sanofi-aventis, Frankfurt, Germany), to name only a few. Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present invention include, but certainly are not limited to the SOLOSTAR.TM. pen (sanofi-aventis), the FLEXPEN.TM. (Novo Nordisk), and the KWIKPEN.TM. (Eli Lilly), the SURECLICK.TM. Autoinjector (Amgen, Thousands Oaks, Calif.), the PENLET.TM. (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L. P.) and the HUMIRA.TM.Pen (Abbott Labs, Abbott Park, Ill.), to name only a few.

[0224] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the aforesaid antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose; especially in the form of injection, it is preferred that the aforesaid antibody is contained in about 5 to about 100 mg and in about 10 to about 250 mg for the other dosage forms.

Administration Regimens

[0225] According to certain embodiments of the present invention, multiple doses of an antibody to toxin A and/or B of Clostridium difficile may be administered to a subject over a defined time course. The methods according to this aspect of the invention comprise sequentially administering to a subject multiple doses of an antibody to toxin A and/or B. As used herein, "sequentially administering" means that each dose of antibody to toxin A and/or B is administered to the subject at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks or months). The present invention includes methods which comprise sequentially administering to the patient a single initial dose of an antibody to toxin A and/or B, followed by one or more secondary doses of the antibody to toxin A and/or B, and optionally followed by one or more tertiary doses of the antibody to toxin A and/or B.

[0226] The terms "initial dose," "secondary doses," and "tertiary doses," refer to the temporal sequence of administration of the antibody to toxin A and/or B. Thus, the "initial dose" is the dose which is administered at the beginning of the treatment regimen (also referred to as the "baseline dose"); the "secondary doses" are the doses which are administered after the initial dose; and the "tertiary doses" are the doses which are administered after the secondary doses. The initial, secondary, and tertiary doses may all contain the same amount of antibody to toxin A and/or B, but generally may differ from one another in terms of frequency of administration. In certain embodiments, however, the amount of antibody to toxin A and/or B contained in the initial, secondary and/or tertiary doses vary from one another (e.g., adjusted up or down as appropriate) during the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as "loading doses" followed by subsequent doses that are administered on a less frequent basis (e.g., "maintenance doses").

[0227] In one exemplary embodiment of the present invention, each secondary and/or tertiary dose is administered 1 to 26 (e.g., 1, 11/2, 2, 21/2, 3, 31/2, 4, 41/2, 5, 51/2, 6, 61/2, 7, 71/2, 8, 81/2, 9, 91/2, 10, 101/2, 11, 111/2, 12, 121/2, 13, 131/2, 14, 141/2, 15, 151/2, 16, 161/2, 17, 171/2, 18, 181/2, 19, 191/2, 20, 201/2, 21, 211/2, 22, 221/2, 23, 231/2, 24, 241/2, 25, 251/2, 26, 261/2, or more) weeks after the immediately preceding dose. The phrase "the immediately preceding dose," as used herein, means, in a sequence of multiple administrations, the dose of antibody to toxin A and/or B which is administered to a patient prior to the administration of the very next dose in the sequence with no intervening doses.

[0228] The methods according to this aspect of the invention may comprise administering to a patient any number of secondary and/or tertiary doses of an antibody to toxin A and/or B. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Likewise, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.

[0229] In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2 weeks after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2 to 4 weeks after the immediately preceding dose. Alternatively, the frequency at which the secondary and/or tertiary doses are administered to a patient can vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by a physician depending on the needs of the individual patient following clinical examination.

Therapeutic Uses of the Antibodies

[0230] Due to their interaction with toxin A and/or toxin B of C. difficile, the present antibodies are useful for treating the primary C. difficile disease or condition, or at least one symptom associated with the disease or condition, such as diarrhea or colitis, or for preventing a relapse of the disease, or for lessening the severity, duration, and/or frequency of recurrences of the disease. The antibodies of the invention are also contemplated for prophylactic use in patients at risk for developing or acquiring a C. difficile infection. These patients include the elderly (for example, in anyone 65 years of age or older), or patients immunocompromised due to illness or treatment with immunosuppressive therapeutics, or patients who may have an underlying medical condition that predisposes them to a C. difficile infection (for example, cancer, inflammatory bowel disease, pre-liver transplant patients with ascites accumulation), or patients that are hospitalized for long periods of time (for example, in some cases this time period may vary from as little as two or three days, but generally can be from one week, to two weeks or longer), making them prone to acquiring nosocomial infections, or patients on long term treatment (.gtoreq.14 days) with broad spectrum antibiotics (in some instances, patients may acquire the infection within 24 hours if the gut is disregulated, but in other instances this may take much longer, for example, one week or longer), or patients on therapy with proton pump inhibitors for treatment of gastrointestinal disorders. It is contemplated that the antibodies of the invention may be used alone, or in conjunction with a second agent, or third agent for treating the C. difficile infection, or for alleviating at least one symptom or complication associated with the C. difficile infection, such as the diarrhea or colitis associated with, or resulting from such an infection. The second or third agents may be delivered concurrently with the antibodies of the invention, or they may be administered separately, either before or after the antibodies of the invention.

[0231] In yet a further embodiment of the invention the present antibodies are used for the preparation of a pharmaceutical composition for treating patients suffering from a C. difficile infection, including those infections caused by a clinically relevant hypervirulent strain of Clostridium difficile, or the diarrhea and colitis associated with a C. difficile infection. In yet another embodiment of the invention the present antibodies are used for the preparation of a pharmaceutical composition for reducing the severity of a primary infection with C. difficile, or for reducing the severity, duration of, and/or number of recurrences with C. difficile. In a further embodiment of the invention the present antibodies are used as adjunct therapy with any other agent useful for treating C. difficile infections, including probiotics, antibiotics, toxoids, vaccines, or any other palliative therapy known to those skilled in the art.

Combination Therapies

[0232] The methods of the present invention, according to certain embodiments, comprise administering to the subject one or more additional therapeutic agents in combination with an antibody to toxin A and/or toxin B of Clostridium difficile. As used herein, the expression "in combination with" means that the additional therapeutic agents are administered before, after, or concurrent with the pharmaceutical composition comprising the anti-toxin A and/or B antibodies. The term "in combination with" also includes sequential or concomitant administration of the anti-toxin A and/or B antibodies and a second therapeutic agent.

[0233] For example, when administered "before" the pharmaceutical composition comprising the anti-toxin A and/or B antibodies, the additional therapeutic agent may be administered about 72 hours, about 60 hours, about 48 hours, about 36 hours, about 24 hours, about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, about 1 hour, about 30 minutes, about 15 minutes or about 10 minutes prior to the administration of the pharmaceutical composition comprising the anti-toxin A and/or B antibodies. When administered "after" the pharmaceutical composition comprising the anti-toxin A and/or B antibodies, the additional therapeutic agent may be administered about 10 minutes, about 15 minutes, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours or about 72 hours after the administration of the pharmaceutical composition comprising the anti-toxin A and/or B antibodies. Administration "concurrent" or with the pharmaceutical composition comprising the anti-toxin A and/or B antibodies means that the additional therapeutic agent is administered to the subject in a separate dosage form within less than 5 minutes (before, after, or at the same time) of administration of the pharmaceutical composition comprising the anti-toxin A and/or B antibodies, or administered to the subject as a single combined dosage formulation comprising both the additional therapeutic agent and the anti-toxin A and/or B antibodies.

[0234] Combination therapies may include an anti-toxin A or anti-toxin B antibody of the invention and any additional therapeutic agent that may be advantageously combined with an antibody of the invention, or with a biologically active fragment of an antibody of the invention.

[0235] For example, a second or third therapeutic agent may be employed to aid in reducing the bacterial load in the gut, such as an antibiotic that is bacteriostatic or bacteriocidal with respect to C. difficile. Exemplary antibiotics include vancomycin, metronidazole, or fidaxomicin. The antibodies may also be used in conjunction with other therapies, such as toxoids, vaccines specific for C. difficile, or probiotic agents, such as Saccharomyces boulardii.

Diagnostic Uses of the Antibodies

[0236] The anti-toxin A or anti-toxin B antibodies of the present invention may also be used to detect and/or measure toxin A or B in a sample, e.g., for diagnostic purposes. It is envisioned that confirmation of an infection thought to be caused by C. difficile may be made by measuring the presence of either toxin A or toxin B through use of any one or more of the antibodies of the invention. Exemplary diagnostic assays for toxin A or toxin B may comprise, e.g., contacting a sample, obtained from a patient, with an anti-toxin A or anti-toxin B antibody of the invention, wherein the anti-toxin A or anti-toxin B antibody is labeled with a detectable label or reporter molecule or used as a capture ligand to selectively isolate toxin A or toxin B protein from patient samples. Alternatively, an unlabeled anti-toxin A or anti-toxin B antibody can be used in diagnostic applications in combination with a secondary antibody which is itself detectably labeled. The detectable label or reporter molecule can be a radioisotope, such as .sup.3H, .sup.14C, .sup.32 P, .sup.35S, or .sup.125I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate, or rhodamine; or an enzyme such as alkaline phosphatase, .beta.-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure toxin A or toxin B in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0237] Samples that can be used in C. difficile diagnostic assays according to the present invention include any tissue or fluid sample obtainable from a patient, which contains detectable quantities of either C. difficile toxin A or toxin B protein, or fragments thereof, under normal or pathological conditions. Generally, levels of toxin A or toxin B in a particular sample obtained from a healthy patient (e.g., a patient not afflicted with a disease or condition associated with the presence of C. difficile) will be measured to initially establish a baseline, or standard, level of toxin A or toxin B from C. difficile. This baseline level of toxin A or toxin B can then be compared against the levels of toxin A or toxin B measured in samples obtained from individuals suspected of having a C. difficile related disease or condition, or symptoms associated with such disease or condition.

EXAMPLES

[0238] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Example 1

Generation of Human Antibodies to Clostridium difficile Toxin a and/or Toxin B

[0239] An immunogen comprising any one of the following can be used to generate antibodies to C. difficile toxin A and/or toxin B. In certain embodiments, the antibodies of the invention are obtained from mice immunized with a primary immunogen, such as a full length, native, inactivated, toxin A (See GenBank accession number CAA63564 (SEQ ID NO: 378)), and/or toxin B (See GenBank accession number CAJ67492 (SEQ ID NO: 380)) from C. difficile, or with a recombinant, but inactivated form of the toxins, or toxin fragments, or a toxoid, followed by immunization with a secondary immunogen, or with an immunogenically active fragment of the native toxin. Animals may be immunized with either inactivated toxin A alone or inactivated toxin B alone, or with both inactivated toxin A and inactivated toxin B, concurrently. The toxins can be inactivated prior to use as an immunogen using standard procedures for preparing toxoids, including by treatment with formaldehyde, glutaraldehyde, peroxide, or oxygen treatment (Relyveld, et al. Methods in Enzymology, 93:24, 1983, Woodrow and Levine, eds. New Generation Vaccines, Marcel Dekker, Inc., New York, 1990). Another means of inactivation is by use of UDP-dialdehyde (Genth et al., (2000), Infect. Immun. 68(3):1094-1101), which may act to preserve the native structure of the toxin compared to other inactivation methods, thereby enhancing the likelihood of eliciting antibodies that are more reactive with the native toxin. Alternatively, mutant toxins from C. difficile, which exhibit reduced toxicity, may be produced using standard recombinant techniques and used as immunogens (See, for example, U.S. Pat. Nos. 5,085,862; 5,221,618; 5,244,657; 5,332,583; 5,358,868; and 5,433,945). Such mutants may contain deletions or point mutations in the active site of the toxin.

[0240] In certain embodiments, the antibodies of the invention are obtained from mice immunized with a primary immunogen, such as a biologically active and/or immunogenic fragment of native toxin A or toxin B, or DNA encoding the active fragment thereof. In certain embodiments, the immunogen may be a peptide from the N terminal or C terminal end of toxin A and/or toxin B, or a fragment derived from the N or C terminal peptide of toxin A and/or toxin B. In certain embodiments of the invention, the immunogen is the carboxy terminal receptor binding domain of toxin A that ranges from about amino acid residues 1832-2710 of SEQ ID NO: 378. In certain embodiments of the invention, the immunogen is the carboxy terminal receptor binding domain of toxin A that is shown in SEQ ID NO: 375. In certain embodiments of the invention, the immunogen is the carboxy terminal receptor binding domain of toxin B that ranges from about amino acid residues 1834-2366 of SEQ ID NO: 380. In certain embodiments of the invention, the immunogen is the carboxy terminal receptor binding domain of toxin B that is shown in SEQ ID NO: 376.

[0241] Accordingly, in one embodiment, the antibodies of the invention were obtained from mice immunized with either an inactivated full length toxin A (toxoid), or an inactivated full length toxin B (toxoid), or both toxoids. Furthermore, in one embodiment, antibodies were obtained from mice immunized with a polypeptide comprising amino acid sequences from the carboxy-terminal receptor binding domain of C. difficile toxin A, or with a polypeptide comprising amino acid sequences from the carboxy-terminal receptor binding domain of C. difficile toxin B, or both, concurrently.

[0242] In certain embodiments, antibodies that bind specifically to C. difficile toxin A or toxin B may be prepared using fragments of the above-noted regions, or peptides that extend beyond the designated regions by about 5 to about 20 amino acid residues from either, or both, the N or C terminal ends of the regions described herein. In certain embodiments, any combination of the above-noted regions or fragments thereof may be used in the preparation of toxin A or toxin B specific antibodies. In certain embodiments, any one or more of the above-noted regions of toxin A or toxin B, or fragments thereof may be used for preparing monospecific, bispecific, or multispecific antibodies.

[0243] The full length proteins, or carboxy-terminal fragments thereof, that were used as immunogens, as noted above, were administered directly, with an adjuvant to stimulate the immune response, to a VELOCIMMUNE.RTM. mouse comprising DNA encoding human Immunoglobulin heavy and kappa light chain variable regions. The antibody immune response was monitored by a C. difficile toxin A and/or toxin B-specific immunoassay. When a desired immune response was achieved splenocytes were harvested and fused with mouse myeloma cells to preserve their viability and form hybridoma cell lines. The hybridoma cell lines were screened and selected to identify cell lines that produce C. difficile toxin A and/or toxin B-specific antibodies. Using this technique, and the various immunogens described above, several anti-C. difficile toxin A and toxin B, as well as cross-reactive, chimeric antibodies (i.e., antibodies possessing human variable domains and mouse constant domains) were obtained; certain exemplary antibodies generated in this manner were designated as H1H3067N, H1H3134N, H1H3117N, H1M3123N, H1M3121N and H1M3124N.

[0244] Anti-C. difficile toxin A and toxin B antibodies were also isolated directly from antigen-positive B cells without fusion to myeloma cells, as described in U.S. 2007/0280945A1, herein specifically incorporated by reference in its entirety. Using this method, several fully human anti-C. difficile toxin A and toxin B antibodies (i.e., antibodies possessing human variable domains and human constant domains) were obtained; exemplary antibodies generated in this manner were designated as follows: H1H3328P, H1H3324P, H1H3325P, H1H3330P, H1H3350P, H1H3347P, H1H3335P, H1H3344P. H1H3339P, H1H3337P, H1H3343P, H1H3411P, H1H3354P, H1H3317P, H1H3355P, H1H3394P and H1H3401P.

[0245] The biological properties of the exemplary antibodies generated in accordance with the methods of this Example are described in detail in the Examples set forth below.

Example 2

Heavy and Light Chain Variable Region Amino Acid Sequences

[0246] Table 1 sets forth the heavy and light chain variable region amino acid sequence pairs of selected antibodies specific for toxin A and/or toxin B from C. difficile and their corresponding antibody identifiers. Antibodies are typically referred to herein according to the following nomenclature: Fc prefix (e.g. "H4H", "H1M, "H2M"), followed by a numerical identifier (e.g. "3117" as shown in Table 1), followed by a "P" or "N" suffix. Thus, according to this nomenclature, an antibody may be referred to as, e.g. "H1H3117". The H4H, H1M, and H2M prefixes on the antibody designations used herein indicate the particular Fc region of the antibody. For example, an "H2M" antibody has a mouse IgG2 Fc, whereas an "H4H" antibody has a human IgG4 Fc. As will be appreciated by a person of ordinary skill in the art, an H1M or H2M antibody can be converted to an H4H antibody, and vice versa, but in any event, the variable domains (including the CDRs), which are indicated by the numerical identifiers shown in Table 1, will remain the same. Antibodies having the same numerical antibody designation, but differing by a letter suffix of N, B or P refer to antibodies having heavy and light chains with identical CDR sequences but with sequence variations in regions that fall outside of the CDR sequences (i.e., in the framework regions). Thus, N, B and P variants of a particular antibody have identical CDR sequences within their heavy and light chain variable regions but differ from one another within their framework regions.

Antibody Comparators

[0247] Anti-toxin A and anti-toxin B antibody controls were included in the following Examples for comparative purposes. Isotype matched negative controls were also used in the Examples. One anti-toxin A control antibody is designated herein as Control I and is an anti-toxin A antibody with heavy and light chain variable domain sequences of the "3D8" antibody as set forth in U.S. Pat. No. 7,625,559 and US2005/0287150. One anti-toxin B antibody is designated herein as Control II and is an anti-toxin B antibody with heavy and light chain variable domain sequences of the "124-152" antibody as set forth in U.S. Pat. No. 7,625,559 and US2005/0287150. Another anti-toxin A antibody is designated herein as Control Ill and is an anti-toxin A antibody with heavy and light chain variable domain sequences of the "3358" antibody as set forth in US2009/0087478.

TABLE-US-00001 TABLE 1 Antibody SEQ ID NOs: Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H1H3117N 2 4 6 8 10 12 14 16 H1H3134N 18 20 22 24 26 28 30 32 H1H3067N 34 36 38 40 42 44 46 48 H1H3121N 50 52 54 56 58 60 62 64 H1H3123N 66 68 70 72 74 76 78 80 H1H3124N 82 84 86 88 90 92 94 96 H1H3324P 98 100 102 104 106 108 110 112 H1H3325P 114 116 118 120 122 124 126 128 H1H3328P 130 132 134 136 138 140 142 144 H1H3330P 146 148 150 152 154 156 158 160 H1H3350P 162 164 166 168 170 172 174 176 H1H3317P 178 180 182 184 186 188 190 192 H1H3335P 194 196 198 200 202 204 206 208 H1H3337P 210 212 214 216 218 220 222 224 H1H3339P 226 228 230 232 234 236 238 240 H1H3343P 242 244 246 248 250 252 254 256 H1H3344P 258 260 262 264 266 268 270 272 H1H3347P 274 276 278 280 282 284 286 288 H1H3354P 290 292 294 296 298 300 302 304 H1H3355P 306 308 310 312 314 316 318 320 H1H3394P 322 324 326 328 330 332 334 336 H1H3401P 338 340 342 344 346 348 350 352 H1H3411P 354 356 358 360 362 364 366 368

Example 3

Variable Gene Utilization Analysis

[0248] To analyze the structure of antibodies produced, the nucleic acids encoding antibody variable regions were cloned and sequenced. From the nucleic acid sequence and predicted amino acid sequence of the antibodies, gene usage was identified for each Heavy Chain Variable Region (HCVR) and Light Chain Variable Region (LCVR). Table 2 sets forth the gene usage for selected antibodies in accordance with the invention.

TABLE-US-00002 TABLE 2 Antibody Identifier HCVR LCVR Antibody HCVR/LCVR V.sub.H D.sub.H J.sub.H V.sub.K J.sub.K H1H3067N 34/42 3-30 6-6 4 4-1 4 H1H3134N 18/26 3-33 3-10 4 4-1 3 H1H3117N 2/10 3-23 1-7 4 3-20 2 H1H3123N 66/74 3-48 4-11 6 1-5 1 H1H3121N 50/58 3-48 5-18 6 1-5 1 H1H3124N 82/90 3-48 3-22 6 1-5 1 H1H3328P 130/138 3-13 3-10 6 1-27 3 H1H3324P 98/106 3-13 3-10 6 1-27 3 H1H3325P 114/122 3-23 3-10 6 1-5 1 H1H3330P 146/154 3-33 1-7 4 1-39 5 H1H3350P 162/170 3-11 7-27 4 3-15 2 H1H3347P 274/282 3-23 1-26 4 1-16 3 H1H3335P 194/202 3-23 1-26 4 1-16 3 H1H3344P 258/266 3-23 2-15 4 1-16 3 H1H3339P 226/234 3-23 1-26 4 1-16 3 H1H3337P 210/218 3-23 1-26 5 1-16 3 H1H3343P 242/250 3-23 1-26 4 1-16 3 H1H3411P 354/362 3-23 1-1 6 1D-12.sup. 2 H1H3354P 290/298 6-1 2-8 4 3-11 2 H1H3317P 178/186 3-30 3-10 4 1D-12.sup. 4 H1H3355P 306/314 3-9 1-26 6 1-6 3 H1H3394P 322/330 1-2 2-2 4 3-20 4 H1H3401P 338/346 3-30 1-1 4 1D-12.sup. 2

Example 4

Antibody Binding to Toxin A and/or Toxin B from C. difficile as Determined by Surface Plasmon Resonance

[0249] Binding affinities and kinetic constants of human monoclonal anti-Clostridium difficile toxin A and/or B antibodies were determined by surface plasmon resonance at 37.degree. C. (Tables 3-5). Measurements were conducted on a T200 Biacore instrument.

[0250] Antibodies, expressed as human IgG1 Fc (AbPID prefix H1H) or hybridoma (AbPID prefix HxM), were captured onto an anti-human or anti-mouse-Fc sensor surface, respectively (Mab capture format). Soluble full-length toxin A or B (TechLab), ranging from 5 to 10 nM, was injected over the antibody-captured surface. Antibody-antigen association was monitored for 150 seconds while dissociation in buffer was monitored for 480 seconds. Kinetic analysis was performed to calculate K.sub.D and half-life of antigen/antibody complex dissociation using Biacore T200 evaluation software 1.0.

[0251] As seen in Tables 3-5, three types of antibodies were isolated: antibodies that bound both toxin A and toxin B ("dual binders", see Table 3), antibodies that bound only toxin A (Table 4), and antibodies that bound only toxin B (Table 5). Several antibodies were identified that bound both toxin A and toxin B, including those designated as H2M3121N, H2M3123N, H2M3124N, H1H3067N and H1H3134N and thus were classified as dual binders. Isolated anti-toxin A antibodies bound toxin in the sub-nanomolar (nM) range similar to the isotype matched comparator Mab (control I; see US patent U.S. Pat. No. 7,625,559 for comparator sequences for clone 3D8 (A toxin Ab) and clone 124-152 (B toxin Ab)), while only a few anti-toxin B binders showed affinities in the range of control II isotype matched comparator Mab (clone 124-152) (.about.200-300 pM). Binding dissociation equilibrium constants and dissociative half-lives were calculated from the kinetic rate constants as: K.sub.D=k.sub.d/k.sub.a; T.sub.1/2 (min)=(ln 2/k.sub.d)/60

TABLE-US-00003 TABLE 3 Biacore affinities of anti-C. difficile Dual Binding mAbs at 37.degree. C. Binding at 37.degree. C./Mab Capture Format Analyte AbPID (Toxin) ka (Ms.sup.-1) kd (s.sup.-1) K.sub.D (Molar) T1/2 (min) H2M3121N Toxin A 9.69E+05 1.66E-04 1.72E-10 69 Toxin B 6.11E+04 7.58E-05 1.24E-09 152 H2M3123N Toxin A 1.23E+06 5.93E-04 4.81E-10 19 Toxin B 3.97E+04 6.54E-05 1.65E-09 176 H2M3124N Toxin A 1.14E+06 1.98E-04 1.74E-10 58 Toxin B 3.31E+05 1.00E-06 3.02E-12 11550 H1H3067N Toxin A 1.44E+05 3.45E-05 2.40E-10 335 Toxin B 2.54E+03 6.43E-04 2.53E-07 18 H1H3134N Toxin A 1.02E+05 2.82E-06 2.78E-11 4096 Toxin B 2.99E+03 9.73E-04 3.25E-07 12

TABLE-US-00004 TABLE 4 Biacore affinities of anti-C. difficile Toxin A mAbs at 37.degree. C. Binding at 37.degree. C./Mab Capture Format T1/2 AbPID Analyte ka (Ms.sup.-1) kd (s.sup.-1) K.sub.D (Molar) (min) H1H3117N Toxin A 4.38E+05 3.84E-05 7.93E-11 332 H1H3324P Toxin A 2.51E+05 3.50E-06 1.39E-11 3297 H1H3325P Toxin A 5.27E+05 5.51E-05 1.05E-10 209 H1H3328P Toxin A 3.82E+05 3.66E-05 9.57E-11 316 H1H3330P Toxin A 2.50E+05 1.37E-04 5.47E-10 85 H1H3350P Toxin A 4.02E+05 4.05E-06 1.01E-11 2854 Control I Toxin A 3.77E+05 3.24E-05 8.59E-11 58

TABLE-US-00005 TABLE 5 Biacore affinities of anti-C. difficile Toxin B mAbs at 37.degree. C. T1/2 AbPID Analyte ka (Ms.sup.-1) kd (s.sup.-1) K.sub.D (Molar) (min) H1H3317P Toxin B 6.50E+05 7.78E-05 1.20E-10 149 H1H3335P Toxin B 1.77E+05 4.14E-04 2.34E-09 28 H1H3337P Toxin B 2.41E+05 9.45E-04 3.93E-09 12 H1H3339P Toxin B 2.76E+05 5.37E-04 1.95E-09 22 H1H3343P Toxin B 2.84E+05 4.48E-04 1.58E-09 26 H1H3344P Toxin B 2.04E+05 8.65E-04 4.24E-09 13 H1H3347P Toxin B 3.39E+05 8.13E-04 2.40E-09 14 H1H3354P Toxin B NB NB NB H1H3355P Toxin B NB NB NB H1H3394P Toxin B 4.86E+05 1.62E-04 3.33E-10 72 H1H3401P Toxin B 4.20E+05 2.41E-04 5.74E-10 48 H1H3411P Toxin B 2.35E+05 1.59E-04 6.77E-10 73 Control II Toxin B 2.11E+06 4.59E-04 2.18E-10 25 NB = no binding under the conditions tested

Example 5

Determination of the Binding Domain for Anti-Clostridium difficile Toxin A and B Antibodies Using Surface Plasmon Resonance

[0252] Studies were done to determine if anti-Clostridium difficile toxin A and/or B antibodies bound to the C-term receptor-Binding Domain (CBD) of each toxin. In these studies, two experimental Biacore formats were employed. The first utilized captured anti-C. difficile antibody surfaces in which 100 nM of CBD-toxin A-Fc (SEQ ID NO:375) or CBD-toxin B-Fc (SEQ ID NO:376) was flowed over and the responses (RU) recorded. The CBD-toxin reagents were formatted in both human and mouse Fc to enable both hybridoma and human Fc formatted antibody analysis. The second format employed antigen (CBD-Fc) captured surfaces in which 500 nM of anti-C. difficile mAb was flowed over. In this format, hybridoma or human Fc formatted antibodies were flowed over human and mouse Fc captured antigens, respectively. In both formats a response that was significantly above background (>50 RU) was considered binding to the CBD of toxin A or B (see Table 6). For both anti-toxin A and anti-toxin B antibodies, epitopes that were within and outside the CBD were obtained. Both control I (3D8 antibody from U.S. Pat. No. 7,625,559 and US 2005/0287150) and control II (124-152 antibody from U.S. Pat. No. 7,625,559 and US 2005/0287150) were mapped to the CBD of their respective toxins in agreement with previous reports (data not shown; see US 2005/0287150 and U.S. Pat. No. 7,625,559).

TABLE-US-00006 TABLE 6 Determination of the domain of binding for C. difficile antibodies C-term Toxin A Binding C-term Toxin B Binding CBD-A mAB Capture CBD-B mAb Capture Capture 100 nM CBD- Capture 500 nM 100 nM CBD-A 500 nM mAB B Binding mAb binding Domain mAb binding (RU) binding (RU) (RU) (RU) Binding.sup.# H2aM3067N -2 237 25 369 C-Term H1M3117N -3 350 -1 21 C-Term A H2aM3121N 0 23 2 10 Non CBD H2aM3123N 1 23 1 14 Non CBD H2aM3124N 0 29 0 19 Non CBD H1M3134N -1 195 23 394 C-Term H1H3324P 269 224 19 -8 C-Term A H1H3325P 17 3 7 -8 Non CBD H1H3328P 354 227 35 -6 C-Term A H1H3330P 441 515 40 -4 C-Term A H1H3335P 13 5 13 -6 Non CBD H1H3337P -17 8 -24 -2 Non CBD H1H3339P 19 2 14 -2 Non CBD H1H3343P 11 3 9 -4 Non CBD H1H3344P 5 5 4 -2 Non CBD H1H3347P 42 -13 44 7 Non CBD H1H3354P -19 -2 -24 -4 Non CBD .sup.#Non CBD indicates no binding to C-term receptor Binding Domain of Toxin-A or -B.

Example 6

Determination of the Domain of Binding for Anti-Clostridium difficile Toxin A and B Antibodies Using Size Exclusion Chromatography

[0253] As a complimentary method for determining if anti-Clostridium difficile toxin A and/or B antibodies bound the C-term receptor Binding Domain (CBD), size exclusion chromatography (SEC) was utilized. Briefly, the CBD of toxin A (SEQ ID NO: 375) or the CBD of toxin B (SEQ ID NO: 376), at .about.500 nM was mixed with excess antibody at specified molar ratios (1:5 and 1:20; CBD:Mab) in phosphate buffered saline containing 5% glycerol pH 7.4 (PBS/G) and incubated at room temperature for 1 hour.

[0254] Any precipitation visible after 1 hr was recorded as +++ (strong), ++ (moderate), + (minimal), or - (not observed). Following centrifugation (5 min. @ 16,000.times.g), the mixture of antibody and CBD was subjected to SEC analysis using a Superose 6 column (GE Healthcare) with PBS/G as the mobile phase. Protein peaks corresponding to complexes larger than the antibody or CBD alone were interpreted as binding to the C-terminal domain.

[0255] The results demonstrated that CBD binding corresponds well with that predicted from the domain of binding inferred from SPR (Biacore) and CBD studies (see example 5). One notable exception was H1H3134N, where binding to CBD-A was not observed via SEC but K.sub.D values indicated dual binding properties for the antibody.

TABLE-US-00007 TABLE 7 Domain of binding for anti-Clostridium difficile toxin A and B antibodies Observed Observed CBD-A CBD-B Precipitation binding via Precipitation binding via Domain Binding Via mAb with CBD-A SEC with CBD-B SEC Biacore H2M3067N +++ Yes NT NT C-Term A/B H1M3117N +++ Yes NT NT C-Term A H2M3121N - No NT NT Non CBD H2M3123N - No NT NT Non CBD H2M3124N - No NT NT Non CBD H1M3134N - No NT NT C-Term A/B H1H3317P NT NT - Yes NT H1H3324P + Yes NT NT C-Term A H1H3325P - No NT NT Non CBD H1H3328P - Yes NT NT C-Term A H1H3330P - Yes N.D. N.D. C-Term A H1H3335P NT NT - No Non CBD H1H3337P NT NT - No Non CBD H1H3339P NT NT - No Non CBD H1H3343P NT NT - No Non CBD H1H3344P NT NT - No Non CBD H1H3347P NT NT - No Non CBD H1H3350P - Yes NT NT NT H1H3354P NT NT - No Non CBD NT = Not Tested. Non CBD indicates no binding to C-term receptor Binding Domain of Toxin-A or -B.

Example 7

Determination of the Neutralization Potency of Anti-Clostridium difficile Toxin a and/or Toxin B Antibodies

[0256] To determine the neutralization potency (IC.sub.50) of anti-C. difficile antibodies in vitro, a cell viability assay was conducted. Briefly, Vero cells (1.25.times.10.sup.3) cultured in MEM alpha medium, supplemented with 10% FBS, were seeded into 96-well microplates and incubated for 16-18 hours at 37.degree. C., in 5% CO.sub.2. Anti-C. difficile toxin antibodies, at various concentrations (0-66 nM), were added to the cells and subsequently incubated with C. difficile toxin A (32 or 25 pM) or toxin B (0.03 pM or 0.01 pM) for 48 hrs. Controls not containing toxin (100% viability) and controls containing toxin but no antibody (100% relative lethality) were utilized. All dilutions of antibody were conducted in triplicate. Following the 2-day incubation, cell viability was measured by adding tetrazolium salt (WST-1; Roche Biochemicals), waiting for 4 hrs to allow for color development and then recording absorbance at 450 nm. Absorbance values were analyzed by a four-parameter logistic equation over an 11-point response curve (GraphPad Prism).

[0257] The results showed that ten antibodies displayed neutralization against toxin A with IC.sub.50 values ranging from 7 pM to 65 pM at 25-32 pM constant toxin A (Table 8A). Of note, H1H3330P demonstrated neutralization potency equal to that of Control III (isotype matched comparator antibody, clone 3358 as set forth in US2009/0087478) and potency of approximately 20 fold greater than control I (see US2005/0287150 for clone 3D8). Several toxin-B neutralizing antibodies showed significantly greater potency than control II (isotype matched comparator antibody, see clone 124-152 of US2005/0287150) with IC.sub.50s ranging from 25-120 pM at 0.03 pM constant toxin B (Table 8B). Antibodies H1M3067N and H1M3134N, while able to bind both toxin A and B showed only neutralization activity against toxin A. While the reason for this is not yet known, one possible explanation for this finding may be that while antibodies can bind at many sites in the repetitive regions of the C terminal portion of the toxin, other parts of the same toxin domain may still be capable of interacting with the mammalian membrane, thus allowing entry of the toxin into the cell.

TABLE-US-00008 TABLE 8A Neutralization potency (IC.sub.50) for selected mAbs against Toxin A Trial 1 (IC.sub.50) Trial 2 (IC.sub.50) Trial 3 (IC.sub.50) Trial4 (IC.sub.50) Trial5 (IC.sub.50) mAb 32 pM Toxin A 32 pM Toxin A 32 pM Toxin A 32 pM Toxin A 25 pM Toxin A H1M3067N 64 pM 44 pM NT NT NT H1M3117N 29 pM 11 pM NT NT NT H2aM3121N 65 pM 35 pM NT NT NT H2aM3123N 65 pM 24 pM NT NT NT H2aM3124N 41 pM 21 pM NT NT NT HIM3134N NT NT NT 38 pM NT H1H3324P NT NT NT 33 pM NT H1H3325P NT NT NT 33 pM NT H1H3328P NT NT 112 pM NT NT H1H3330P NT NT 7 pM NT 7 pM Control I NT NT NT NT 199 pM Control III 18 pM 6 pM 10 pM 11 pM 9 pM NT: Not tested

TABLE-US-00009 TABLE 8B Neutralization potency (IC.sub.50) for selected Mabs against Toxin B Trial 1 (IC.sub.50) Trial 2 (IC.sub.50) Trial3 (IC.sub.50) mAb 0.1 pM Toxin B 0.1 pM Toxin B 0.03 pM Toxin B H1M3067N No Neutralization HIM3134N No Neutralization HIH3317P No Neutralization NT NT H1H3335P 730 pM 380 pM 120 pM H1H3337P 1730 pM 980 pM 320 pM H1H3339P 480 pM 270 pM 90 pM H1H3343P 280 pM 200 pM 50 pM H1H3344P 580 pM 400 pM 40 pM H1H3347P 130 pM 90 pM 25 pM H1H3350P No Neutralization NT NT H1H3340P NT No Neutralization NT H1H3411P NT .sup. 8 pM.sup.# NT Control II 1800 pM 1500 pM 290 pM .sup.#Antibody only partially protect (40-50%) at highest concentration NT: Not Tested

Example 8

Generation of a Bi-specific Antibody

[0258] Various bi-specific antibodies are generated for use in practicing the methods of the invention. For example, C. difficile toxin A or toxin B-specific antibodies are generated in a bi-specific format (a "bi-specific") in which variable regions binding to distinct domains of toxin A and/or B are linked together to confer dual-domain and/or dual toxin specificity within a single binding molecule. Appropriately designed bi-specifics may enhance overall toxin neutralization efficacy through increasing both specificity and binding avidity. Variable regions with specificity for individual domains, as shown in FIG. 1, (e.g., segments of the N-terminal domain, which is the glucosylating enzymatic domain (designated as domain `A`), or the autocatalytic processing domain (designated as domain `C`), or the translocating domain (designated as domain D'), or the carboxy terminal receptor binding domain (designated as domain `B`) or that can bind to different regions within one domain, are paired on a structural scaffold that allows each region to bind simultaneously to the separate epitopes, or to different regions within one domain. In one example for a bi-specific, heavy chain variable regions (V.sub.H) from a binder with specificity for one domain are recombined with light chain variable regions (V.sub.L) from a series of binders with specificity for a second domain to identify non-cognate V.sub.L partners that can be paired with an original V.sub.H without disrupting the original specificity for that V.sub.H. In this way, a single V.sub.L segment (e.g., V.sub.L1) can be combined with two different V.sub.H domains (e.g., V.sub.H1 and V.sub.H2) to generate a bi-specific comprised of two binding "arms" (V.sub.H1-V.sub.L1 and V.sub.H2-V.sub.L1). Use of a single V.sub.L segment reduces the complexity of the system and thereby simplifies and increases efficiency in cloning, expression, and purification processes used to generate the bi-specific (See, for example, U.S. Ser. No. 13/022,759 and US2010/0331527).

[0259] Alternatively, antibodies that bind both toxin A and/or toxin B and a second target, such as, but not limited to, for example, a second different anti-toxin A or anti-toxin B antibody, or a toxoid, or a vaccine, may be prepared in a bi-specific format using techniques described herein, or other techniques known to those skilled in the art. Antibody variable regions binding to distinct toxin A regions may be linked together with variable regions that bind to relevant sites on, for example, toxin B, to confer dual-antigen specificity within a single binding molecule. Appropriately designed bi-specifics of this nature serve a dual function. For example, in the case of a bi-specific antibody that binds both toxin A and toxin B, one may be able to better neutralize both toxin A and toxin B concurrently, without the need for administration of a composition containing two separate antibodies. Variable regions with specificity for toxin A, are combined with a variable region with specificity for toxin B and are paired on a structural scaffold that allows each variable region to bind to the separate antigens.

[0260] The bi-specific binders are tested for binding and functional blocking of the target antigens, for example, toxin A and toxin B, in any of the assays described above for antibodies. For example, standard methods to measure soluble protein binding are used to assess the bispecific interaction, such as Biacore, ELISA, size exclusion chromatography, multi-angle laser light scattering, direct scanning calorimetry, and other methods. Binding of bi-specific antibodies to both toxin A and toxin B is determined through use of an ELISA binding assay in which synthetic peptides representing the different toxins are coated onto the wells of microtiter plates, and binding of a bi-specific is determined through use of a secondary detection antibody. Binding experiments can also be conducted using surface plasmon resonance experiments, in which real-time binding interaction of peptide to antibody is measured by flowing a peptide or bi-specific across a sensor surface on which bi-specific or peptide, respectively, is captured. Functional in vitro blocking of both toxin A and toxin B by a bi-specific is determined using any bioassay such as the neutralization assay described herein, or by in vivo protection studies in appropriate animal models, such as those described herein.

Example 9

Evaluation of In Vivo Efficacy of Anti-Toxin a and/or Anti-Toxin B Antibodies Against C. difficile Infection (CDI) in a Hamster Relapse Model (A) and in an Acute Hamster Model (B)

[0261] The efficacy of antibodies specific for toxin A and/or toxin B from C. difficile against infection with C. difficile was evaluated in hamsters in two different models, described below. Hamsters, in the presence of clindamycin, are sensitive to C. difficile infection and usually die within 2-4 days after infection.

[0262] (A) Relapse model:

[0263] Male Syrian Golden Hamsters were given an oral suspension containing a mixture of C. difficile spores and vegetative cells (10.sup.6 in total) on day -1. Twenty-four hours after infection (day 0), animals received a single subcutaneous injection of clindamycin (10 mg/kg). On days 1-3, hamsters were administered oral vancomycin (10 mg/kg) once per day to ameliorate the infection. The antibiotic vancomycin is used clinically to treat a C. difficile infection. After the last vancomycin dose, antibodies were administered subcutaneously q.d. for 4 days (days 3-6), or 1 day (day 3) according to their treatment and dosing group (see Tables 9A and 9B below).

[0264] Two different trials (See FIGS. 2 and 3) using the relapse model as a surrogate for clinical efficacy were conducted. Both trials compared two antibody combinations: [0265] 1. Antibodies designated H1H3330P and H1H3347P [0266] 2. Comparator anti-Toxin A (Control I; See U.S. Pat. No. 7,625,559 for clone 3D8 sequence) and comparator anti-Toxin B antibodies (Control II; See U.S. Pat. No. 7,625,559 for clone 124-152 sequence)

[0267] In Trial 1, four doses of antibody were administered at 5 mg/kg of each antibody (a total 10 mg/kg dose). In Trial 2, one dose of antibody was given at either 5 mg/kg or 2 mg/kg of each antibody (a total of either 10 mg/kg or 4 mg/kg).

TABLE-US-00010 TABLE 9A Trial 1 Design: Relapse model: Combination Treatments with H1H3330P + H1H3347P or comparator anti-Toxin A + comparator anti-Toxin B Dose (mg/kg .times. Group Treatment* # doses) n 1 Negative Control (irrelevant) antibody 10 .times. 4 14 2 Comparator anti-Toxin A and [5/5] .times. 4 23 comparator anti-Toxin B 3 (H1H3330P + H1H3347P [5/5] .times. 4 23 combination) 4 No antibody 15 *All animals received vancomycin as noted above.

TABLE-US-00011 TABLE 9B Trial 2 Design: Relapse model: Combination Treatments with H1H3330P + H1H3347P or comparator anti-Toxin A + comparator anti-Toxin B Dose (mg/kg .times. # Group Treatment* doses) n 1 Negative Control (irrelevant) antibody 10 .times. 1 14 2 Comparator anti-Toxin A and comparator [5/5] .times. 1 16 anti-Toxin B 3 (H1H3330P + H1H3347P combination) [5/5] .times. 1 16 4 Comparator anti-Toxin A and comparator [2/2] .times. 1 16 anti-Toxin B 5 (H1H3330P + H1H3347P combination) [2/2] .times. 1 16 *All animals received vancomycin as noted above.

[0268] Animals were observed twice a day for the duration of the experiment. General observations included signs for mortality and morbidity, the presence of diarrhea ("wet tail") and overall appearance (activity, general response to handling, touch, ruffled fur). Animals judged to be in a moribund state were euthanized. Criteria used to assign a moribund state were extended periods (5 days) of weight loss, progression to an emaciated state, prolonged lethargy (more than 3 days), signs of paralysis, skin erosions or trauma, hunched posture, and a distended abdomen. Observations continued, with deaths or euthanasia recorded for a period up to 18 days post-infection for the relapse model.

[0269] (B) Acute model:

[0270] Male Syrian Golden Hamsters were treated with clindamycin intraperitoneally at a dose of 10 mg/kg on day -1. On day 0 C. difficile spores were diluted with sterile PBS to give 100 spores/300 .mu.l and administered by oral gavage. Antibodies were administered every day for 4 days, beginning on day -3 and continuing to day 0, using a subcutaneous route. The dose of the antibodies is indicated in the figure legend. See also Table 9C below for the study outline.

TABLE-US-00012 TABLE 9C Trial 3: Acute model: Combination Treatments with H1H3330P + H1H3347P at various doses Dose (mg/kg .times. # # Group Treatment doses) Animals 1 Uninfected control -- 5 2 Infected control PBS .times. 4 10 3 Negative Control [100] .times. 4 14 (irrelevant) antibody 4 (H1H3330P + H1H3347P [50/50] .times. 4 14 5 combination) [16/16] .times. 4 14 6 [5.5/5.5] .times. 4 14 7 1.85/1.85 .times. 4 14

TABLE-US-00013 TABLE 9D Trial 4: Acute model: Combination Treatments with H1H3330P + H1H3347P at various doses Dose (mg/kg .times. # # Group Treatment doses) Animals 1 Uninfected control -- 5 2 Infected control PBS .times. 4 10 3 Negative Control [100] .times. 4 14 (irrelevant) antibody 4 (H1H3330P + H1H3347P [20/20] .times. 4 14 5 combination) [5/5] .times. 4 14 6 Comparator anti-Toxin A and [20/20] .times. 4 14 7 comparator anti-Toxin B [5/5] .times. 4 14

[0271] Animals were observed twice a day for the duration of the experiment. General observations included signs for mortality and morbidity, the presence of diarrhea ("wet tail") and overall appearance (activity, general response to handling, touch, ruffled fur). Animals judged to be in a moribund state were euthanized. Criteria used to assign a moribund state were extended periods (5 days) of weight loss, progression to an emaciated state, prolonged lethargy (more than 3 days), signs of paralysis, skin erosions or trauma, hunched posture, and a distended abdomen. Observations continued, with deaths or euthanasia recorded for a period up to10 days for the acute model.

[0272] Results

[0273] Statistical analysis of data from hamster models was done using the Log-Rank (Mantel Cox) test. For pairwise comparisons the Bonferroni correction was applied to the critical P value.

[0274] In the first trial, which was a multi-dose study using a hamster relapse model, (see FIG. 2), combination treatment with H1H3330P plus H1H3347P, or combination treatment with the comparator antibodies, showed an increase in overall survival vs isotype control, or vancomycin alone, (74-78%; combination of anti-toxin A and anti-toxin B antibodies vs 27-43%; control arms). Specifically, by day 19, 27% of the hamsters receiving PBS alone survived; 43% receiving the isotype control survived; 74% receiving the anti-toxin A plus anti-toxin B comparator antibody combination survived; and 78% receiving the H1H3330P (anti-A antibody) plus H1H3347P (anti-B antibody) combination survived.

[0275] In the second trial, which was a single-dose study using a hamster relapse model (see FIG. 3), combination treatment with either H1H3330P plus H1H3347P, or the comparator antibodies, increased survival as compared to the isotype control (negative control antibody), although there was no discrimination between the 2 mg/kg and 5 mg/kg doses.

[0276] In the first acute model study in hamsters (See FIG. 4), treatment with the H1H3330P plus H1H3347P combination showed significant protection of the hamsters from death in a titratable manner compared to the negative controls (p<0.0001 for all groups vs isotype controls). The doses titrated from 50 mg/kg to 1.85 mg/kg (of each antibody given as a combination), with the high dose providing protection for all of the animals until day 7 compared to day 1 for the lowest dose.

[0277] In further studies using the acute hamster model (see FIG. 5), combination treatment with either H1H3330P plus H1H3347P, or a combination of the comparator antibodies, significantly increased survival as compared to the isotype control (FIG. 5; Isotype control at 40 mg/kg vs Control I/Control II at 20 mg/kg each, p<0.0001; isotype control at 40 mg/kg vs Control I/Control II at 5 mg/kg each, p=0.0003; isotype control at 40 mg/kg vs H1H3330P/H1H3347P at 20 mg/kg each, p<0.0001; isotype control at 40 mg/kg vs H1H3330P/H1H3347P at 5 mg/kg each, p<0.0001). However, treatment with a combination of H1H3330P plus H1H3347P protected the hamsters from death in a manner superior to comparator antibody controls when tested at the low dose of 5 mg/kg of each antibody (p<0.0001), whereas there was no significant difference between the combination of H1H3330P plus H1H3347P vs the combination of the comparator antibodies at the higher dose of 20 mg/kg of each antibody (p=0.73). This result clearly demonstrates superiority at a low dose in the acute hamster model and suggests that doses of the H1H3330P plus H1H3347P antibodies could be effective in the clinic at lower concentrations compared to the comparator antibodies.

Example 10

The Effect of Anti-Toxin A and Anti-Toxin B Antibodies on Blocking the Cytotoxicity Induced by Culture Supernatant from Several Group BI Hypervirulent C. difficile Strains: Comparison with Comparator mAbs

[0278] Patients infected with clinically-hypervirulent BI/NAP1/027 strains have lower cure rates than patients infected with non-BI strains when treated with either fidaxomicin or vancomycin (Petrella, L A, et al. (2012), Clinical Infectious Diseases, 55(3): 351-357). Furthermore, BI/NAP1/027 strains are associated with a higher CDI recurrence rate and higher expected mortality rate when compared to prototypic strains (Loo, V G, et al. (2005), N Engl J Med, 353:23; Petrella, L A, et al. (2012), Clinical Infectious Diseases, 55(3): 351-357. These hypervirulent strains are characterized by an increase in toxin A and B production, the presence of binary toxin and increased resistance to fluoroquinolones. The increase in toxin A and B production is most likely caused by a loss-of-function mutation in tcdC, a putative negative regulator of tcdA and tcdB expression, resulting in sustained toxin production throughout the lifecycle.

[0279] The ability of a 1:1 molar ratio mix of H1H3330P and H1H3347P to neutralize toxin from four clinically-isolated C. difficile BI/NAP1/027 strains was tested in a cell-based neutralization assay. The VA5 and VA17 clinically isolated hypervirulent strains were obtained from Case Western Reserve University, Cleveland, Ohio. The 6336 and 6443 clinically isolated hypervirulent strains were obtained from the Dept. of Veterans Affairs, Edward Hines, Jr. Hospital, Hines, Ill. Neutralization assays utilized Vero cells, a monkey kidney epithelial cell line, due to their susceptibility to both C. difficile toxins. Cells were incubated with varying amounts of antibody cocktail and a fixed amount of culture supernatant isolated from several C. difficile strains for 48 hours. Cytotoxicity was determined by addition of WST-1 reagent, a redox indicator that yields a colorimetric change when reduced; metabolic activity during cell growth reduces WST-1, resulting in increased absorbance at 450 nm.

[0280] Culture supernatant from several clinically isolated BI strains exhibited a wide range of cytotoxic activity on Vero cells, with EC.sub.50 values for inducing cell cytotoxicity ranging from a 3700-fold dilution for the VA5 strain to 88200-fold dilution for the 6443 strain. A 1:1 molar ratio mix of H1H3330P and H1H3347P blocked cytotoxicity induced by culture supernatants from all group BI strains tested with a more than 34-fold better neutralization potency compared to a 1:1 molar ratio mix of comparator anti-Toxin A (control I; See U.S. Pat. No. 7,625,559 for clone 3D8 sequence) and comparator anti-Toxin B (control II; See U.S. Pat. No. 7,625,559 for clone 124-152 sequence). These data demonstrate that the H1H3330P/H1H3347P antibody pair was able to neutralize culture cytotoxicity with IC.sub.50 values in the picomolar range (31-45 pM) for tested hypervirulent BI strains, compared to the comparator mAb cocktail (IC.sub.50 range: 1200-1700 pM; see Table 10).

TABLE-US-00014 TABLE 10 EC.sub.50 Neutralization Assay (Fold [Supernatant] H1H3330P/ Comparator Fold Dilu- (Fold H1H3347P mAb 1/2 pair/ Strain tion) Dilution) IC.sub.50 (pM) IC.sub.50 (pM) control VA5 6900 4700 36 1400 39 VA17 3700 3000 31 1400 45 6336 15200 12100 45 1700 38 6443 88200 57500 35 1200 34

Example 11

Epitope Mapping of the Anti-Toxin A Antibody H1H3330P

[0281] The epitope of the C-term receptor-Binding Domain (CBD) of toxin A (SEQ ID:375) bound by H1H3330P was determined using mass spectrometry based proteomics. Briefly, the CBD of toxin A was subjected to trypsin digestion over a 12 hour period and the samples run on 10-14% gradient SDS-PAGE, followed by transfer to nitrocellulose membranes and Western blot analysis using either the H1H3330P antibody, or the control I antibody (See U.S. Pat. No. 7,625,559 for 3D8 antibody sequence) as primary antibodies. Further analysis of the peptide sequences that bound to the H1H3330P antibody was done using 2D electrophoresis followed by MALDI-TOF MS analysis. The procedures are described in greater detail below.

Limited trypsin digestion of Toxin A

[0282] Recombinant C-term receptor-Binding Domain (CBD) of toxin A (0.4 .mu.g/.mu.l in PBS) was added with sequence grade modified trypsin (Promega, Cat # V511C) at a 1:80 mass ratio and incubated at 37.degree. C. for 0-12 hr. The enzyme was inactivated by adding 2 volumes of 1.times. Laemmli sample buffer and heated at 95.degree. C. for 5 min. The samples were stored at -20.degree. C. until analysis.

Western Blot Analysis

[0283] The extent of the proteolysis was first examined by 10-14% gradient SDS-PAGE. The amount of each sample loaded was equivalent to 1 .mu.g initial CBD of Toxin A, and the separated proteins in the gel were visualized with SimplyBlue coomassie stain (Invitrogen, Cat# LC6065).

[0284] The samples digested for 0 hour and 12 hour were then selected for 10-14% gradient SDS-PAGE separation with an equivalent to 50 ng initial CBD of toxin A loaded for each sample. Separated proteins were transferred to a nitrocellose membrane, and probed with primary antibody H1H3330P or Control Antibody I at a concentration of 1 .mu.g/ml in TBST (Tris-buffer saline solution containing 0.05% Tween-20) overnight at 4.degree. C. followed by anti-human IgG HRP conjugated secondary antibody (Pierce, Cat #31412; at 1:15,000 dilution in TBST). Both H1H3330P & Control 1 antibodies had a human IgG1 constant domain. The membrane was incubated with chemiluminescence substrate (Perkin Elmer, Cat # NEL103E001EA) and the image was captured onto X-ray film.

2D Gel Electrophoresis

[0285] To determine which amino acids were represented in the protein band unique to Western blots performed using the H1H3330P antibody, a two-dimensional (2D) gel was performed using the 12 hour trypsin digest of the CBD of toxin A.

In-Gel Trypsin Digestion and Peptide Mapping by MALDI-TOF MS

[0286] The 2D-gel analysis revealed 4 protein spots, closely clustered in the pH (pl values .about.9) dimension, which composed the 50 kDa band that was visualized by Western blot using H1H3330P. The 4 protein spots with corresponding molecular weights of .about.50 kDa from the 2D-gel using the 12 hour trypsin digestion were excised, destained by 50% acetonitrile, reduced by 65 mM DTT, and alkylated by 135 mM iodoacetamide. After dehydration by acetonitrile, 20 ul of 2.5 ng/.mu.l sequence grade trypsin (Promega, Cat # V5111) was added to cover the gel bands and the in-gel digestion was carried out by overnight incubation at 37.degree. C.

[0287] The resulting peptides were desalted by Ziptip C18 (Millipore, Cat# ZTC18S096) and analyzed by Bruker UltrafleXtreme MALDI-TOF-TOF MS. The spectrum was processed by FlexAnalysis software and internally calibrated with autolytic trypsin fragment peaks. The calibrated peak lists were searched against an in-house database containing the sequence of the CBD Toxin A at a mass accuracy of 10 ppm.

Results

[0288] The results showed that blotting with H1H3330P revealed a major protein band at around 50 kDa at 12 hours post trypsin digestion, while no protein band having a molecular weight of around 50 kDa was observed when blotting was carried out with the control 1 antibody at 12 hours post trypsin digestion.

[0289] To determine which amino acids were represented in the 50 kDa protein band, unique to Western blots performed using H1H3330P, a two-dimensional (2D) gel was performed using the 12 hour trypsin digest, as described above. As noted, 2D-gel analysis revealed 4 protein spots, closely clustered in the pH (pl values .about.9) dimension, which composed the 50 kDa band visualized by Western blot using H1H3330P. Mass spectrometry analysis of these 4 protein spots identified 17 matching peptides covering amino acid residues 468-863 of the CBD of toxin A (SEQ ID: 375). This fragment of toxin A (amino acids spanning residue 468 to 863) has a predicted molecular weight of 45 kDa and a predicted isoelectric point of 9.01 corresponding well to the values obtained from 2-D gel analysis.

[0290] This example illustrates that the anti-toxin A antibody H1H3330P has an epitope unique from that of Control 1 (3D8 in U.S. Pat. No. 7,625,559) and binds the CBD of toxin A within amino acids 468 to 863. Particular amino acid sequences were identified within this region which interacted with the H1H3330P antibody and these amino acid sequences were residues 468-488 of SEQ ID NO: 375, residues 510-530 of SEQ ID NO: 375, residues 602-610 of SEQ ID NO: 375, residues 644-703 of SEQ ID NO: 724-794 of SEQ ID NO: 375, residues 799-814 of SEQ ID NO: 375 and residues 858-863 of SEQ ID NO: 375.

Sequence CWU 1

1

3981357DNAArtificial SequenceSynthetic 1gaggtgcaac tgttggagtc tgggggaggc ttggcacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt caactttggc acccatgaca tgagctgggt ccgccaggct 120ccagggaagg gactagagtg ggtctcaggt cttacaagta ctggtggtag cgcttcctcc 180gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa tattctgtat 240ttacaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaaacgttt 300aactggaact cctactttga ctactggggc cagggaaccc tggtcaccgt ctcctca 3572119PRTArtificial SequenceSynthetic 2Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Ala Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Gly Thr His 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Leu Thr Ser Thr Gly Gly Ser Ala Ser Ser Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ile Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Thr Phe Asn Trp Asn Ser Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 324DNAArtificial SequenceSynthetic 3ggattcaact ttggcaccca tgac 2448PRTArtificial SequenceSynthetic 4Gly Phe Asn Phe Gly Thr His Asp1 5 524DNAArtificial SequenceSynthetic 5cttacaagta ctggtggtag cgct 2468PRTArtificial SequenceSynthetic 6Leu Thr Ser Thr Gly Gly Ser Ala1 5 736DNAArtificial SequenceSynthetic 7gcgaaaacgt ttaactggaa ctcctacttt gactac 36812PRTArtificial SequenceSynthetic 8Ala Lys Thr Phe Asn Trp Asn Ser Tyr Phe Asp Tyr1 5 10 9324DNAArtificial SequenceSynthetic 9gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtattagc acctactact tagcctggta ccagcagaaa 120cctgaccagc ctcccaggct cctcatctat ggtacatcca gcagggccac tggcatccca 180gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggac 240cctgaagatt ttgcagtgta ttactgtcaa cagtatggta actcactgta cacttttggc 300caggggacca agctggagat caaa 32410108PRTArtificial SequenceSynthetic 10Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Thr Tyr 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Asp Gln Pro Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Thr Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Asp65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Asn Ser Leu 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 1121DNAArtificial SequenceSynthetic 11cagagtatta gcacctacta c 21127PRTArtificial SequenceSynthetic 12Gln Ser Ile Ser Thr Tyr Tyr1 5 139DNAArtificial SequenceSynthetic 13ggtacatcc 9143PRTArtificial SequenceSynthetic 14Gly Thr Ser1 1527DNAArtificial SequenceSynthetic 15caacagtatg gtaactcact gtacact 27169PRTArtificial SequenceSynthetic 16Gln Gln Tyr Gly Asn Ser Leu Tyr Thr1 5 17357DNAArtificial SequenceSynthetic 17caggtacaac tggtggaatc tgggggagac gtggttcagc ctgggaggtc cctgagactc 60tcctgtgcag catctggatt caccttcagt ggccacggca tgcactgggt ccgccaggct 120ccaggcaagg gtctagagtg ggtggcactt atattgtatg atggaagtag tgaagactat 180acagactccg tgaagggccg ctttaccgtc tccagagaca attccaagaa caccctgtat 240ttgcaaatga acagtctgag agccgaagac acggctgtct attactgtgc gcgagggagt 300attttaaatc gcccgtttga ttactggggc cagggaaccc tggtcaccgt ctcctca 35718119PRTArtificial SequenceSynthetic 18Gln Val Gln Leu Val Glu Ser Gly Gly Asp Val Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly His 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Leu Ile Leu Tyr Asp Gly Ser Ser Glu Asp Tyr Thr Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Ile Leu Asn Arg Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 1924DNAArtificial SequenceSynthetic 19ggattcacct tcagtggcca cggc 24208PRTArtificial SequenceSynthetic 20Gly Phe Thr Phe Ser Gly His Gly1 5 2124DNAArtificial SequenceSynthetic 21atattgtatg atggaagtag tgaa 24228PRTArtificial SequenceSynthetic 22Ile Leu Tyr Asp Gly Ser Ser Glu1 5 2336DNAArtificial SequenceSynthetic 23gcgcgaggga gtattttaaa tcgcccgttt gattac 362412PRTArtificial SequenceSynthetic 24Ala Arg Gly Ser Ile Leu Asn Arg Pro Phe Asp Tyr1 5 10 25339DNAArtificial SequenceSynthetic 25gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60atcaactgca agtccagcca gagtatttta ttcagttcca acaataagat ctacttagct 120tggttccagc agaaaccagg acagcctcct aaactactca tttactggac atctacccgg 180gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcact 240atcagtagtc tgcaggctga agatgtggca gtttactact gtcaacaata ttatactctt 300ccattcactt tcggccctgg gaccaaagtg gatatcaaa 33926113PRTArtificial SequenceSynthetic 26Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Ile Leu Phe Ser 20 25 30 Ser Asn Asn Lys Ile Tyr Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Thr Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Thr Leu Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile 100 105 110 Lys2736DNAArtificial SequenceSynthetic 27cagagtattt tattcagttc caacaataag atctac 362812PRTArtificial SequenceSynthetic 28Gln Ser Ile Leu Phe Ser Ser Asn Asn Lys Ile Tyr1 5 10 299DNAArtificial SequenceSynthetic 29tggacatct 9303PRTArtificial SequenceSynthetic 30Trp Thr Ser1 3127DNAArtificial SequenceSynthetic 31caacaatatt atactcttcc attcact 27329PRTArtificial SequenceSynthetic 32Gln Gln Tyr Tyr Thr Leu Pro Phe Thr1 5 33357DNAArtificial SequenceSynthetic 33cagatacagc tggtggagtc tgggggaggc gtggtccagc ctggaaggtc cctgagactc 60tcctgtgtag cctctgggtt caccctcagt ggacatggca tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg ggtggcattt atatcatttg atggaggtca tcaagactac 180acagacgccg cggagggccg attcaccatc tccagagaca attccaagaa cacgttgtat 240ctggaaatgg tcagcctgag acctgcagac acggctatat attattgtgt gaaagggagc 300gactcgtcgc gaggttttgg ctactggggc cggggaatcc tggtcaccgt ctcctca 35734119PRTArtificial SequenceSynthetic 34Gln Ile Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Leu Ser Gly His 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Ser Phe Asp Gly Gly His Gln Asp Tyr Thr Asp Ala Ala 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Glu Met Val Ser Leu Arg Pro Ala Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Val Lys Gly Ser Asp Ser Ser Arg Gly Phe Gly Tyr Trp Gly Arg Gly 100 105 110 Ile Leu Val Thr Val Ser Ser 115 3524DNAArtificial SequenceSynthetic 35gggttcaccc tcagtggaca tggc 24368PRTArtificial SequenceSynthetic 36Gly Phe Thr Leu Ser Gly His Gly1 5 3724DNAArtificial SequenceSynthetic 37atatcatttg atggaggtca tcaa 24388PRTArtificial SequenceSynthetic 38Ile Ser Phe Asp Gly Gly His Gln1 5 3936DNAArtificial SequenceSynthetic 39gtgaaaggga gcgactcgtc gcgaggtttt ggctac 364012PRTArtificial SequenceSynthetic 40Val Lys Gly Ser Asp Ser Ser Arg Gly Phe Gly Tyr1 5 10 41339DNAArtificial SequenceSynthetic 41gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60atcaactgca agtccagcca gagtgtttta ttcagttccg acaataagaa ctacttggct 120tggtaccagc tgaaaccagg tcagcctcct cacctactta tttactgggc atctattcgt 180gattccgggg tccctgaccg atttagtggc agcgggtctg ggacagattt cacgctcacc 240atcagcagcc tgcaggctga ggatgtggca gtttactcct gtcatcaata ttatagtgct 300ccactcacct tcggcggagg gaccaaggtg gagatcaaa 33942113PRTArtificial SequenceSynthetic 42Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Phe Ser 20 25 30 Ser Asp Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Leu Lys Pro Gly Gln 35 40 45 Pro Pro His Leu Leu Ile Tyr Trp Ala Ser Ile Arg Asp Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Ser Cys His Gln 85 90 95 Tyr Tyr Ser Ala Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 100 105 110 Lys4336DNAArtificial SequenceSynthetic 43cagagtgttt tattcagttc cgacaataag aactac 364412PRTArtificial SequenceSynthetic 44Gln Ser Val Leu Phe Ser Ser Asp Asn Lys Asn Tyr1 5 10 459DNAArtificial SequenceSynthetic 45tgggcatct 9463PRTArtificial SequenceSynthetic 46Trp Ala Ser1 4727DNAArtificial SequenceSynthetic 47catcaatatt atagtgctcc actcacc 27489PRTArtificial SequenceSynthetic 48His Gln Tyr Tyr Ser Ala Pro Leu Thr1 5 49372DNAArtificial SequenceSynthetic 49gaggtgcagc tggtggagtc tgggggggac ttggtacaac ctggagggtc cctgagactc 60tcctgtgcag cctctggagt caccttcagg acatatgaaa tgaattgggt ccgccaggct 120ccagggaagg gactggagtg gatttcacac attagtagca gtggtgatat tatatactat 180acaaagtctg tgaagggccg attcaccatc tccagagata acgccaagaa ctcactgttt 240ctgcaaatga caagtctgag agccgaggac acggctgtat attactgtgc gagagaaagg 300tacagtcaat acggttatta ttacttcgga atggatgtct ggggccaagg gaccacggtc 360accgtctcct ca 37250124PRTArtificial SequenceSynthetic 50Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Thr Phe Arg Thr Tyr 20 25 30 Glu Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Ser His Ile Ser Ser Ser Gly Asp Ile Ile Tyr Tyr Thr Lys Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Phe65 70 75 80 Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Arg Tyr Ser Gln Tyr Gly Tyr Tyr Tyr Phe Gly Met Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 5124DNAArtificial SequenceSynthetic 51ggagtcacct tcaggacata tgaa 24528PRTArtificial SequenceSynthetic 52Gly Val Thr Phe Arg Thr Tyr Glu1 5 5324DNAArtificial SequenceSynthetic 53attagtagca gtggtgatat tata 24548PRTArtificial SequenceSynthetic 54Ile Ser Ser Ser Gly Asp Ile Ile1 5 5551DNAArtificial SequenceSynthetic 55gcgagagaaa ggtacagtca atacggttat tattacttcg gaatggatgt c 515617PRTArtificial SequenceSynthetic 56Ala Arg Glu Arg Tyr Ser Gln Tyr Gly Tyr Tyr Tyr Phe Gly Met Asp1 5 10 15 Val57321DNAArtificial SequenceSynthetic 57gacatccaga tgacccagtc tccttccacc ctgtctgcat ctataggaga cagagtcacc 60atcacttgcc gggccagtca gaatactgat aagtggatgg cctggtatca gcagaaagca 120gggaaagccc ctaaactcct gatctataag gcgtctattt tagaaagtgg ggtcccttca 180aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag cctgcagcct 240gatgattttg caacttatta ctgccaagaa tataatactt attttcgggc gttcggccaa 300gggaccaagg tggaaaccag a 32158107PRTArtificial SequenceSynthetic 58Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Ile Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asn Thr Asp Lys Trp 20 25 30 Met Ala Trp Tyr Gln Gln Lys Ala Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ile Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Glu Tyr Asn Thr Tyr Phe Arg 85 90 95 Ala Phe Gly Gln Gly Thr Lys Val Glu Thr Arg 100 105 5918DNAArtificial SequenceSynthetic 59cagaatactg ataagtgg 18606PRTArtificial SequenceSynthetic 60Gln Asn Thr Asp Lys Trp1 5 619DNAArtificial SequenceSynthetic 61aaggcgtct 9623PRTArtificial SequenceSynthetic 62Lys Ala Ser1 6327DNAArtificial SequenceSynthetic 63caagaatata atacttattt tcgggcg 27649PRTArtificial SequenceSynthetic 64Gln Glu Tyr Asn Thr Tyr Phe Arg Ala1 5 65372DNAArtificial SequenceSynthetic 65gacgtgcagc tggtggagtc tgggggagac tttgtacaac ctggagggtc cctgagactc 60tcctgtgcag cctctggagt cgccttcaat gattatgaaa tgaattggat ccgccaggct 120ccagggaaga gactggagtg gatttcacac attgatagta gtggtactat tatatattac 180gcagactctg tgaagggccg attcaccatc tccagagaca gcgccaagaa ctcactgttt 240ctgcaaatgg acagtctgag agccgaggac acggctgttt attactgtgc gagagaaagg 300tacagtcact acggatatta ctacttcggt atggatgtct ggggccaagg gaccacggtc 360accgtctcct ca 37266124PRTArtificial SequenceSynthetic 66Asp Val Gln Leu Val Glu Ser Gly Gly Asp Phe Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Ala Phe Asn Asp Tyr 20 25 30 Glu Met Asn Trp Ile Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Ile 35 40 45 Ser His Ile Asp Ser Ser Gly Thr Ile Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Ser Ala Lys Asn Ser Leu Phe65 70 75 80 Leu Gln Met Asp Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Arg Tyr Ser His Tyr Gly Tyr Tyr Tyr Phe

Gly Met Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 6724DNAArtificial SequenceSynthetic 67ggagtcgcct tcaatgatta tgaa 24688PRTArtificial SequenceSynthetic 68Gly Val Ala Phe Asn Asp Tyr Glu1 5 6924DNAArtificial SequenceSynthetic 69attgatagta gtggtactat tata 24708PRTArtificial SequenceSynthetic 70Ile Asp Ser Ser Gly Thr Ile Ile1 5 7151DNAArtificial SequenceSynthetic 71gcgagagaaa ggtacagtca ctacggatat tactacttcg gtatggatgt c 517217PRTArtificial SequenceSynthetic 72Ala Arg Glu Arg Tyr Ser His Tyr Gly Tyr Tyr Tyr Phe Gly Met Asp1 5 10 15 Val73321DNAArtificial SequenceSynthetic 73gacatccaga tgacccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggccagtca gaatattgat aactggttgg cctggtatca gcagaaaaca 120ggtaaagccc ctaacctcct gatctataag gcgtctactt tggaaagtgg ggtcccttca 180aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcatcag cctgcagcct 240gatgattttg caacttatta ctgccaagaa tataatactt attctcggac gttcggccaa 300ggcaccaagg tggaaatcaa a 32174107PRTArtificial SequenceSynthetic 74Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asn Ile Asp Asn Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Thr Gly Lys Ala Pro Asn Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ile Ser Leu Gln Pro65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Glu Tyr Asn Thr Tyr Ser Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 7518DNAArtificial SequenceSynthetic 75cagaatattg ataactgg 18766PRTArtificial SequenceSynthetic 76Gln Asn Ile Asp Asn Trp1 5 779DNAArtificial SequenceSynthetic 77aaggcgtct 9783PRTArtificial SequenceSynthetic 78Lys Ala Ser1 7927DNAArtificial SequenceSynthetic 79caagaatata atacttattc tcggacg 27809PRTArtificial SequenceSynthetic 80Gln Glu Tyr Asn Thr Tyr Ser Arg Thr1 5 81372DNAArtificial SequenceSynthetic 81gagatacaat tgatagagtc tgggggagac atggtacaac ctggagggtc cctgagactc 60tcctgtgcag cctctggaat ctcccttaat agttatgaaa tgaattgggt ccgccagact 120ccagggatgg ggctggagtg gatttcacac ataagtagta gtggaacttc tatatattat 180gcaaactctg tgaagggccg attcaccata ttcagagaca gcgccaagaa ctcactgttg 240ctgcaaatga acagtctgag agccgaggac acggctattt attactgtgc aagagaaaga 300tacgatcact ccgggtatta ctacctcgga atggatgtct ggggcctagg gaccacggtc 360accgtctcgt ca 37282124PRTArtificial SequenceSynthetic 82Glu Ile Gln Leu Ile Glu Ser Gly Gly Asp Met Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Ser Leu Asn Ser Tyr 20 25 30 Glu Met Asn Trp Val Arg Gln Thr Pro Gly Met Gly Leu Glu Trp Ile 35 40 45 Ser His Ile Ser Ser Ser Gly Thr Ser Ile Tyr Tyr Ala Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Phe Arg Asp Ser Ala Lys Asn Ser Leu Leu65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Glu Arg Tyr Asp His Ser Gly Tyr Tyr Tyr Leu Gly Met Asp 100 105 110 Val Trp Gly Leu Gly Thr Thr Val Thr Val Ser Ser 115 120 8324DNAArtificial SequenceSynthetic 83ggaatctccc ttaatagtta tgaa 24848PRTArtificial SequenceSynthetic 84Gly Ile Ser Leu Asn Ser Tyr Glu1 5 8524DNAArtificial SequenceSynthetic 85ataagtagta gtggaacttc tata 24868PRTArtificial SequenceSynthetic 86Ile Ser Ser Ser Gly Thr Ser Ile1 5 8751DNAArtificial SequenceSynthetic 87gcaagagaaa gatacgatca ctccgggtat tactacctcg gaatggatgt c 518817PRTArtificial SequenceSynthetic 88Ala Arg Glu Arg Tyr Asp His Ser Gly Tyr Tyr Tyr Leu Gly Met Asp1 5 10 15 Val89321DNAArtificial SequenceSynthetic 89gacatccaga tgacccagtc tccttccacc ctgtctgcat ctttaggaga cagagtcacc 60atcacttgcc gggccagtca gaatattgat aactggatgg cctggtatca gcagaaagtt 120gggaaagccc ctaaactctt gatatatagg gcgtctactt tagaaactgg ggtcccttca 180aggttcggcg gcagtggatt tgggacagaa ttcactctca ccatcagcag cctgcagcct 240ggtgattttg cgacttacta ctgccaagaa tataatagtt attttcggac gttcggccaa 300gggaccaagg tggagatcaa a 32190107PRTArtificial SequenceSynthetic 90Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Leu Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asn Ile Asp Asn Trp 20 25 30 Met Ala Trp Tyr Gln Gln Lys Val Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Arg Ala Ser Thr Leu Glu Thr Gly Val Pro Ser Arg Phe Gly Gly 50 55 60 Ser Gly Phe Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Gly Asp Phe Ala Thr Tyr Tyr Cys Gln Glu Tyr Asn Ser Tyr Phe Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 9118DNAArtificial SequenceSynthetic 91cagaatattg ataactgg 18926PRTArtificial SequenceSynthetic 92Gln Asn Ile Asp Asn Trp1 5 939DNAArtificial SequenceSynthetic 93agggcgtct 9943PRTArtificial SequenceSynthetic 94Arg Ala Ser1 9527DNAArtificial SequenceSynthetic 95caagaatata atagttattt tcggacg 27969PRTArtificial SequenceSynthetic 96Gln Glu Tyr Asn Ser Tyr Phe Arg Thr1 5 97372DNAArtificial SequenceSynthetic 97gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt caccttcagt agttacgaca tgcactgggt ccgccaagtt 120ataggaaaag gtctggagtg ggtctcagct attggtactg ttggtgacac atactatgca 180ggctccgtga agggccgatt caccatctcc agagaaaatg ccaagaattc cttgtacctt 240caaatgaaca gcctgagagc cggggacacg gctgtgtatt actgtgcaag agatcggggg 300ggtgcgaata tttatagttt ctactacggt atggacgtct ggggccaagg gaccacggtc 360accgtctcct ca 37298124PRTArtificial SequenceSynthetic 98Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Asp Met His Trp Val Arg Gln Val Ile Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Gly Thr Val Gly Asp Thr Tyr Tyr Ala Gly Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu Tyr Leu65 70 75 80 Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Arg Gly Gly Ala Asn Ile Tyr Ser Phe Tyr Tyr Gly Met Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 9924DNAArtificial SequenceSynthetic 99ggattcacct tcagtagtta cgac 241008PRTArtificial SequenceSynthetic 100Gly Phe Thr Phe Ser Ser Tyr Asp1 5 10121DNAArtificial SequenceSynthetic 101attggtactg ttggtgacac a 211027PRTArtificial SequenceSynthetic 102Ile Gly Thr Val Gly Asp Thr1 5 10354DNAArtificial SequenceSynthetic 103gcaagagatc gggggggtgc gaatatttat agtttctact acggtatgga cgtc 5410418PRTArtificial SequenceSynthetic 104Ala Arg Asp Arg Gly Gly Ala Asn Ile Tyr Ser Phe Tyr Tyr Gly Met1 5 10 15 Asp Val105324DNAArtificial SequenceSynthetic 105gacatccaga tgacccagtc tccatcctcc ctgtctgtat ctgtaggaga cagagtcacc 60atcacttgcc gggcgagtca ggacattagc aattatttag cctggtatca gcagaaacca 120gggaaagttc ctaagctcct gatctatgct gcatccactt tgcaatcagg ggtcccatct 180cggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240gaagatgttg caacttattt ctgtcaaaag tataacagtg ccccattcac tttcggccct 300gggaccaaag tggatatcaa acga 324106108PRTArtificial SequenceSynthetic 106Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Val Ala Thr Tyr Phe Cys Gln Lys Tyr Asn Ser Ala Pro Phe 85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg 100 105 10718DNAArtificial SequenceSynthetic 107caggacatta gcaattat 181086PRTArtificial SequenceSynthetic 108Gln Asp Ile Ser Asn Tyr1 5 1099DNAArtificial SequenceSynthetic 109gctgcatcc 91103PRTArtificial SequenceSynthetic 110Ala Ala Ser1 11127DNAArtificial SequenceSynthetic 111caaaagtata acagtgcccc attcact 271129PRTArtificial SequenceSynthetic 112Gln Lys Tyr Asn Ser Ala Pro Phe Thr1 5 113393DNAArtificial SequenceSynthetic 113gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctttaac agttttgtca tgagctgggt ccgtcaggct 120ccagggaagg ggctggagtg ggtctcagct attagtggtt atggtggtag cacatactac 180gcagactcca tgaagggccg gttcaccgtc tccagagaca attccaagaa tacgctgtat 240ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagatcac 300aaggatttct atgcttcggg gagttatttt aaccgggact actactacgg tatggacgtc 360tggggccaag ggaccacggt caccgtctcc tca 393114131PRTArtificial SequenceSynthetic 114Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Ser Phe 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Met 50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp His Lys Asp Phe Tyr Ala Ser Gly Ser Tyr Phe Asn Arg 100 105 110 Asp Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr 115 120 125 Val Ser Ser 130 11524DNAArtificial SequenceSynthetic 115ggattcacct ttaacagttt tgtc 241168PRTArtificial SequenceSynthetic 116Gly Phe Thr Phe Asn Ser Phe Val1 5 11724DNAArtificial SequenceSynthetic 117attagtggtt atggtggtag caca 241188PRTArtificial SequenceSynthetic 118Ile Ser Gly Tyr Gly Gly Ser Thr1 5 11972DNAArtificial SequenceSynthetic 119gcgaaagatc acaaggattt ctatgcttcg gggagttatt ttaaccggga ctactactac 60ggtatggacg tc 7212024PRTArtificial SequenceSynthetic 120Ala Lys Asp His Lys Asp Phe Tyr Ala Ser Gly Ser Tyr Phe Asn Arg1 5 10 15 Asp Tyr Tyr Tyr Gly Met Asp Val 20 121324DNAArtificial SequenceSynthetic 121gacatccaga tgacccagtc tccttccacc ctgtctgcat ctgttggaga cagagtcacc 60atcacttgcc gggccagtca gagtattagt agctggttgg cctggtatca gcagaaacca 120gggaaagccc ctaaggtcct gatctataag gcgtctagtt tagaaagtgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag cctgcagcct 240gatgattttg caacttatta ctgccaacag tataatagtt attctcggac gttcggccaa 300gggaccaagg tggaaatcaa acga 324122108PRTArtificial SequenceSynthetic 122Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Ser Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 12318DNAArtificial SequenceSynthetic 123cagagtatta gtagctgg 181246PRTArtificial SequenceSynthetic 124Gln Ser Ile Ser Ser Trp1 5 1259DNAArtificial SequenceSynthetic 125aaggcgtct 91263PRTArtificial SequenceSynthetic 126Lys Ala Ser1 12727DNAArtificial SequenceSynthetic 127caacagtata atagttattc tcggacg 271289PRTArtificial SequenceSynthetic 128Gln Gln Tyr Asn Ser Tyr Ser Arg Thr1 5 129372DNAArtificial SequenceSynthetic 129gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgacactc 60tcctgcgcag cctctagatt caccttcagt aactacgaca tgcactgggt ccgccaagcc 120acaggaaaag gtctggagtg ggtctcagct attggtactg tcggtgacac atactatgca 180ggctctgtga agggccgatt caccatctcc agagacgatg ccaagaattc cctttatctc 240caaatgaaca gcctgagagc cggggacacg gctgtttatt actgtgcaag agatcggggg 300ggtgcgggga cttatagttt ctattacggt atggacgtct ggggccaagg gaccacggtc 360accgtctcct ca 372130124PRTArtificial SequenceSynthetic 130Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Thr Leu Ser Cys Ala Ala Ser Arg Phe Thr Phe Ser Asn Tyr 20 25 30 Asp Met His Trp Val Arg Gln Ala Thr Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Gly Thr Val Gly Asp Thr Tyr Tyr Ala Gly Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Ser Leu Tyr Leu65 70 75 80 Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Arg Gly Gly Ala Gly Thr Tyr Ser Phe Tyr Tyr Gly Met Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 13124DNAArtificial SequenceSynthetic 131agattcacct tcagtaacta cgac 241328PRTArtificial SequenceSynthetic 132Arg Phe Thr Phe Ser Asn Tyr Asp1 5 13321DNAArtificial SequenceSynthetic 133attggtactg tcggtgacac a 211347PRTArtificial SequenceSynthetic 134Ile Gly Thr Val Gly Asp Thr1 5 13554DNAArtificial SequenceSynthetic 135gcaagagatc gggggggtgc ggggacttat agtttctatt acggtatgga cgtc 5413618PRTArtificial SequenceSynthetic 136Ala Arg Asp Arg Gly Gly Ala Gly Thr Tyr Ser Phe Tyr Tyr Gly Met1 5 10 15

Asp Val137324DNAArtificial SequenceSynthetic 137gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggcgagtca ggacattagc aattatttag cctggtatca gcagaaacca 120gggaaagttc ctaaactcct gatctatgct gcttccactt tgcaatcagg ggtcccatct 180cggttcagtg gtagtggatc tgggacagat ttcactctca ccgtcagcag cctgcagcct 240gaagatgttg caacttatta ctgtcaaaag tataccagtg ccccattcac tttcggccct 300gggaccaaag tggatatcaa acga 324138108PRTArtificial SequenceSynthetic 138Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Val Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Thr Ser Ala Pro Phe 85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg 100 105 13918DNAArtificial SequenceSynthetic 139caggacatta gcaattat 181406PRTArtificial SequenceSynthetic 140Gln Asp Ile Ser Asn Tyr1 5 1419DNAArtificial SequenceSynthetic 141gctgcttcc 91423PRTArtificial SequenceSynthetic 142Ala Ala Ser1 14327DNAArtificial SequenceSynthetic 143caaaagtata ccagtgcccc attcact 271449PRTArtificial SequenceSynthetic 144Gln Lys Tyr Thr Ser Ala Pro Phe Thr1 5 145369DNAArtificial SequenceSynthetic 145caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctggggggtc cctgagactc 60tcctgtgcag cgtctggatt caccttcagt agctatgcca tgcactgggt ccgccaggct 120ccaggcaagg gactggagtg ggtggcaatt atatggtttg atggaagtaa tgaagattat 180gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa catggtatat 240ctgcaaataa acagcctgag agccgaggac acggctgtgt attactgtgc gagatctgcc 300aactggaact acgaaggggg acccctcttt gactactggg gccagggaac cctggtcacc 360gtctcctca 369146123PRTArtificial SequenceSynthetic 146Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ile Ile Trp Phe Asp Gly Ser Asn Glu Asp Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Met Val Tyr65 70 75 80 Leu Gln Ile Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ala Asn Trp Asn Tyr Glu Gly Gly Pro Leu Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 14724DNAArtificial SequenceSynthetic 147ggattcacct tcagtagcta tgcc 241488PRTArtificial SequenceSynthetic 148Gly Phe Thr Phe Ser Ser Tyr Ala1 5 14924DNAArtificial SequenceSynthetic 149atatggtttg atggaagtaa tgaa 241508PRTArtificial SequenceSynthetic 150Ile Trp Phe Asp Gly Ser Asn Glu1 5 15148DNAArtificial SequenceSynthetic 151gcgagatctg ccaactggaa ctacgaaggg ggacccctct ttgactac 4815216PRTArtificial SequenceSynthetic 152Ala Arg Ser Ala Asn Trp Asn Tyr Glu Gly Gly Pro Leu Phe Asp Tyr1 5 10 15 153324DNAArtificial SequenceSynthetic 153gacatccaga tgacccagtc tccatcctcc ctgtctgctt ctgtaggaga ccgagtcacc 60atcacttgcc gggcaagtca gaccattagc acctttttaa attggtatca gcagaagcca 120gggaaaggcc ctgaactcct gatctacact gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagtg gcagtggatc tgggacagat ttcgctctca ccatcagcag tctgcaacct 240gaagattttg cgacttacta ctgtcaacag aattacaatg accctcccac cttcggccaa 300gggacacgac tggagattaa acga 324154108PRTArtificial SequenceSynthetic 154Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Ser Thr Phe 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Gly Pro Glu Leu Leu Ile 35 40 45 Tyr Thr Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Ala Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Tyr Asn Asp Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg 100 105 15518DNAArtificial SequenceSynthetic 155cagaccatta gcaccttt 181566PRTArtificial SequenceSynthetic 156Gln Thr Ile Ser Thr Phe1 5 1579DNAArtificial SequenceSynthetic 157actgcatcc 91583PRTArtificial SequenceSynthetic 158Thr Ala Ser1 15927DNAArtificial SequenceSynthetic 159caacagaatt acaatgaccc tcccacc 271609PRTArtificial SequenceSynthetic 160Gln Gln Asn Tyr Asn Asp Pro Pro Thr1 5 161348DNAArtificial SequenceSynthetic 161caggtgcagc tggtggagtc tgggggaggc ttggtcaagc ctggagggtc cctgacactc 60tcctgtgtag cctctggatt caccttcact gactactaca ttagttggat ccgccaggct 120ccggggaagg gactggagtg gatttcatac attggtactg gtggtgctgc caaatactac 180gcagactctg ttaagggccg attcaccgtc tccagggaca acgccaagaa ctcactgtat 240ctactaatga acaacctgag agccgaggac acggccgtat attattgtgc gagagatctg 300gggatctttg acttatgggg ccagggaacc ctggtcaccg tctcctca 348162116PRTArtificial SequenceSynthetic 162Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15 Ser Leu Thr Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Tyr Ile Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Ser Tyr Ile Gly Thr Gly Gly Ala Ala Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80 Leu Leu Met Asn Asn Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Leu Gly Ile Phe Asp Leu Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 16324DNAArtificial SequenceSynthetic 163ggattcacct tcactgacta ctac 241648PRTArtificial SequenceSynthetic 164Gly Phe Thr Phe Thr Asp Tyr Tyr1 5 16524DNAArtificial SequenceSynthetic 165attggtactg gtggtgctgc caaa 241668PRTArtificial SequenceSynthetic 166Ile Gly Thr Gly Gly Ala Ala Lys1 5 16727DNAArtificial SequenceSynthetic 167gcgagagatc tggggatctt tgactta 271689PRTArtificial SequenceSynthetic 168Ala Arg Asp Leu Gly Ile Phe Asp Leu1 5 169327DNAArtificial SequenceSynthetic 169gaaattgtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc 60ctctcctgta gggccagtca gagtgttagt agtagtttag cctggtacca ccagaaacct 120ggccaggctc ccaggctcct catccatggt gtttccacca gggccactgg tatcccagcc 180aggttcagtg gcactgggtc tgggacagaa ttcactctca ccatcagcag cctgcagtct 240gaagattttg cagtttatta ctgtcaacag tatcataact ggcctccgta cacttttggc 300caggggacca agctggagat caaacga 327170109PRTArtificial SequenceSynthetic 170Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Leu Ala Trp Tyr His Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 His Gly Val Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Thr Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr His Asn Trp Pro Pro 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 17118DNAArtificial SequenceSynthetic 171cagagtgtta gtagtagt 181726PRTArtificial SequenceSynthetic 172Gln Ser Val Ser Ser Ser1 5 1739DNAArtificial SequenceSynthetic 173ggtgtttcc 91743PRTArtificial SequenceSynthetic 174Gly Val Ser1 17530DNAArtificial SequenceSynthetic 175caacagtatc ataactggcc tccgtacact 3017610PRTArtificial SequenceSynthetic 176Gln Gln Tyr His Asn Trp Pro Pro Tyr Thr1 5 10 177363DNAArtificial SequenceSynthetic 177caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cctctggatt caccttcagt agttttggca tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg ggtgtcaatg atatcaaccg atggaagtaa gaaaaattat 180gcagactccg tgaagggccg attcaccatc accagagaca attcaaagaa cacgctgtat 240ttggaaatga acagcctgag agctgaggac acggctgtgt attacggtgt gagagttggg 300tactatgatt cggggagtta ttataactat tggggccagg gaaccctggt caccgtctcc 360tca 363178121PRTArtificial SequenceSynthetic 178Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Met Ile Ser Thr Asp Gly Ser Lys Lys Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Thr Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Glu Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Gly 85 90 95 Val Arg Val Gly Tyr Tyr Asp Ser Gly Ser Tyr Tyr Asn Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 17924DNAArtificial SequenceSynthetic 179ggattcacct tcagtagttt tggc 241808PRTArtificial SequenceSynthetic 180Gly Phe Thr Phe Ser Ser Phe Gly1 5 18124DNAArtificial SequenceSynthetic 181atatcaaccg atggaagtaa gaaa 241828PRTArtificial SequenceSynthetic 182Ile Ser Thr Asp Gly Ser Lys Lys1 5 18342DNAArtificial SequenceSynthetic 183gtgagagttg ggtactatga ttcggggagt tattataact at 4218414PRTArtificial SequenceSynthetic 184Val Arg Val Gly Tyr Tyr Asp Ser Gly Ser Tyr Tyr Asn Tyr1 5 10 185324DNAArtificial SequenceSynthetic 185gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtaggtga cagagtcacc 60atcacttgtc gggcgagtca gggtattcgc agctggttag cctggtttca gcagagacca 120gggaaagccc ctaacctcct gatctatgct gcatccagtt tgcaaagtgg ggtctcatcc 180aggttcagcg gcagtggctc tgggacagaa ttcactctca gcatcagcag cctgcagcct 240gaagattttg caacttacta ttgtcaacag gcttacagtt ttccgctcac tttcggcgga 300gggaccaagg tggagatcaa acga 324186108PRTArtificial SequenceSynthetic 186Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Ser Trp 20 25 30 Leu Ala Trp Phe Gln Gln Arg Pro Gly Lys Ala Pro Asn Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Ser Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Ser Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Tyr Ser Phe Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 18718DNAArtificial SequenceSynthetic 187cagggtattc gcagctgg 181886PRTArtificial SequenceSynthetic 188Gln Gly Ile Arg Ser Trp1 5 1899DNAArtificial SequenceSynthetic 189gctgcatcc 91903PRTArtificial SequenceSynthetic 190Ala Ala Ser1 19127DNAArtificial SequenceSynthetic 191caacaggctt acagttttcc gctcact 271929PRTArtificial SequenceSynthetic 192Gln Gln Ala Tyr Ser Phe Pro Leu Thr1 5 193348DNAArtificial SequenceSynthetic 193gaggtgcagc tggtggagtc tgggggaggc ttggtgcggc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctttagg atctatgcca tgagctgggt ccgccaggct 120ccagggaagg ggctggagtg ggtctcaggt attagtggta gtggtgataa tacatactat 180acagactccg tgaagggccg gttcatcatc tccagagaca attccaagag cacgctgtat 240ctgcaaatga acagcctgag agccgaagat acggccgtat attactgtgc gagagggtgg 300gagttactga actactgggg ccagggaacc ctggtcaccg tctcctca 348194116PRTArtificial SequenceSynthetic 194Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Arg Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ile Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Gly Ser Gly Asp Asn Thr Tyr Tyr Thr Asp Ser Val 50 55 60 Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Ser Lys Ser Thr Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Trp Glu Leu Leu Asn Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 19524DNAArtificial SequenceSynthetic 195ggattcacct ttaggatcta tgcc 241968PRTArtificial SequenceSynthetic 196Gly Phe Thr Phe Arg Ile Tyr Ala1 5 19724DNAArtificial SequenceSynthetic 197attagtggta gtggtgataa taca 241988PRTArtificial SequenceSynthetic 198Ile Ser Gly Ser Gly Asp Asn Thr1 5 19927DNAArtificial SequenceSynthetic 199gcgagagggt gggagttact gaactac 272009PRTArtificial SequenceSynthetic 200Ala Arg Gly Trp Glu Leu Leu Asn Tyr1 5 201324DNAArtificial SequenceSynthetic 201gacatccaga tgacccagtc tccatcctca ctgtctgcat ttgtaggaga cagagtcacc 60atcacttgtc gggcgagtca ggacattagc aatcatttag cctggtttca gcagaaacca 120gggaaagtcc ctaagtccct gatctatgct gcgtccagtt tgcaaagtgg ggtcccatca 180aaattcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240gaagattttg caacttatta ctgccaacag tatggtcttt atcctcccac tttcggccct 300gggaccaaag tggatatcaa acga 324202108PRTArtificial SequenceSynthetic 202Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Phe Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn His 20 25 30 Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Val Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Lys Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Gly Leu Tyr Pro Pro 85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg 100 105 20318DNAArtificial SequenceSynthetic 203caggacatta gcaatcat

182046PRTArtificial SequenceSynthetic 204Gln Asp Ile Ser Asn His1 5 2059DNAArtificial SequenceSynthetic 205gctgcgtcc 92063PRTArtificial SequenceSynthetic 206Ala Ala Ser1 20727DNAArtificial SequenceSynthetic 207caacagtatg gtctttatcc tcccact 272089PRTArtificial SequenceSynthetic 208Gln Gln Tyr Gly Leu Tyr Pro Pro Thr1 5 209348DNAArtificial SequenceSynthetic 209gaggtgcagc tggtggagtc tgggggaggc ttggtacagc cgggggggtc cctgagactc 60tcctgtgcag cctctggatt cacttttagc atctatgcca tgagctgggt ccgccaggct 120ccagggaagg ggctggagtg ggtctcaggt attagtggta gtggtggtag aacatactac 180gcagactccg ttaagggccg gttcaccatc tctagagaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agtcgaggac acggccgttt attactgtgc gagagggtgg 300gagcttctta acttctgggg ccagggaacc ctggtcaccg tctcctca 348210116PRTArtificial SequenceSynthetic 210Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Gly Ser Gly Gly Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Trp Glu Leu Leu Asn Phe Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 21124DNAArtificial SequenceSynthetic 211ggattcactt ttagcatcta tgcc 242128PRTArtificial SequenceSynthetic 212Gly Phe Thr Phe Ser Ile Tyr Ala1 5 21324DNAArtificial SequenceSynthetic 213attagtggta gtggtggtag aaca 242148PRTArtificial SequenceSynthetic 214Ile Ser Gly Ser Gly Gly Arg Thr1 5 21527DNAArtificial SequenceSynthetic 215gcgagagggt gggagcttct taacttc 272169PRTArtificial SequenceSynthetic 216Ala Arg Gly Trp Glu Leu Leu Asn Phe1 5 217324DNAArtificial SequenceSynthetic 217gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgtc gggcgagtca gggcattagt aataatttag cctggtttca gcagaaacca 120gggaaagccc ctaagtccct gatctatgct gcatccagtt tgaaaagtgg ggtcccatca 180aagttcagcg gcagtggatc tgggacagat ttcactctca ccatcaacag cctgcagcct 240gaagattttg caacttatta ctgccaccag tataatagtt atcctcccac tttcggccct 300gggaccaaag tggatatcaa acga 324218108PRTArtificial SequenceSynthetic 218Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Asn 20 25 30 Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Lys Ser Gly Val Pro Ser Lys Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Tyr Asn Ser Tyr Pro Pro 85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg 100 105 21918DNAArtificial SequenceSynthetic 219cagggcatta gtaataat 182206PRTArtificial SequenceSynthetic 220Gln Gly Ile Ser Asn Asn1 5 2219DNAArtificial SequenceSynthetic 221gctgcatcc 92223PRTArtificial SequenceSynthetic 222Ala Ala Ser1 22327DNAArtificial SequenceSynthetic 223caccagtata atagttatcc tcccact 272249PRTArtificial SequenceSynthetic 224His Gln Tyr Asn Ser Tyr Pro Pro Thr1 5 225348DNAArtificial SequenceSynthetic 225gaggtgcagc tggtggagtc tgggggaggc ttggtgcagc ctggggggtc cctgagactc 60tcctgtgcag tctctggatt cacctttagc atctatgcca tgagctgggt ccgccaggct 120ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtgataa gacatactac 180gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac acggccgtat tttactgtgc gagagggtgg 300gagctcctaa actactgggg ccagggaacc ctggtcaccg tctcctca 348226116PRTArtificial SequenceSynthetic 226Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Asp Lys Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Phe Tyr Cys 85 90 95 Ala Arg Gly Trp Glu Leu Leu Asn Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 22724DNAArtificial SequenceSynthetic 227ggattcacct ttagcatcta tgcc 242288PRTArtificial SequenceSynthetic 228Gly Phe Thr Phe Ser Ile Tyr Ala1 5 22924DNAArtificial SequenceSynthetic 229attagtggta gtggtgataa gaca 242308PRTArtificial SequenceSynthetic 230Ile Ser Gly Ser Gly Asp Lys Thr1 5 23127DNAArtificial SequenceSynthetic 231gcgagagggt gggagctcct aaactac 272329PRTArtificial SequenceSynthetic 232Ala Arg Gly Trp Glu Leu Leu Asn Tyr1 5 233324DNAArtificial SequenceSynthetic 233gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgtc gggcgagtct ggacattagt aattttttag cctggtttca gcagaaacca 120gggacagccc ctaagtccct gatctattct gcatccagtt tgcggactgg ggtcccatca 180aagttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240gaagattttg caacttatta ctgccagcag tatagttctt accctcccac tttcggccct 300gggaccaaag tggatatcaa acga 324234108PRTArtificial SequenceSynthetic 234Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Leu Asp Ile Ser Asn Phe 20 25 30 Leu Ala Trp Phe Gln Gln Lys Pro Gly Thr Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ser Ala Ser Ser Leu Arg Thr Gly Val Pro Ser Lys Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr Pro Pro 85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg 100 105 23518DNAArtificial SequenceSynthetic 235ctggacatta gtaatttt 182366PRTArtificial SequenceSynthetic 236Leu Asp Ile Ser Asn Phe1 5 2379DNAArtificial SequenceSynthetic 237tctgcatcc 92383PRTArtificial SequenceSynthetic 238Ser Ala Ser1 23927DNAArtificial SequenceSynthetic 239cagcagtata gttcttaccc tcccact 272409PRTArtificial SequenceSynthetic 240Gln Gln Tyr Ser Ser Tyr Pro Pro Thr1 5 241348DNAArtificial SequenceSynthetic 241gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ccggggggtc cctgagactc 60tcctgtgtag cctctggatt caactttaga atctatgcca tgagctgggt ccgccaggct 120ccagggaagg ggccggagtg ggtctcaggt attagtggta gtggtgataa cacatactac 180gcagcctccg tgaagggccg gttcaccgtc tccagagaca attccaagaa cacgctgtat 240ctgcaaatga ccagcctgag agccgaggac acggccgtat tttactgtgc gagagggtgg 300gagctcctaa actattgggg ccagggaacc ctggtcaccg tctcctca 348242116PRTArtificial SequenceSynthetic 242Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Asn Phe Arg Ile Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Gly Ile Ser Gly Ser Gly Asp Asn Thr Tyr Tyr Ala Ala Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Phe Tyr Cys 85 90 95 Ala Arg Gly Trp Glu Leu Leu Asn Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 24324DNAArtificial SequenceSynthetic 243ggattcaact ttagaatcta tgcc 242448PRTArtificial SequenceSynthetic 244Gly Phe Asn Phe Arg Ile Tyr Ala1 5 24524DNAArtificial SequenceSynthetic 245attagtggta gtggtgataa caca 242468PRTArtificial SequenceSynthetic 246Ile Ser Gly Ser Gly Asp Asn Thr1 5 24727DNAArtificial SequenceSynthetic 247gcgagagggt gggagctcct aaactat 272489PRTArtificial SequenceSynthetic 248Ala Arg Gly Trp Glu Leu Leu Asn Tyr1 5 249324DNAArtificial SequenceSynthetic 249gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgtc gggcgagtct ggacattggc aattttttag cctggtttca gcagaaacca 120gggacagccc ctaagtccct gatctattct gcatccagtc tgcagactgg ggtcccatca 180aagttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240gaagattttg caacttatta ctgccaacag tataattctt atcctcccac tttcggccct 300gggaccaaag tggatatcaa acga 324250108PRTArtificial SequenceSynthetic 250Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Leu Asp Ile Gly Asn Phe 20 25 30 Leu Ala Trp Phe Gln Gln Lys Pro Gly Thr Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ser Ala Ser Ser Leu Gln Thr Gly Val Pro Ser Lys Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Pro 85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg 100 105 25118DNAArtificial SequenceSynthetic 251ctggacattg gcaatttt 182526PRTArtificial SequenceSynthetic 252Leu Asp Ile Gly Asn Phe1 5 2539DNAArtificial SequenceSynthetic 253tctgcatcc 92543PRTArtificial SequenceSynthetic 254Ser Ala Ser1 25527DNAArtificial SequenceSynthetic 255caacagtata attcttatcc tcccact 272569PRTArtificial SequenceSynthetic 256Gln Gln Tyr Asn Ser Tyr Pro Pro Thr1 5 257348DNAArtificial SequenceSynthetic 257gaggtgcagc tggtggagtc tgggggaggc ttggtacagc cgggggggtc cctgagactc 60tcctgtgcag cctctggatt tacctttaaa atctatgcca tgagttgggt ccgccagggc 120ccagggaagg ggctggagtg ggtctcggct attagtggaa atggtgacaa aacatactat 180acagactccg tgcagggccg gttcaccatc tccagagaca attccaagaa cacactcttt 240ctccaaatga acagcctgag agccgaggac acggccatat attactgtgc gcgagggtgg 300gaactgctaa attactgggg ccagggaacc ctggtcaccg tctcctca 348258116PRTArtificial SequenceSynthetic 258Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Lys Ile Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Gly Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Asn Gly Asp Lys Thr Tyr Tyr Thr Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Gly Trp Glu Leu Leu Asn Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 25924DNAArtificial SequenceSynthetic 259ggatttacct ttaaaatcta tgcc 242608PRTArtificial SequenceSynthetic 260Gly Phe Thr Phe Lys Ile Tyr Ala1 5 26124DNAArtificial SequenceSynthetic 261attagtggaa atggtgacaa aaca 242628PRTArtificial SequenceSynthetic 262Ile Ser Gly Asn Gly Asp Lys Thr1 5 26327DNAArtificial SequenceSynthetic 263gcgcgagggt gggaactgct aaattac 272649PRTArtificial SequenceSynthetic 264Ala Arg Gly Trp Glu Leu Leu Asn Tyr1 5 265324DNAArtificial SequenceSynthetic 265gacatccaga tgacccagtc tccatcctca ctgtctgcat ctataggaga cagagtcacc 60atcacttgtc gggcgagtca ggacattagc aattctttag cctggtttca gcagaaacca 120gggaaagccc ctaagtccct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagat ttcactctca ccatctccag cctgcagcct 240gaagattttg caacttatta ctgccaacaa tatattcctt tccctcccac tttcggccct 300gggaccaaag tggatatcaa acga 324266108PRTArtificial SequenceSynthetic 266Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Ile Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Ser 20 25 30 Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ile Pro Phe Pro Pro 85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg 100 105 26718DNAArtificial SequenceSynthetic 267caggacatta gcaattct 182686PRTArtificial SequenceSynthetic 268Gln Asp Ile Ser Asn Ser1 5 2699DNAArtificial SequenceSynthetic 269gctgcatcc 92703PRTArtificial SequenceSynthetic 270Ala Ala Ser1 27127DNAArtificial SequenceSynthetic 271caacaatata ttcctttccc tcccact 272729PRTArtificial SequenceSynthetic 272Gln Gln Tyr Ile Pro Phe Pro Pro Thr1 5 273348DNAArtificial SequenceSynthetic 273gaggtgcagc tggtggagtc tgggggaggc ctggtacagc cgggggggtc cctgagactc 60tcctgtgtag cttctggatt cacctttacc agctatgcca tgagctgggt ccgccaggct 120ccagggaggg ggctgcagtg ggtctcagct attggtggta gtggtgatag tatatattac 180gcagactccg tcaagggccg gttcaccatc tccagagaca actccaagaa tacgctgtat 240ctgcaaatgg acagcctgag agccgaggac acggccgtat attactgtgc aagaggatgg 300gagttactca attactgggg ccagggaacc ctggtcaccg tctcctca 348274116PRTArtificial SequenceSynthetic 274Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Thr Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Gln Trp Val 35 40 45 Ser Ala Ile Gly Gly Ser Gly Asp Ser Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln Met Asp Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Trp Glu Leu Leu Asn Tyr Trp Gly Gln Gly Thr Leu Val 100

105 110 Thr Val Ser Ser 115 27524DNAArtificial SequenceSynthetic 275ggattcacct ttaccagcta tgcc 242768PRTArtificial SequenceSynthetic 276Gly Phe Thr Phe Thr Ser Tyr Ala1 5 27724DNAArtificial SequenceSynthetic 277attggtggta gtggtgatag tata 242788PRTArtificial SequenceSynthetic 278Ile Gly Gly Ser Gly Asp Ser Ile1 5 27927DNAArtificial SequenceSynthetic 279gcaagaggat gggagttact caattac 272809PRTArtificial SequenceSynthetic 280Ala Arg Gly Trp Glu Leu Leu Asn Tyr1 5 281324DNAArtificial SequenceSynthetic 281gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgtc gggcgagtca ggacattggc aattttttag cctggtttca gcagaaacca 120gggaaagccc ctaagtccct gatctatgct gcatccagtt tgaaaagtgg ggtcccatca 180aagatcagcg gcagtggatc tgggacagat ttcactctca ccatcaacag cctgcagcct 240gaagattttg caacttatta ctgccaacag tataatattt accctcccac tttcggccct 300gggaccaaag tggatatcaa acga 324282108PRTArtificial SequenceSynthetic 282Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Asn Phe 20 25 30 Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Lys Ser Gly Val Pro Ser Lys Ile Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ile Tyr Pro Pro 85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg 100 105 28318DNAArtificial SequenceSynthetic 283caggacattg gcaatttt 182846PRTArtificial SequenceSynthetic 284Gln Asp Ile Gly Asn Phe1 5 2859DNAArtificial SequenceSynthetic 285gctgcatcc 92863PRTArtificial SequenceSynthetic 286Ala Ala Ser1 28727DNAArtificial SequenceSynthetic 287caacagtata atatttaccc tcccact 272889PRTArtificial SequenceSynthetic 288Gln Gln Tyr Asn Ile Tyr Pro Pro Thr1 5 289360DNAArtificial SequenceSynthetic 289caggtacagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac cctctcactc 60acctgtgcca tctccgggga cagtgtctct agcaacagtg ctgcttggaa ctggatcagg 120cagtccccat cgagaggcct tgagtggctg ggaaggacat actacaggtc caagtggtat 180catgattatg ctttttctgt gaaaagtcga atacttatca atccagacac atccaagaac 240ctgttctccc tgcaagtgaa ctctgtgact cccgaggaca cggctgtgta ttactgtgca 300agagataggc gatcctactt tgactactgg ggccagggaa ccctggtcac cgtctcctca 360290120PRTArtificial SequenceSynthetic 290Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30 Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45 Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr His Asp Tyr Ala 50 55 60 Phe Ser Val Lys Ser Arg Ile Leu Ile Asn Pro Asp Thr Ser Lys Asn65 70 75 80 Leu Phe Ser Leu Gln Val Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Ala Arg Asp Arg Arg Ser Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 29130DNAArtificial SequenceSynthetic 291ggggacagtg tctctagcaa cagtgctgct 3029210PRTArtificial SequenceSynthetic 292Gly Asp Ser Val Ser Ser Asn Ser Ala Ala1 5 10 29327DNAArtificial SequenceSynthetic 293acatactaca ggtccaagtg gtatcat 272949PRTArtificial SequenceSynthetic 294Thr Tyr Tyr Arg Ser Lys Trp Tyr His1 5 29530DNAArtificial SequenceSynthetic 295gcaagagata ggcgatccta ctttgactac 3029610PRTArtificial SequenceSynthetic 296Ala Arg Asp Arg Arg Ser Tyr Phe Asp Tyr1 5 10 297324DNAArtificial SequenceSynthetic 297gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca gggccagtcg gagtgttagc agttccttag cctggtacca acagaaacct 120ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg catcccagcc 180aggttcagtg gcggtgggtc tgggacagac ttcactctca ccatcagcag cctagagcct 240gaagattttg cagtttatta ctgtcagcag cgtaacaact ggcctcccac ttttggccag 300gggaccaagc tggagatcaa acga 324298108PRTArtificial SequenceSynthetic 298Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Arg Ser Val Ser Ser Ser 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Gly Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Asn Asn Trp Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 29918DNAArtificial SequenceSynthetic 299cggagtgtta gcagttcc 183006PRTArtificial SequenceSynthetic 300Arg Ser Val Ser Ser Ser1 5 3019DNAArtificial SequenceSynthetic 301gatgcatcc 93023PRTArtificial SequenceSynthetic 302Asp Ala Ser1 30327DNAArtificial SequenceSynthetic 303cagcagcgta acaactggcc tcccact 273049PRTArtificial SequenceSynthetic 304Gln Gln Arg Asn Asn Trp Pro Pro Thr1 5 305369DNAArtificial SequenceSynthetic 305gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggcaggtc cctgagactc 60tcctgtacag cctctggatt cgtttttgaa gattatgcca tgcactgggt ccggcaagct 120ccagggaagg gcctggagtg ggtctcaggt attagttgga atagtggtag gataggctat 180acggactctg tgaagggccg attcaccgtc tccagagaca acgccaagaa ctccttgtat 240ctgcaaatga acagtctgac aactgaggac acggccttgt attattgtgc aaaagataaa 300tcgccctcta agtggaactt actaggtatg gacgtctggg gccaagggac cacggtcacc 360gtctcctca 369306123PRTArtificial SequenceSynthetic 306Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Val Phe Glu Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Arg Ile Gly Tyr Thr Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Thr Thr Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Lys Ser Pro Ser Lys Trp Asn Leu Leu Gly Met Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 30724DNAArtificial SequenceSynthetic 307ggattcgttt ttgaagatta tgcc 243088PRTArtificial SequenceSynthetic 308Gly Phe Val Phe Glu Asp Tyr Ala1 5 30924DNAArtificial SequenceSynthetic 309attagttgga atagtggtag gata 243108PRTArtificial SequenceSynthetic 310Ile Ser Trp Asn Ser Gly Arg Ile1 5 31148DNAArtificial SequenceSynthetic 311gcaaaagata aatcgccctc taagtggaac ttactaggta tggacgtc 4831216PRTArtificial SequenceSynthetic 312Ala Lys Asp Lys Ser Pro Ser Lys Trp Asn Leu Leu Gly Met Asp Val1 5 10 15 313324DNAArtificial SequenceSynthetic 313gccatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca ggacattaga aatgatttag gctggtttca gcagaaacca 120gggagagccc ctaacctcct aatctttggt gcatccagtt tacaaagtgg ggtcccatca 180aggttcagcg gcagtggatc tggcacagat ttcactctca ccatcagcgg cctgcagcct 240gaagattttt caacttatta ctgtctacaa gattacactt acccattcac tttcggccct 300gggaccaaag tggatatcaa acga 324314108PRTArtificial SequenceSynthetic 314Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Asp 20 25 30 Leu Gly Trp Phe Gln Gln Lys Pro Gly Arg Ala Pro Asn Leu Leu Ile 35 40 45 Phe Gly Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Gln Pro65 70 75 80 Glu Asp Phe Ser Thr Tyr Tyr Cys Leu Gln Asp Tyr Thr Tyr Pro Phe 85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg 100 105 31518DNAArtificial SequenceSynthetic 315caggacatta gaaatgat 183166PRTArtificial SequenceSynthetic 316Gln Asp Ile Arg Asn Asp1 5 3179DNAArtificial SequenceSynthetic 317ggtgcatcc 93183PRTArtificial SequenceSynthetic 318Gly Ala Ser1 31927DNAArtificial SequenceSynthetic 319ctacaagatt acacttaccc attcact 273209PRTArtificial SequenceSynthetic 320Leu Gln Asp Tyr Thr Tyr Pro Phe Thr1 5 321363DNAArtificial SequenceSynthetic 321caggtgcagc tggtgcagtc tggggctgag gtacagaagc ccggggcgtc agtgaaagtc 60tcctgcaagg cttctggata caccttcacc gactactata ttcattgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggatgg atcaacccta aaactggtgg cacaaactat 180gcaccgaagt ttcagggcag ggtcaccatg accagggact cgtccatcat cacagcctac 240atggacttga ccagactgac ctctgacgac acggccgtgt tttactgtgc gagacgggga 300tataatagta ggtggtccgt ttttgactac tggggccagg gaaccctggt caccgtctcc 360tca 363322121PRTArtificial SequenceSynthetic 322Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Gln Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Lys Thr Gly Gly Thr Asn Tyr Ala Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Ser Ser Ile Ile Thr Ala Tyr65 70 75 80 Met Asp Leu Thr Arg Leu Thr Ser Asp Asp Thr Ala Val Phe Tyr Cys 85 90 95 Ala Arg Arg Gly Tyr Asn Ser Arg Trp Ser Val Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 32324DNAArtificial SequenceSynthetic 323ggatacacct tcaccgacta ctat 243248PRTArtificial SequenceSynthetic 324Gly Tyr Thr Phe Thr Asp Tyr Tyr1 5 32524DNAArtificial SequenceSynthetic 325atcaacccta aaactggtgg caca 243268PRTArtificial SequenceSynthetic 326Ile Asn Pro Lys Thr Gly Gly Thr1 5 32742DNAArtificial SequenceSynthetic 327gcgagacggg gatataatag taggtggtcc gtttttgact ac 4232814PRTArtificial SequenceSynthetic 328Ala Arg Arg Gly Tyr Asn Ser Arg Trp Ser Val Phe Asp Tyr1 5 10 329327DNAArtificial SequenceSynthetic 329gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgta gggccagtca gagtgtttac agcaactact tagcctggta ccagcagaaa 120cgtggcctgg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240cctgaagatt ttgcagtgta ttactgtcag cagcatggtg gctcaccggt cactttcggc 300ggagggacca aggtggagat caaacga 327330109PRTArtificial SequenceSynthetic 330Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Tyr Ser Asn 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Arg Gly Leu Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Gly Gly Ser Pro 85 90 95 Val Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 33121DNAArtificial SequenceSynthetic 331cagagtgttt acagcaacta c 213327PRTArtificial SequenceSynthetic 332Gln Ser Val Tyr Ser Asn Tyr1 5 3339DNAArtificial SequenceSynthetic 333ggtgcatcc 93343PRTArtificial SequenceSynthetic 334Gly Ala Ser1 33527DNAArtificial SequenceSynthetic 335cagcagcatg gtggctcacc ggtcact 273369PRTArtificial SequenceSynthetic 336Gln Gln His Gly Gly Ser Pro Val Thr1 5 337366DNAArtificial SequenceSynthetic 337caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgaag cctctggatt caccttcagt agctatggca tgcactgggt ccgccaggct 120ccaggcaacg ggctggagtg gatcgcagtt atatcatctg atggaaataa taaatattat 180atagaatccg tgaagggccg attcaccatg tccagagaca attccaagaa cacgctgtat 240ctgcaattga acagcctgag aactgaggac acggctgtgt attactgtgc gacttacaac 300tggaacgacg acggggacgg ggtttttgac tactggggcc agggaaccct ggtcaccgtc 360tcctca 366338122PRTArtificial SequenceSynthetic 338Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Asn Gly Leu Glu Trp Ile 35 40 45 Ala Val Ile Ser Ser Asp Gly Asn Asn Lys Tyr Tyr Ile Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Met Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln Leu Asn Ser Leu Arg Thr Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Tyr Asn Trp Asn Asp Asp Gly Asp Gly Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 33924DNAArtificial SequenceSynthetic 339ggattcacct tcagtagcta tggc 243408PRTArtificial SequenceSynthetic 340Gly Phe Thr Phe Ser Ser Tyr Gly1 5 34124DNAArtificial SequenceSynthetic 341atatcatctg atggaaataa taaa 243428PRTArtificial SequenceSynthetic 342Ile Ser Ser Asp Gly Asn Asn Lys1 5 34345DNAArtificial SequenceSynthetic 343gcgacttaca actggaacga cgacggggac ggggtttttg actac 4534415PRTArtificial SequenceSynthetic 344Ala Thr Tyr Asn Trp Asn Asp Asp Gly Asp Gly Val Phe Asp Tyr1 5 10 15 345324DNAArtificial SequenceSynthetic 345gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtaggaga cagagtcacc 60atcacttgtc gggcgagtca gggtattagc aactggttag cctggtatca gcagaaacca 120gggaaagccc ctaagctcct gatctatggt acatccagtt tgcaaagtgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240gaagattttg caacttacta ttgtcaacag gttaagagtt tcccgtacac ttttggccag 300gggaccaagc tggagatcaa acga 324346108PRTArtificial

SequenceSynthetic 346Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Thr Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Lys Ser Phe Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 34718DNAArtificial SequenceSynthetic 347cagggtatta gcaactgg 183486PRTArtificial SequenceSynthetic 348Gln Gly Ile Ser Asn Trp1 5 3499DNAArtificial SequenceSynthetic 349ggtacatcc 93503PRTArtificial SequenceSynthetic 350Gly Thr Ser1 35127DNAArtificial SequenceSynthetic 351caacaggtta agagtttccc gtacact 273529PRTArtificial SequenceSynthetic 352Gln Gln Val Lys Ser Phe Pro Tyr Thr1 5 353354DNAArtificial SequenceSynthetic 353gaggtgcagc tggtggagtc ggggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctttagc agatatggca tgaactgggt ccgccaggct 120ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacataccac 180gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacactgtat 240ctgcaaatga atagcctgag agccgcggac acggccatat atttctgtgc gtcttacaat 300tggaacgacg gggtggacgt ctggggccaa gggaccacgg tcaccgtctc ctca 354354117PRTArtificial SequenceSynthetic 354Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr His Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Ala Asp Thr Ala Ile Tyr Phe Cys 85 90 95 Ala Ser Tyr Asn Trp Asn Asp Gly Val Asp Val Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser 115 35524DNAArtificial SequenceSynthetic 355ggattcacct ttagcagata tggc 243568PRTArtificial SequenceSynthetic 356Gly Phe Thr Phe Ser Arg Tyr Gly1 5 35724DNAArtificial SequenceSynthetic 357attagtggta gtggtggtag caca 243588PRTArtificial SequenceSynthetic 358Ile Ser Gly Ser Gly Gly Ser Thr1 5 35933DNAArtificial SequenceSynthetic 359gcgtcttaca attggaacga cggggtggac gtc 3336011PRTArtificial SequenceSynthetic 360Ala Ser Tyr Asn Trp Asn Asp Gly Val Asp Val1 5 10 361324DNAArtificial SequenceSynthetic 361gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctataggaga cagggtcacc 60atcacttgtc gggcgagtca gggtattagc aactggttag cctggtatca gcagaaacca 120gggaaagccc ctaagctcct gatctatggt gcatccagtt tgcaaagtgg agtctcatca 180aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcatcag ccttcagcct 240gaagattttg caacttacta ttgtcaacag gctaacagtt tcccgtacac ttttggccag 300gggaccaagc tggagatcaa acga 324362108PRTArtificial SequenceSynthetic 362Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Ile Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Ser Leu Gln Ser Gly Val Ser Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ile Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 36318DNAArtificial SequenceSynthetic 363cagggtatta gcaactgg 183646PRTArtificial SequenceSynthetic 364Gln Gly Ile Ser Asn Trp1 5 3659DNAArtificial SequenceSynthetic 365ggtgcatcc 93663PRTArtificial SequenceSynthetic 366Gly Ala Ser1 36727DNAArtificial SequenceSynthetic 367caacaggcta acagtttccc gtacact 273689PRTArtificial SequenceSynthetic 368Gln Gln Ala Asn Ser Phe Pro Tyr Thr1 5 369214PRTArtificial SequenceSynthetic 369Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln His Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 370452PRTArtificial SequenceSynthetic 370Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Asn Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Leu Ile Trp Tyr Asp Gly Ser Asn Glu Asp Tyr Thr Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Gly Met Val Arg Gly Val Ile Asp Val Phe Asp Ile Trp 100 105 110 Gly Gln Gly Thr Val Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly Lys 450 371215PRTArtificial SequenceSynthetic 371Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Thr 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 372449PRTArtificial SequenceSynthetic 372Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Ser Gly Glu1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Phe Tyr Pro Gly Asp Ser Ser Thr Arg Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Val Asn Thr Ala Tyr65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Arg Arg Asn Trp Gly Asn Ala Phe Asp Ile Trp Gly Gln Gly 100 105 110 Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys 373213PRTArtificial SequenceSynthetic 373Asn Ile Val Met Thr Gln Ser Pro Lys Ser Met Ser Met Ser Val Gly1 5 10 15 Glu Arg Val Thr Phe Asn Cys Arg Ala Ser Glu Asn Val Gly Thr Tyr 20 25 30 Val Phe Trp Tyr Gln Gln Lys Pro Glu Gln Ser Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Gly Val Gln Ala65 70 75 80 Glu Asp Leu Ala Asp Tyr His Cys Gly Gln Ser Tyr Arg His Leu Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro 100 105 110 Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly 115 120 125 Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn 130 135 140 Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn145 150 155 160 Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser 165 170 175 Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr 180 185 190 Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 374449PRTArtificial SequenceSynthetic 374Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala1 5 10 15 Ser Val Arg Leu Ser Cys Lys Ala Gly Gly Tyr Thr Phe

Thr Ser Tyr 20 25 30 Trp Leu His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Tyr Asp Tyr Ser Glu Thr Phe 50 55 60 Arg Thr Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asp Thr Ala Tyr65 70 75 80 Met Gln Leu Thr Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Ser Asn Tyr Asp Ile Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr 115 120 125 Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu 130 135 140 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp145 150 155 160 Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser 180 185 190 Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser 195 200 205 Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys 210 215 220 Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro225 230 235 240 Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser 245 250 255 Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp 260 265 270 Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr 275 280 285 Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val 290 295 300 Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu305 310 315 320 Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg 325 330 335 Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val 340 345 350 Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr 355 360 365 Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr 370 375 380 Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu385 390 395 400 Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys 405 410 415 Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu 420 425 430 Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly 435 440 445 Lys 375863PRTArtificial SequenceSynthetic 375Phe Asn Leu Val Thr Gly Trp Gln Thr Ile Asn Gly Lys Lys Tyr Tyr1 5 10 15 Phe Asp Ile Asn Thr Gly Ala Ala Leu Ile Ser Tyr Lys Ile Ile Asn 20 25 30 Gly Lys His Phe Tyr Phe Asn Asn Asp Gly Val Met Gln Leu Gly Val 35 40 45 Phe Lys Gly Pro Asp Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Gln 50 55 60 Asn Asn Asn Ile Glu Gly Gln Ala Ile Val Tyr Gln Ser Lys Phe Leu65 70 75 80 Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Ala Val 85 90 95 Thr Gly Trp Arg Ile Ile Asn Asn Glu Lys Tyr Tyr Phe Asn Pro Asn 100 105 110 Asn Ala Ile Ala Ala Val Gly Leu Gln Val Ile Asp Asn Asn Lys Tyr 115 120 125 Tyr Phe Asn Pro Asp Thr Ala Ile Ile Ser Lys Gly Trp Gln Thr Val 130 135 140 Asn Gly Ser Arg Tyr Tyr Phe Asp Thr Asp Thr Ala Ile Ala Phe Asn145 150 155 160 Gly Tyr Lys Thr Ile Asp Gly Lys His Phe Tyr Phe Asp Ser Asp Cys 165 170 175 Val Val Lys Ile Gly Val Phe Ser Thr Ser Asn Gly Phe Glu Tyr Phe 180 185 190 Ala Pro Ala Asn Thr Tyr Asn Asn Asn Ile Glu Gly Gln Ala Ile Val 195 200 205 Tyr Gln Ser Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Asp 210 215 220 Asn Asn Ser Lys Ala Val Thr Gly Trp Gln Thr Ile Asp Ser Lys Lys225 230 235 240 Tyr Tyr Phe Asn Thr Asn Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr 245 250 255 Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ala Glu Ala Ala 260 265 270 Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn 275 280 285 Thr Ala Ile Ala Ser Thr Gly Tyr Thr Ile Ile Asn Gly Lys His Phe 290 295 300 Tyr Phe Asn Thr Asp Gly Ile Met Gln Ile Gly Val Phe Lys Gly Pro305 310 315 320 Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile 325 330 335 Glu Gly Gln Ala Ile Leu Tyr Gln Asn Glu Phe Leu Thr Leu Asn Gly 340 345 350 Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Ala Val Thr Gly Trp Arg 355 360 365 Ile Ile Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Ala Ile Ala 370 375 380 Ala Ile His Leu Cys Thr Ile Asn Asn Asp Lys Tyr Tyr Phe Ser Tyr385 390 395 400 Asp Gly Ile Leu Gln Asn Gly Tyr Ile Thr Ile Glu Arg Asn Asn Phe 405 410 415 Tyr Phe Asp Ala Asn Asn Glu Ser Lys Met Val Thr Gly Val Phe Lys 420 425 430 Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr His Asn Asn 435 440 445 Asn Ile Glu Gly Gln Ala Ile Val Tyr Gln Asn Lys Phe Leu Thr Leu 450 455 460 Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Ala Val Thr Gly465 470 475 480 Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn Thr Ala 485 490 495 Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe 500 505 510 Asn Leu Asn Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly 515 520 525 Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe Ile Ala Ser Thr Gly Tyr 530 535 540 Thr Ser Ile Asn Gly Lys His Phe Tyr Phe Asn Thr Asp Gly Ile Met545 550 555 560 Gln Ile Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro 565 570 575 Ala Asn Thr His Asn Asn Asn Ile Glu Gly Gln Ala Ile Leu Tyr Gln 580 585 590 Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp 595 600 605 Ser Lys Ala Val Thr Gly Leu Arg Thr Ile Asp Gly Lys Lys Tyr Tyr 610 615 620 Phe Asn Thr Asn Thr Ala Val Ala Val Thr Gly Trp Gln Thr Ile Asn625 630 635 640 Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser Ile Ala Ser Thr Gly 645 650 655 Tyr Thr Ile Ile Ser Gly Lys His Phe Tyr Phe Asn Thr Asp Gly Ile 660 665 670 Met Gln Ile Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr Phe Ala 675 680 685 Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Arg Tyr 690 695 700 Gln Asn Arg Phe Leu Tyr Leu His Asp Asn Ile Tyr Tyr Phe Gly Asn705 710 715 720 Asn Ser Lys Ala Ala Thr Gly Trp Val Thr Ile Asp Gly Asn Arg Tyr 725 730 735 Tyr Phe Glu Pro Asn Thr Ala Met Gly Ala Asn Gly Tyr Lys Thr Ile 740 745 750 Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gln Ile Gly Val 755 760 765 Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp 770 775 780 Ala Asn Asn Ile Glu Gly Gln Ala Ile Arg Tyr Gln Asn Arg Phe Leu785 790 795 800 His Leu Leu Gly Lys Ile Tyr Tyr Phe Gly Asn Asn Ser Lys Ala Val 805 810 815 Thr Gly Trp Gln Thr Ile Asn Gly Lys Val Tyr Tyr Phe Met Pro Asp 820 825 830 Thr Ala Met Ala Ala Ala Gly Gly Leu Phe Glu Ile Asp Gly Val Ile 835 840 845 Tyr Phe Phe Gly Val Asp Gly Val Lys Ala Pro Gly Ile Tyr Gly 850 855 860 376516PRTArtificial SequenceSynthetic 376Asn Leu Ile Thr Gly Phe Val Thr Val Gly Asp Asp Lys Tyr Tyr Phe1 5 10 15 Asn Pro Ile Asn Gly Gly Ala Ala Ser Ile Gly Glu Thr Ile Ile Asp 20 25 30 Asp Lys Asn Tyr Tyr Phe Asn Gln Ser Gly Val Leu Gln Thr Gly Val 35 40 45 Phe Ser Thr Glu Asp Gly Phe Lys Tyr Phe Ala Pro Ala Asn Thr Leu 50 55 60 Asp Glu Asn Leu Glu Gly Glu Ala Ile Asp Phe Thr Gly Lys Leu Ile65 70 75 80 Ile Asp Glu Asn Ile Tyr Tyr Phe Asp Asp Asn Tyr Arg Gly Ala Val 85 90 95 Glu Trp Lys Glu Leu Asp Gly Glu Met His Tyr Phe Ser Pro Glu Thr 100 105 110 Gly Lys Ala Phe Lys Gly Leu Asn Gln Ile Gly Asp Tyr Lys Tyr Tyr 115 120 125 Phe Asn Ser Asp Gly Val Met Gln Lys Gly Phe Val Ser Ile Asn Asp 130 135 140 Asn Lys His Tyr Phe Asp Asp Ser Gly Val Met Lys Val Gly Tyr Thr145 150 155 160 Glu Ile Asp Gly Lys His Phe Tyr Phe Ala Glu Asn Gly Glu Met Gln 165 170 175 Ile Gly Val Phe Asn Thr Glu Asp Gly Phe Lys Tyr Phe Ala His His 180 185 190 Asn Glu Asp Leu Gly Asn Glu Glu Gly Glu Glu Ile Ser Tyr Ser Gly 195 200 205 Ile Leu Asn Phe Asn Asn Lys Ile Tyr Tyr Phe Asp Asp Ser Phe Thr 210 215 220 Ala Val Val Gly Trp Lys Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe225 230 235 240 Asp Glu Asp Thr Ala Glu Ala Tyr Ile Gly Leu Ser Leu Ile Asn Asp 245 250 255 Gly Gln Tyr Tyr Phe Asn Asp Asp Gly Ile Met Gln Val Gly Phe Val 260 265 270 Thr Ile Asn Asp Lys Val Phe Tyr Phe Ser Asp Ser Gly Ile Ile Glu 275 280 285 Ser Gly Val Gln Asn Ile Asp Asp Asn Tyr Phe Tyr Ile Asp Asp Asn 290 295 300 Gly Ile Val Gln Ile Gly Val Phe Asp Thr Ser Asp Gly Tyr Lys Tyr305 310 315 320 Phe Ala Pro Ala Asn Thr Val Asn Asp Asn Ile Tyr Gly Gln Ala Val 325 330 335 Glu Tyr Ser Gly Leu Val Arg Val Gly Glu Asp Val Tyr Tyr Phe Gly 340 345 350 Glu Thr Tyr Thr Ile Glu Thr Gly Trp Ile Tyr Asp Met Glu Asn Glu 355 360 365 Ser Asp Lys Tyr Tyr Phe Asn Pro Glu Thr Lys Lys Ala Cys Lys Gly 370 375 380 Ile Asn Leu Ile Asp Asp Ile Lys Tyr Tyr Phe Asp Glu Lys Gly Ile385 390 395 400 Met Arg Thr Gly Leu Ile Ser Phe Glu Asn Asn Asn Tyr Tyr Phe Asn 405 410 415 Glu Asn Gly Glu Met Gln Phe Gly Tyr Ile Asn Ile Glu Asp Lys Met 420 425 430 Phe Tyr Phe Gly Glu Asp Gly Val Met Gln Ile Gly Val Phe Asn Thr 435 440 445 Pro Asp Gly Phe Lys Tyr Phe Ala His Gln Asn Thr Leu Asp Glu Asn 450 455 460 Phe Glu Gly Glu Ser Ile Asn Tyr Thr Gly Trp Leu Asp Leu Asp Glu465 470 475 480 Lys Arg Tyr Tyr Phe Thr Asp Glu Tyr Ile Ala Ala Thr Gly Ser Val 485 490 495 Ile Ile Asp Gly Glu Glu Tyr Tyr Phe Asp Pro Asp Thr Ala Gln Leu 500 505 510 Val Ile Ser Glu 515 3778133DNAClostridium difficile 377atgtctttaa tatctaaaga agagttaata aaactcgcat atagcattag accaagagaa 60aatgagtata aaactatact aactaattta gacgaatata ataagttaac tacaaacaat 120aatgaaaata aatatttaca attaaaaaaa ctaaatgaat caattgatgt ttttatgaat 180aaatataaaa cttcaagcag aaatagagca ctctctaatc taaaaaaaga tatattaaaa 240gaagtaattc ttattaaaaa ttccaataca agccctgtag aaaaaaattt acattttgta 300tggataggtg gagaagtcag tgatattgct cttgaataca taaaacaatg ggctgatatt 360aatgcagaat ataatattaa actgtggtat gatagtgaag cattcttagt aaatacacta 420aaaaaggcta tagttgaatc ttctaccact gaagcattac agctactaga ggaagagatt 480caaaatcctc aatttgataa tatgaaattt tacaaaaaaa ggatggaatt tatatatgat 540agacaaaaaa ggtttataaa ttattataaa tctcaaatca ataaacctac agtacctaca 600atagatgata ttataaagtc tcatctagta tctgaatata atagagatga aactgtatta 660gaatcatata gaacaaattc tttgagaaaa ataaatagta atcatgggat agatatcagg 720gctaatagtt tgtttacaga acaagagtta ttaaatattt atagtcagga gttgttaaat 780cgtggaaatt tagctgcagc atctgacata gtaagattat tagccctaaa aaattttggc 840ggagtatatt tagatgttga tatgcttcca ggtattcact ctgatttatt taaaacaata 900tctagaccta gctctattgg actagaccgt tgggaaatga taaaattaga ggctattatg 960aagtataaaa aatatataaa taattataca tcagaaaact ttgataaact tgatcaacaa 1020ttaaaagata attttaaact cattatagaa agtaaaagtg aaaaatctga gatattttct 1080aaattagaaa atttaaatgt atctgatctt gaaattaaaa tagctttcgc tttaggcagt 1140gttataaatc aagccttgat atcaaaacaa ggttcatatc ttactaacct agtaatagaa 1200caagtaaaaa atagatatca atttttaaac caacacctta acccagccat agagtctgat 1260aataacttca cagatactac taaaattttt catgattcat tatttaattc agctaccgca 1320gaaaactcta tgtttttaac aaaaatagca ccatacttac aagtaggttt tatgccagaa 1380gctcgctcca caataagttt aagtggtcca ggagcttatg cgtcagctta ctatgatttc 1440ataaatttac aagaaaatac tatagaaaaa actttaaaag catcagattt aatagaattt 1500aaattcccag aaaataatct atctcaattg acagaacaag aaataaatag tctatggagc 1560tttgatcaag caagtgcaaa atatcaattt gagaaatatg taagagatta tactggtgga 1620tctctttctg aagacaatgg ggtagacttt aataaaaata ctgccctcga caaaaactat 1680ttattaaata ataaaattcc atcaaacaat gtagaagaag ctggaagtaa aaattatgtt 1740cattatatca tacagttaca aggagatgat ataagttatg aagcaacatg caatttattt 1800tctaaaaatc ctaaaaatag tattattata caacgaaata tgaatgaaag tgcaaaaagc 1860tactttttaa gtgatgatgg agaatctatt ttagaattaa ataaatatag gatacctgaa 1920agattaaaaa ataaggaaaa agtaaaagta acctttattg gacatggtaa agatgaattc 1980aacacaagcg aatttgctag attaagtgta gattcacttt ccaatgagat aagttcattt 2040ttagatacca taaaattaga tatatcacct aaaaatgtag aagtaaactt acttggatgt 2100aatatgttta gttatgattt taatgttgaa gaaacttatc ctgggaagtt gctattaagt 2160attatggaca aaattacttc cactttacct gatgtaaata aaaattctat tactatagga 2220gcaaatcaat atgaagtaag aattaatagt gagggaagaa aagaacttct ggctcactca 2280ggtaaatgga taaataaaga agaagctatt atgagcgatt tatctagtaa agaatacatt 2340ttttttgatt ctatagataa taagctaaaa gcaaagtcca agaatattcc aggattagca 2400tcaatatcag aagatataaa aacattatta cttgatgcaa gtgttagtcc tgatacaaaa 2460tttattttaa ataatcttaa gcttaatatt gaatcttcta ttggtgatta catttattat 2520gaaaaattag agcctgttaa aaatataatt cacaattcta tagatgattt aatagatgag 2580ttcaatctac ttgaaaatgt atctgatgaa ttatatgaat taaaaaaatt aaataatcta 2640gatgagaagt atttaatatc ttttgaagat atctcaaaaa ataattcaac ttactctgta 2700agatttatta acaaaagtaa tggtgagtca gtttatgtag aaacagaaaa agaaattttt 2760tcaaaatata gcgaacatat tacaaaagaa ataagtacta taaagaatag tataattaca 2820gatgttaatg gtaatttatt ggataatata cagttagatc atacttctca agttaataca 2880ttaaacgcag cattctttat tcaatcatta atagattata gtagcaataa agatgtactg 2940aatgatttaa gtacctcagt taaggttcaa ctttatgctc aactatttag tacaggttta 3000aatactatat atgactctat ccaattagta aatttaatat caaatgcagt aaatgatact 3060ataaatgtac tacctacaat aacagagggg atacctattg tatctactat attagacgga 3120ataaacttag gtgcagcaat taaggaatta ctagacgaac atgacccatt actaaaaaaa 3180gaattagaag ctaaggtggg tgttttagca ataaatatgt cattatctat

agctgcaact 3240gtagcttcaa ttgttggaat aggtgctgaa gttactattt tcttattacc tatagctggt 3300atatctgcag gaataccttc attagttaat aatgaattaa tattgcatga taaggcaact 3360tcagtggtaa actattttaa tcatttgtct gaatctaaaa aatatggccc tcttaaaaca 3420gaagatgata aaattttagt tcctattgat gatttagtaa tatcagaaat agattttaat 3480aataattcga taaaactagg aacatgtaat atattagcaa tggagggggg atcaggacac 3540acagtgactg gtaatataga tcactttttc tcatctccat ctataagttc tcatattcct 3600tcattatcaa tttattctgc aataggtata gaaacagaaa atctagattt ttcaaaaaaa 3660ataatgatgt tacctaatgc tccttcaaga gtgttttggt gggaaactgg agcagttcca 3720ggtttaagat cattggaaaa tgacggaact agattacttg attcaataag agatttatac 3780ccaggtaaat tttactggag attctatgct tttttcgatt atgcaataac tacattaaaa 3840ccagtttatg aagacactaa tattaaaatt aaactagata aagatactag aaacttcata 3900atgccaacta taactactaa cgaaattaga aacaaattat cttattcatt tgatggagca 3960ggaggaactt actctttatt attatcttca tatccaatat caacgaatat aaatttatct 4020aaagatgatt tatggatatt taatattgat aatgaagtaa gagaaatatc tatagaaaat 4080ggtactatta aaaaaggaaa gttaataaaa gatgttttaa gtaaaattga tataaataaa 4140aataaactta ttataggcaa tcaaacaata gatttttcag gcgatataga taataaagat 4200agatatatat tcttgacttg tgagttagat gataaaatta gtttaataat agaaataaat 4260cttgttgcaa aatcttatag tttgttattg tctggggata aaaattattt gatatccaat 4320ttatctaata ttattgagaa aatcaatact ttaggcctag atagtaaaaa tatagcgtac 4380aattacactg atgaatctaa taataaatat tttggagcta tatctaaaac aagtcaaaaa 4440agcataatac attataaaaa agacagtaaa aatatattag aattttataa tgacagtaca 4500ttagaattta acagtaaaga ttttattgct gaagatataa atgtatttat gaaagatgat 4560attaatacta taacaggaaa atactatgtt gataataata ctgataaaag tatagatttc 4620tctatttctt tagttagtaa aaatcaagta aaagtaaatg gattatattt aaatgaatcc 4680gtatactcat cttaccttga ttttgtgaaa aattcagatg gacaccataa tacttctaat 4740tttatgaatt tatttttgga caatataagt ttctggaaat tgtttgggtt tgaaaatata 4800aattttgtaa tcgataaata ctttaccctt gttggtaaaa ctaatcttgg atatgtagaa 4860tttatttgtg acaataataa aaatatagat atatattttg gtgaatggaa aacatcgtca 4920tctaaaagca ctatatttag cggaaatggt agaaatgttg tagtagagcc tatatataat 4980cctgatacgg gtgaagatat atctacttca ctagattttt cctatgaacc tctctatgga 5040atagatagat atatcaataa agtattgata gcacctgatt tatatacaag tttaataaat 5100attaatacca attattattc aaatgagtac taccctgaga ttatagttct taacccaaat 5160acattccaca aaaaagtaaa tataaattta gatagttctt cttttgagta taaatggtct 5220acagaaggaa gtgactttat tttagttaga tacttagaag aaagtaataa aaaaatatta 5280caaaaaataa gaatcaaagg tatcttatct aatactcaat catttaataa aatgagtata 5340gattttaaag atattaaaaa actatcatta ggatatataa tgagtaattt taaatcattt 5400aattctgaaa atgaattaga tagagatcat ttaggattta aaataataga taataaaact 5460tattactatg atgaagatag taaattagtt aaaggattaa tcaatataaa taattcatta 5520ttctattttg atcctataga atttaactta gtaactggat ggcaaactat caatggtaaa 5580aaatattatt ttgatataaa tactggagca gctttaatta gttataaaat tattaatggt 5640aaacactttt attttaataa tgatggtgtg atgcagttgg gagtatttaa aggacctgat 5700ggatttgaat attttgcacc tgccaatact caaaataata acatagaagg tcaggctata 5760gtttatcaaa gtaaattctt aactttgaat ggcaaaaaat attattttga taatgactca 5820aaagcagtca ctggatggag aattattaac aatgagaaat attactttaa tcctaataat 5880gctattgctg cagtcggatt gcaagtaatt gacaataata agtattattt caatcctgac 5940actgctatca tctcaaaagg ttggcagact gttaatggta gtagatacta ctttgatact 6000gataccgcta ttgcctttaa tggttataaa actattgatg gtaaacactt ttattttgat 6060agtgattgtg tagtgaaaat aggtgtgttt agtacctcta atggatttga atattttgca 6120cctgctaata cttataataa taacatagaa ggtcaggcta tagtttatca aagtaaattc 6180ttaactttga atggtaaaaa atattacttt gataataact caaaagcagt taccggatgg 6240caaactattg atagtaaaaa atattacttt aatactaaca ctgctgaagc agctactgga 6300tggcaaacta ttgatggtaa aaaatattac tttaatacta acactgctga agcagctact 6360ggatggcaaa ctattgatgg taaaaaatat tactttaata ctaacactgc tatagcttca 6420actggttata caattattaa tggtaaacat ttttatttta atactgatgg tattatgcag 6480ataggagtgt ttaaaggacc taatggattt gaatattttg cacctgctaa tacggatgct 6540aacaacatag aaggtcaagc tatactttac caaaatgaat tcttaacttt gaatggtaaa 6600aaatattact ttggtagtga ctcaaaagca gttactggat ggagaattat taacaataag 6660aaatattact ttaatcctaa taatgctatt gctgcaattc atctatgcac tataaataat 6720gacaagtatt actttagtta tgatggaatt cttcaaaatg gatatattac tattgaaaga 6780aataatttct attttgatgc taataatgaa tctaaaatgg taacaggagt atttaaagga 6840cctaatggat ttgagtattt tgcacctgct aatactcaca ataataacat agaaggtcag 6900gctatagttt accagaacaa attcttaact ttgaatggca aaaaatatta ttttgataat 6960gactcaaaag cagttactgg atggcaaacc attgatggta aaaaatatta ctttaatctt 7020aacactgctg aagcagctac tggatggcaa actattgatg gtaaaaaata ttactttaat 7080cttaacactg ctgaagcagc tactggatgg caaactattg atggtaaaaa atattacttt 7140aatactaaca ctttcatagc ctcaactggt tatacaagta ttaatggtaa acatttttat 7200tttaatactg atggtattat gcagatagga gtgtttaaag gacctaatgg atttgaatac 7260tttgcacctg ctaatactca taataataac atagaaggtc aagctatact ttaccaaaat 7320aaattcttaa ctttgaatgg taaaaaatat tactttggta gtgactcaaa agcagttacc 7380ggattgcgaa ctattgatgg taaaaaatat tactttaata ctaacactgc tgttgcagtt 7440actggatggc aaactattaa tggtaaaaaa tactacttta atactaacac ttctatagct 7500tcaactggtt atacaattat tagtggtaaa catttttatt ttaatactga tggtattatg 7560cagataggag tgtttaaagg acctgatgga tttgaatact ttgcacctgc taatacagat 7620gctaacaata tagaaggtca agctatacgt tatcaaaata gattcctata tttacatgac 7680aatatatatt attttggtaa taattcaaaa gcagctactg gttgggtaac tattgatggt 7740aatagatatt acttcgagcc taatacagct atgggtgcga atggttataa aactattgat 7800aataaaaatt tttactttag aaatggttta cctcagatag gagtgtttaa agggtctaat 7860ggatttgaat actttgcacc tgctaatacg gatgctaaca atatagaagg tcaagctata 7920cgttatcaaa atagattcct acatttactt ggaaaaatat attactttgg taataattca 7980aaagcagtta ctggatggca aactattaat ggtaaagtat attactttat gcctgatact 8040gctatggctg cagctggtgg acttttcgag attgatggtg ttatatattt ctttggtgtt 8100gatggagtaa aagcccctgg gatatatggc taa 81333782710PRTClostridium difficile 378Met Ser Leu Ile Ser Lys Glu Glu Leu Ile Lys Leu Ala Tyr Ser Ile1 5 10 15 Arg Pro Arg Glu Asn Glu Tyr Lys Thr Ile Leu Thr Asn Leu Asp Glu 20 25 30 Tyr Asn Lys Leu Thr Thr Asn Asn Asn Glu Asn Lys Tyr Leu Gln Leu 35 40 45 Lys Lys Leu Asn Glu Ser Ile Asp Val Phe Met Asn Lys Tyr Lys Thr 50 55 60 Ser Ser Arg Asn Arg Ala Leu Ser Asn Leu Lys Lys Asp Ile Leu Lys65 70 75 80 Glu Val Ile Leu Ile Lys Asn Ser Asn Thr Ser Pro Val Glu Lys Asn 85 90 95 Leu His Phe Val Trp Ile Gly Gly Glu Val Ser Asp Ile Ala Leu Glu 100 105 110 Tyr Ile Lys Gln Trp Ala Asp Ile Asn Ala Glu Tyr Asn Ile Lys Leu 115 120 125 Trp Tyr Asp Ser Glu Ala Phe Leu Val Asn Thr Leu Lys Lys Ala Ile 130 135 140 Val Glu Ser Ser Thr Thr Glu Ala Leu Gln Leu Leu Glu Glu Glu Ile145 150 155 160 Gln Asn Pro Gln Phe Asp Asn Met Lys Phe Tyr Lys Lys Arg Met Glu 165 170 175 Phe Ile Tyr Asp Arg Gln Lys Arg Phe Ile Asn Tyr Tyr Lys Ser Gln 180 185 190 Ile Asn Lys Pro Thr Val Pro Thr Ile Asp Asp Ile Ile Lys Ser His 195 200 205 Leu Val Ser Glu Tyr Asn Arg Asp Glu Thr Val Leu Glu Ser Tyr Arg 210 215 220 Thr Asn Ser Leu Arg Lys Ile Asn Ser Asn His Gly Ile Asp Ile Arg225 230 235 240 Ala Asn Ser Leu Phe Thr Glu Gln Glu Leu Leu Asn Ile Tyr Ser Gln 245 250 255 Glu Leu Leu Asn Arg Gly Asn Leu Ala Ala Ala Ser Asp Ile Val Arg 260 265 270 Leu Leu Ala Leu Lys Asn Phe Gly Gly Val Tyr Leu Asp Val Asp Met 275 280 285 Leu Pro Gly Ile His Ser Asp Leu Phe Lys Thr Ile Ser Arg Pro Ser 290 295 300 Ser Ile Gly Leu Asp Arg Trp Glu Met Ile Lys Leu Glu Ala Ile Met305 310 315 320 Lys Tyr Lys Lys Tyr Ile Asn Asn Tyr Thr Ser Glu Asn Phe Asp Lys 325 330 335 Leu Asp Gln Gln Leu Lys Asp Asn Phe Lys Leu Ile Ile Glu Ser Lys 340 345 350 Ser Glu Lys Ser Glu Ile Phe Ser Lys Leu Glu Asn Leu Asn Val Ser 355 360 365 Asp Leu Glu Ile Lys Ile Ala Phe Ala Leu Gly Ser Val Ile Asn Gln 370 375 380 Ala Leu Ile Ser Lys Gln Gly Ser Tyr Leu Thr Asn Leu Val Ile Glu385 390 395 400 Gln Val Lys Asn Arg Tyr Gln Phe Leu Asn Gln His Leu Asn Pro Ala 405 410 415 Ile Glu Ser Asp Asn Asn Phe Thr Asp Thr Thr Lys Ile Phe His Asp 420 425 430 Ser Leu Phe Asn Ser Ala Thr Ala Glu Asn Ser Met Phe Leu Thr Lys 435 440 445 Ile Ala Pro Tyr Leu Gln Val Gly Phe Met Pro Glu Ala Arg Ser Thr 450 455 460 Ile Ser Leu Ser Gly Pro Gly Ala Tyr Ala Ser Ala Tyr Tyr Asp Phe465 470 475 480 Ile Asn Leu Gln Glu Asn Thr Ile Glu Lys Thr Leu Lys Ala Ser Asp 485 490 495 Leu Ile Glu Phe Lys Phe Pro Glu Asn Asn Leu Ser Gln Leu Thr Glu 500 505 510 Gln Glu Ile Asn Ser Leu Trp Ser Phe Asp Gln Ala Ser Ala Lys Tyr 515 520 525 Gln Phe Glu Lys Tyr Val Arg Asp Tyr Thr Gly Gly Ser Leu Ser Glu 530 535 540 Asp Asn Gly Val Asp Phe Asn Lys Asn Thr Ala Leu Asp Lys Asn Tyr545 550 555 560 Leu Leu Asn Asn Lys Ile Pro Ser Asn Asn Val Glu Glu Ala Gly Ser 565 570 575 Lys Asn Tyr Val His Tyr Ile Ile Gln Leu Gln Gly Asp Asp Ile Ser 580 585 590 Tyr Glu Ala Thr Cys Asn Leu Phe Ser Lys Asn Pro Lys Asn Ser Ile 595 600 605 Ile Ile Gln Arg Asn Met Asn Glu Ser Ala Lys Ser Tyr Phe Leu Ser 610 615 620 Asp Asp Gly Glu Ser Ile Leu Glu Leu Asn Lys Tyr Arg Ile Pro Glu625 630 635 640 Arg Leu Lys Asn Lys Glu Lys Val Lys Val Thr Phe Ile Gly His Gly 645 650 655 Lys Asp Glu Phe Asn Thr Ser Glu Phe Ala Arg Leu Ser Val Asp Ser 660 665 670 Leu Ser Asn Glu Ile Ser Ser Phe Leu Asp Thr Ile Lys Leu Asp Ile 675 680 685 Ser Pro Lys Asn Val Glu Val Asn Leu Leu Gly Cys Asn Met Phe Ser 690 695 700 Tyr Asp Phe Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu Leu Leu Ser705 710 715 720 Ile Met Asp Lys Ile Thr Ser Thr Leu Pro Asp Val Asn Lys Asn Ser 725 730 735 Ile Thr Ile Gly Ala Asn Gln Tyr Glu Val Arg Ile Asn Ser Glu Gly 740 745 750 Arg Lys Glu Leu Leu Ala His Ser Gly Lys Trp Ile Asn Lys Glu Glu 755 760 765 Ala Ile Met Ser Asp Leu Ser Ser Lys Glu Tyr Ile Phe Phe Asp Ser 770 775 780 Ile Asp Asn Lys Leu Lys Ala Lys Ser Lys Asn Ile Pro Gly Leu Ala785 790 795 800 Ser Ile Ser Glu Asp Ile Lys Thr Leu Leu Leu Asp Ala Ser Val Ser 805 810 815 Pro Asp Thr Lys Phe Ile Leu Asn Asn Leu Lys Leu Asn Ile Glu Ser 820 825 830 Ser Ile Gly Asp Tyr Ile Tyr Tyr Glu Lys Leu Glu Pro Val Lys Asn 835 840 845 Ile Ile His Asn Ser Ile Asp Asp Leu Ile Asp Glu Phe Asn Leu Leu 850 855 860 Glu Asn Val Ser Asp Glu Leu Tyr Glu Leu Lys Lys Leu Asn Asn Leu865 870 875 880 Asp Glu Lys Tyr Leu Ile Ser Phe Glu Asp Ile Ser Lys Asn Asn Ser 885 890 895 Thr Tyr Ser Val Arg Phe Ile Asn Lys Ser Asn Gly Glu Ser Val Tyr 900 905 910 Val Glu Thr Glu Lys Glu Ile Phe Ser Lys Tyr Ser Glu His Ile Thr 915 920 925 Lys Glu Ile Ser Thr Ile Lys Asn Ser Ile Ile Thr Asp Val Asn Gly 930 935 940 Asn Leu Leu Asp Asn Ile Gln Leu Asp His Thr Ser Gln Val Asn Thr945 950 955 960 Leu Asn Ala Ala Phe Phe Ile Gln Ser Leu Ile Asp Tyr Ser Ser Asn 965 970 975 Lys Asp Val Leu Asn Asp Leu Ser Thr Ser Val Lys Val Gln Leu Tyr 980 985 990 Ala Gln Leu Phe Ser Thr Gly Leu Asn Thr Ile Tyr Asp Ser Ile Gln 995 1000 1005 Leu Val Asn Leu Ile Ser Asn Ala Val Asn Asp Thr Ile Asn Val Leu 1010 1015 1020 Pro Thr Ile Thr Glu Gly Ile Pro Ile Val Ser Thr Ile Leu Asp Gly1025 1030 1035 1040 Ile Asn Leu Gly Ala Ala Ile Lys Glu Leu Leu Asp Glu His Asp Pro 1045 1050 1055 Leu Leu Lys Lys Glu Leu Glu Ala Lys Val Gly Val Leu Ala Ile Asn 1060 1065 1070 Met Ser Leu Ser Ile Ala Ala Thr Val Ala Ser Ile Val Gly Ile Gly 1075 1080 1085 Ala Glu Val Thr Ile Phe Leu Leu Pro Ile Ala Gly Ile Ser Ala Gly 1090 1095 1100 Ile Pro Ser Leu Val Asn Asn Glu Leu Ile Leu His Asp Lys Ala Thr1105 1110 1115 1120 Ser Val Val Asn Tyr Phe Asn His Leu Ser Glu Ser Lys Lys Tyr Gly 1125 1130 1135 Pro Leu Lys Thr Glu Asp Asp Lys Ile Leu Val Pro Ile Asp Asp Leu 1140 1145 1150 Val Ile Ser Glu Ile Asp Phe Asn Asn Asn Ser Ile Lys Leu Gly Thr 1155 1160 1165 Cys Asn Ile Leu Ala Met Glu Gly Gly Ser Gly His Thr Val Thr Gly 1170 1175 1180 Asn Ile Asp His Phe Phe Ser Ser Pro Ser Ile Ser Ser His Ile Pro1185 1190 1195 1200 Ser Leu Ser Ile Tyr Ser Ala Ile Gly Ile Glu Thr Glu Asn Leu Asp 1205 1210 1215 Phe Ser Lys Lys Ile Met Met Leu Pro Asn Ala Pro Ser Arg Val Phe 1220 1225 1230 Trp Trp Glu Thr Gly Ala Val Pro Gly Leu Arg Ser Leu Glu Asn Asp 1235 1240 1245 Gly Thr Arg Leu Leu Asp Ser Ile Arg Asp Leu Tyr Pro Gly Lys Phe 1250 1255 1260 Tyr Trp Arg Phe Tyr Ala Phe Phe Asp Tyr Ala Ile Thr Thr Leu Lys1265 1270 1275 1280 Pro Val Tyr Glu Asp Thr Asn Ile Lys Ile Lys Leu Asp Lys Asp Thr 1285 1290 1295 Arg Asn Phe Ile Met Pro Thr Ile Thr Thr Asn Glu Ile Arg Asn Lys 1300 1305 1310 Leu Ser Tyr Ser Phe Asp Gly Ala Gly Gly Thr Tyr Ser Leu Leu Leu 1315 1320 1325 Ser Ser Tyr Pro Ile Ser Thr Asn Ile Asn Leu Ser Lys Asp Asp Leu 1330 1335 1340 Trp Ile Phe Asn Ile Asp Asn Glu Val Arg Glu Ile Ser Ile Glu Asn1345 1350 1355 1360 Gly Thr Ile Lys Lys Gly Lys Leu Ile Lys Asp Val Leu Ser Lys Ile 1365 1370 1375 Asp Ile Asn Lys Asn Lys Leu Ile Ile Gly Asn Gln Thr Ile Asp Phe 1380 1385 1390 Ser Gly Asp Ile Asp Asn Lys Asp Arg Tyr Ile Phe Leu Thr Cys Glu 1395 1400 1405 Leu Asp Asp Lys Ile Ser Leu Ile Ile Glu Ile Asn Leu Val Ala Lys 1410 1415 1420 Ser Tyr Ser Leu Leu Leu Ser Gly Asp Lys Asn Tyr Leu Ile Ser Asn1425 1430 1435 1440 Leu Ser Asn Thr Ile Glu Lys Ile Asn Thr Leu Gly Leu Asp Ser Lys 1445 1450 1455 Asn Ile Ala Tyr Asn Tyr Thr Asp Glu Ser Asn Asn Lys Tyr Phe Gly 1460 1465 1470 Ala Ile Ser Lys Thr Ser Gln Lys Ser Ile Ile His Tyr Lys Lys Asp 1475 1480 1485 Ser Lys Asn Ile Leu Glu Phe Tyr Asn Asp Ser Thr Leu Glu Phe Asn 1490 1495 1500 Ser Lys Asp Phe Ile Ala Glu Asp Ile Asn Val Phe Met Lys Asp Asp1505 1510 1515 1520 Ile Asn Thr Ile Thr Gly Lys Tyr Tyr Val Asp Asn Asn Thr Asp Lys 1525 1530 1535 Ser Ile Asp Phe Ser Ile Ser Leu Val Ser Lys Asn Gln Val Lys Val 1540 1545 1550 Asn Gly Leu

Tyr Leu Asn Glu Ser Val Tyr Ser Ser Tyr Leu Asp Phe 1555 1560 1565 Val Lys Asn Ser Asp Gly His His Asn Thr Ser Asn Phe Met Asn Leu 1570 1575 1580 Phe Leu Asp Asn Ile Ser Phe Trp Lys Leu Phe Gly Phe Glu Asn Ile1585 1590 1595 1600 Asn Phe Val Ile Asp Lys Tyr Phe Thr Leu Val Gly Lys Thr Asn Leu 1605 1610 1615 Gly Tyr Val Glu Phe Ile Cys Asp Asn Asn Lys Asn Ile Asp Ile Tyr 1620 1625 1630 Phe Gly Glu Trp Lys Thr Ser Ser Ser Lys Ser Thr Ile Phe Ser Gly 1635 1640 1645 Asn Gly Arg Asn Val Val Val Glu Pro Ile Tyr Asn Pro Asp Thr Gly 1650 1655 1660 Glu Asp Ile Ser Thr Ser Leu Asp Phe Ser Tyr Glu Pro Leu Tyr Gly1665 1670 1675 1680 Ile Asp Arg Tyr Ile Asn Lys Val Leu Ile Ala Pro Asp Leu Tyr Thr 1685 1690 1695 Ser Leu Ile Asn Ile Asn Thr Asn Tyr Tyr Ser Asn Glu Tyr Tyr Pro 1700 1705 1710 Glu Ile Ile Val Leu Asn Pro Asn Thr Phe His Lys Lys Val Asn Ile 1715 1720 1725 Asn Leu Asp Ser Ser Ser Phe Glu Tyr Lys Trp Ser Thr Glu Gly Ser 1730 1735 1740 Asp Phe Ile Leu Val Arg Tyr Leu Glu Glu Ser Asn Lys Lys Ile Leu1745 1750 1755 1760 Gln Lys Ile Arg Ile Lys Gly Ile Leu Ser Asn Thr Gln Ser Phe Asn 1765 1770 1775 Lys Met Ser Ile Asp Phe Lys Asp Ile Lys Lys Leu Ser Leu Gly Tyr 1780 1785 1790 Ile Met Ser Asn Phe Lys Ser Phe Asn Ser Glu Asn Glu Leu Asp Arg 1795 1800 1805 Asp His Leu Gly Phe Lys Ile Ile Asp Asn Lys Thr Tyr Tyr Tyr Asp 1810 1815 1820 Glu Asp Ser Lys Leu Val Lys Gly Leu Ile Asn Ile Asn Asn Ser Leu1825 1830 1835 1840 Phe Tyr Phe Asp Pro Ile Glu Phe Asn Leu Val Thr Gly Trp Gln Thr 1845 1850 1855 Ile Asn Gly Lys Lys Tyr Tyr Phe Asp Ile Asn Thr Gly Ala Ala Leu 1860 1865 1870 Thr Ser Tyr Lys Ile Ile Asn Gly Lys His Phe Tyr Phe Asn Asn Asp 1875 1880 1885 Gly Val Met Gln Leu Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr 1890 1895 1900 Phe Ala Pro Ala Asn Thr Gln Asn Asn Asn Ile Glu Gly Gln Ala Ile1905 1910 1915 1920 Val Tyr Gln Ser Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe 1925 1930 1935 Asp Asn Asn Ser Lys Ala Val Thr Gly Trp Arg Ile Ile Asn Asn Glu 1940 1945 1950 Lys Tyr Tyr Phe Asn Pro Asn Asn Ala Ile Ala Ala Val Gly Leu Gln 1955 1960 1965 Val Ile Asp Asn Asn Lys Tyr Tyr Phe Asn Pro Asp Thr Ala Ile Ile 1970 1975 1980 Ser Lys Gly Trp Gln Thr Val Asn Gly Ser Arg Tyr Tyr Phe Asp Thr1985 1990 1995 2000 Asp Thr Ala Ile Ala Phe Asn Gly Tyr Lys Thr Ile Asp Gly Lys His 2005 2010 2015 Phe Tyr Phe Asp Ser Asp Cys Val Val Lys Ile Gly Val Phe Ser Thr 2020 2025 2030 Ser Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Tyr Asn Asn Asn 2035 2040 2045 Ile Glu Gly Gln Ala Ile Val Tyr Gln Ser Lys Phe Leu Thr Leu Asn 2050 2055 2060 Gly Lys Lys Tyr Tyr Phe Asp Asn Asn Ser Lys Ala Val Thr Gly Leu2065 2070 2075 2080 Gln Thr Ile Asp Ser Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ala Glu 2085 2090 2095 Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn 2100 2105 2110 Thr Asn Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys 2115 2120 2125 Lys Tyr Tyr Phe Asn Thr Asn Thr Ala Ile Ala Ser Thr Gly Tyr Thr 2130 2135 2140 Ile Ile Asn Gly Lys His Phe Tyr Phe Asn Thr Asp Gly Ile Met Gln2145 2150 2155 2160 Ile Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala 2165 2170 2175 Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Leu Tyr Gln Asn 2180 2185 2190 Glu Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp Ser 2195 2200 2205 Lys Ala Val Thr Gly Trp Arg Ile Ile Asn Asn Lys Lys Tyr Tyr Phe 2210 2215 2220 Asn Pro Asn Asn Ala Ile Ala Ala Ile His Leu Cys Thr Ile Asn Asn2225 2230 2235 2240 Asp Lys Tyr Tyr Phe Ser Tyr Asp Gly Ile Leu Gln Asn Gly Tyr Ile 2245 2250 2255 Thr Ile Glu Arg Asn Asn Phe Tyr Phe Asp Ala Asn Asn Glu Ser Lys 2260 2265 2270 Met Val Thr Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala 2275 2280 2285 Pro Ala Asn Thr His Asn Asn Asn Ile Glu Gly Gln Ala Ile Val Tyr 2290 2295 2300 Gln Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Asp Asn2305 2310 2315 2320 Asp Ser Lys Ala Val Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr 2325 2330 2335 Tyr Phe Asn Leu Asn Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile 2340 2345 2350 Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn Thr Ala Glu Ala Ala Thr 2355 2360 2365 Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr 2370 2375 2380 Phe Ile Ala Ser Thr Gly Tyr Thr Ser Ile Asn Gly Lys His Phe Tyr2385 2390 2395 2400 Phe Asn Thr Asp Gly Ile Met Gln Ile Gly Val Phe Lys Gly Pro Asn 2405 2410 2415 Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu 2420 2425 2430 Gly Gln Ala Ile Leu Tyr Gln Asn Lys Phe Leu Thr Leu Asn Gly Lys 2435 2440 2445 Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Ala Val Thr Gly Leu Arg Thr 2450 2455 2460 Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ala Val Ala Val2465 2470 2475 2480 Thr Gly Trp Gln Thr Ile Asn Gly Lys Lys Tyr Tyr Phe Asn Thr Asn 2485 2490 2495 Thr Ser Ile Ala Ser Thr Gly Tyr Thr Ile Ile Ser Gly Lys His Phe 2500 2505 2510 Tyr Phe Asn Thr Asp Gly Ile Met Gln Ile Gly Val Phe Lys Gly Pro 2515 2520 2525 Asp Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile 2530 2535 2540 Glu Gly Gln Ala Ile Arg Tyr Gln Asn Arg Phe Leu Tyr Leu His Asp2545 2550 2555 2560 Asn Ile Tyr Tyr Phe Gly Asn Asn Ser Lys Ala Ala Thr Gly Trp Val 2565 2570 2575 Thr Ile Asp Gly Asn Arg Tyr Tyr Phe Glu Pro Asn Thr Ala Met Gly 2580 2585 2590 Ala Asn Gly Tyr Lys Thr Ile Asp Asn Lys Asn Phe Tyr Phe Arg Asn 2595 2600 2605 Gly Leu Pro Gln Ile Gly Val Phe Lys Gly Ser Asn Gly Phe Glu Tyr 2610 2615 2620 Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile2625 2630 2635 2640 Arg Tyr Gln Asn Arg Phe Leu His Leu Leu Gly Lys Ile Tyr Tyr Phe 2645 2650 2655 Gly Asn Asn Ser Lys Ala Val Thr Gly Trp Gln Thr Ile Asn Gly Lys 2660 2665 2670 Val Tyr Tyr Phe Met Pro Asp Thr Ala Met Ala Ala Ala Gly Gly Leu 2675 2680 2685 Phe Glu Ile Asp Gly Val Ile Tyr Phe Phe Gly Val Asp Gly Val Lys 2690 2695 2700 Ala Pro Gly Ile Tyr Gly2705 27103797101DNAClostridium difficile 379atgagtttag ttaatagaaa acagttagaa aaaatggcaa atgtaagatt tcgtactcaa 60gaagatgaat atgttgcaat attggatgct ttagaagaat atcataatat gtcagagaat 120actgtagtcg aaaaatattt aaaattaaaa gatataaata gtttaacaga tatttatata 180gatacatata aaaaatctgg tagaaataaa gccttaaaaa aatttaagga atatctagtt 240acagaagtat tagagctaaa gaataataat ttaactccag ttgagaaaaa tttacatttt 300gtttggattg gaggtcaaat aaatgacact gctattaatt atataaatca atggaaagat 360gtaaatagtg attataatgt taatgttttt tatgatagta atgcattttt gataaacaca 420ttgaaaaaaa ctgtagtaga atcagcaata aatgatacac ttgaatcatt tagagaaaac 480ttaaatgacc ctagatttga ctataataaa ttcttcagaa aacgtatgga aataatttat 540gataaacaga aaaatttcat aaactactat aaagctcaaa gagaagaaaa tcctgaactt 600ataattgatg atattgtaaa gacatatctt tcaaatgagt attcaaagga gatagatgaa 660cttaatacct atattgaaga atccttaaat aaaattacac agaatagtgg aaatgatgtt 720agaaactttg aagaatttaa aaatggagag tcattcaact tatatgaaca agagttggta 780gaaaggtgga atttagctgc tgcttctgac atattaagaa tatctgcatt aaaagaaatt 840ggtggtatgt atttagatgt tgatatgtta ccaggaatac aaccagactt atttgagtct 900atagagaaac ctagttcagt aacagtggat ttttgggaaa tgacaaagtt agaagctata 960atgaaataca aagaatatat accagaatat acctcagaac attttgacat gttagacgaa 1020gaagttcaaa gtagttttga atctgttcta gcttctaagt cagataaatc agaaatattc 1080tcatcacttg gtgatatgga ggcatcacca ctagaagtta aaattgcatt taatagtaag 1140ggtattataa atcaagggct aatttctgtg aaagactcat attgtagcaa tttaatagta 1200aaacaaatcg agaatagata taaaatattg aataatagtt taaatccagc tattagcgag 1260gataatgatt ttaatactac aacgaatacc tttattgata gtataatggc tgaagctaat 1320gcagataatg gtagatttat gatggaacta ggaaagtatt taagagttgg tttcttccca 1380gatgttaaaa ctactattaa cttaagtggc cctgaagcat atgcggcagc ttatcaagat 1440ttattaatgt ttaaagaagg cagtatgaat atccatttga tagaagctga tttaagaaac 1500tttgaaatct ctaaaactaa tatttctcaa tcaactgaac aagaaatggc tagcttatgg 1560tcatttgacg atgcaagagc taaagctcaa tttgaagaat ataaaaggaa ttattttgaa 1620ggttctcttg gtgaagatga taatcttgat ttttctcaaa atatagtagt tgacaaggag 1680tatcttttag aaaaaatatc ttcattagca agaagttcag agagaggata tatacactat 1740attgttcagt tacaaggaga taaaattagt tatgaagcag catgtaactt atttgcaaag 1800actccttatg atagtgtact gtttcagaaa aatatagaag attcagaaat tgcatattat 1860tataatcctg gagatggtga aatacaagaa atagacaagt ataaaattcc aagtataatt 1920tctgatagac ctaagattaa attaacattt attggtcatg gtaaagatga atttaatact 1980gatatatttg caggttttga tgtagattca ttatccacag aaatagaagc agcaatagat 2040ttagctaaag aggatatttc tcctaagtca atagaaataa atttattagg atgtaatatg 2100tttagctact ctatcaacgt agaggagact tatcctggaa aattattact taaagttaaa 2160gataaaatat cagaattaat gccatctata agtcaagact ctattatagt aagtgcaaat 2220caatatgaag ttagaataaa tagtgaagga agaagagaat tattggatca ttctggtgaa 2280tggataaata aagaagaaag tattataaag gatatttcat caaaagaata tatatcattt 2340aatcctaaag aaaataaaat tacagtaaaa tctaaaaatt tacctgagct atctacatta 2400ttacaagaaa ttagaaataa ttctaattca agtgatattg aactagaaga aaaagtaatg 2460ttaacagaat gtgagataaa tgttatttca aatatagata cgcaaattgt tgaggaaagg 2520attgaagaag ctaagaattt aacttctgac tctattaatt atataaaaga tgaatttaaa 2580ctaatagaat ctatttctga tgcactatgt gacttaaaac aacagaatga attagaagat 2640tctcatttta tatcttttga ggacatatca gagactgatg agggatttag tataagattt 2700attaataaag aaactggaga atctatattt gtagaaactg aaaaaacaat attctctgaa 2760tatgctaatc atataactga agagatttct aagataaaag gtactatatt tgatactgta 2820aatggtaagt tagtaaaaaa agtaaattta gatactacac acgaagtaaa tactttaaat 2880gctgcatttt ttatacaatc attaatagaa tataatagtt ctaaagaatc tcttagtaat 2940ttaagtgtag caatgaaagt ccaagtttac gctcaattat ttagtactgg tttaaatact 3000attacagatg cagccaaagt tgttgaatta gtatcaactg cattagatga aactatagac 3060ttacttccta cattatctga aggattacct ataattgcaa ctattataga tggtgtaagt 3120ttaggtgcag caatcaaaga gctaagtgaa acgagtgacc cattattaag acaagaaata 3180gaagctaaga taggtataat ggcagtaaat ttaacaacag ctacaactgc aatcattact 3240tcatctttgg ggatagctag tggatttagt atacttttag ttcctttagc aggaatttca 3300gcaggtatac caagcttagt aaacaatgaa cttgtacttc gagataaggc aacaaaggtt 3360gtagattatt ttaaacatgt ttcattagtt gaaactgaag gagtatttac tttattagat 3420gataaaataa tgatgccaca agatgattta gtgatatcag aaatagattt taataataat 3480tcaatagttt taggtaaatg tgaaatctgg agaatggaag gtggttcagg tcatactgta 3540actgatgata tagatcactt cttttcagca ccatcaataa catatagaga gccacactta 3600tctatatatg acgtattgga agtacaaaaa gaagaacttg atttgtcaaa agatttaatg 3660gtattaccta atgctccaaa tagagtattt gcttgggaaa caggatggac accaggttta 3720agaagcttag aaaatgatgg cacaaaactg ttagaccgta taagagataa ctatgaaggt 3780gagttttatt ggagatattt tgcttttata gctgatgctt taataacaac attaaaacca 3840agatatgaag atactaatat aagaataaat ttagatagta atactagaag ttttatagtt 3900ccaataataa ctacagaata tataagagaa aaattatcat attctttcta tggttcagga 3960ggaacttatg cattgtctct ttctcaatat aatatgggta taaatataga attaagtgaa 4020agtgatgttt ggattataga tgttgataat gttgtgagag atgtaactat agaatctgat 4080aaaattaaaa aaggtgattt aatagaaggt attttatcta cactaagtat tgaagagaat 4140aaaattatct taaatagcca tgagattaat ttttctggtg aggtaaatgg aagtaatgga 4200tttgtttctt taacattttc aattttagaa ggaataaatg caattataga agttgattta 4260ttatctaaat catataaatt acttatttct ggcgaattaa aaatattgat gttaaattca 4320aatcatattc aacagaaaat agattatata ggattcaata gcgaattaca gaaaaatata 4380ccatatagct ttgtagatag tgaaggaaaa gagaatggtt ttattaatgg ttcaacaaaa 4440gaaggtttat ttgtatctga attacctgat gtagttctta taagtaaggt ttatatggat 4500gatagtaagc cttcatttgg atattatagt aataatttga aagatgtcaa agttataact 4560aaagataatg ttaatatatt aacaggttat tatcttaagg atgatataaa aatctctctt 4620tctttgactc tacaagatga aaaaactata aagttaaata gtgtgcattt agatgaaagt 4680ggagtagctg agattttgaa gttcatgaat agaaaaggta atacaaatac ttcagattct 4740ttaatgagct ttttagaaag tatgaatata aaaagtattt tcgttaattt cttacaatct 4800aatattaagt ttatattaga tgctaatttt ataataagtg gtactacttc tattggccaa 4860tttgagttta tttgtgatga aaatgataat atacaaccat atttcattaa gtttaataca 4920ctagaaacta attatacttt atatgtagga aatagacaaa atatgatagt ggaaccaaat 4980tatgatttag atgattctgg agatatatct tcaactgtta tcaatttctc tcaaaagtat 5040ctttatggaa tagacagttg tgttaataaa gttgtaattt caccaaatat ttatacagat 5100gaaataaata taacgcctgt atatgaaaca aataatactt atccagaagt tattgtatta 5160gatgcaaatt atataaatga aaaaataaat gttaatatca atgatctatc tatacgatat 5220gtatggagta atgatggtaa tgattttatt cttatgtcaa ctagtgaaga aaataaggtg 5280tcacaagtta aaataagatt cgttaatgtt tttaaagata agactttggc aaataagcta 5340tcttttaact ttagtgataa acaagatgta cctgtaagtg aaataatctt atcatttaca 5400ccttcatatt atgaggatgg attgattggc tatgatttgg gtctagtttc tttatataat 5460gagaaatttt atattaataa ctttggaatg atggtatctg gattaatata tattaatgat 5520tcattatatt attttaaacc accagtaaat aatttgataa ctggatttgt gactgtaggc 5580gatgataaat actactttaa tccaattaat ggtggagctg cttcaattgg agagacaata 5640attgatgaca aaaattatta tttcaaccaa agtggagtgt tacaaacagg tgtatttagt 5700acagaagatg gatttaaata ttttgcccca gctaatacac ttgatgaaaa cctagaagga 5760gaagcaattg attttactgg aaaattaatt attgacgaaa atatttatta ttttgatgat 5820aattatagag gagctgtaga atggaaagaa ttagatggtg aaatgcacta ttttagccca 5880gaaacaggta aagcttttaa aggtctaaat caaataggtg attataaata ctatttcaat 5940tctgatggag ttatgcaaaa aggatttgtt agtataaatg ataataaaca ctattttgat 6000gattctggtg ttatgaaagt aggttacact gaaatagatg gcaagcattt ctactttgct 6060gaaaacggag aaatgcaaat aggagtattt aatacagaag atggatttaa atattttgct 6120catcataatg aagatttagg aaatgaagaa ggtgaagaaa tctcatattc tggtatatta 6180aatttcaata ataaaattta ctattttgat gattcattta cagctgtagt tggatggaaa 6240gatttagagg atggttcaaa gtattatttt gatgaagata cagcagaagc atatataggt 6300ttgtcattaa taaatgatgg tcaatattat tttaatgatg atggaattat gcaagttgga 6360tttgtcacta taaatgataa agtcttctac ttctctgact ctggaattat agaatctgga 6420gtacaaaaca tagatgacaa ttatttctat atagatgata atggtatagt tcaaattggt 6480gtatttgata cttcagatgg atataaatat tttgcacctg ctaatactgt aaatgataat 6540atttacggac aagcagttga atatagtggt ttagttagag ttggtgaaga tgtatattat 6600tttggagaaa catatacaat tgagactgga tggatatatg atatggaaaa tgaaagtgat 6660aaatattatt tcaatccaga aactaaaaaa gcatgcaaag gtattaattt aattgatgat 6720ataaaatatt attttgatga gaagggcata atgagaacgg gtcttatatc atttgaaaat 6780aataattatt actttaatga gaatggtgaa atgcaatttg gttatataaa tatagaagat 6840aagatgttct attttggtga agatggtgtc atgcagattg gagtatttaa tacaccagat 6900ggatttaaat actttgcaca tcaaaatact ttggatgaga attttgaggg agaatcaata 6960aactatactg gttggttaga tttagatgaa aagagatatt attttacaga tgaatatatt 7020gcagcaactg gttcagttat tattgatggt gaggagtatt attttgatcc tgatacagct 7080caattagtga ttagtgaata g 71013802366PRTClostridium difficile 380Met Ser Leu Val Asn Arg Lys Gln Leu Glu Lys Met Ala Asn Val Arg1 5 10 15 Phe Arg Thr Gln Glu Asp Glu Tyr Val Ala Ile Leu Asp Ala Leu Glu 20 25 30 Glu Tyr His Asn Met Ser Glu Asn Thr Val Val Glu Lys Tyr Leu Lys 35 40 45 Leu Lys Asp Ile

Asn Ser Leu Thr Asp Ile Tyr Ile Asp Thr Tyr Lys 50 55 60 Lys Ser Gly Arg Asn Lys Ala Leu Lys Lys Phe Lys Glu Tyr Leu Val65 70 75 80 Thr Glu Val Leu Glu Leu Lys Asn Asn Asn Leu Thr Pro Val Glu Lys 85 90 95 Asn Leu His Phe Val Trp Ile Gly Gly Gln Ile Asn Asp Thr Ala Ile 100 105 110 Asn Tyr Ile Asn Gln Trp Lys Asp Val Asn Ser Asp Tyr Asn Val Asn 115 120 125 Val Phe Tyr Asp Ser Asn Ala Phe Leu Ile Asn Thr Leu Lys Lys Thr 130 135 140 Val Val Glu Ser Ala Ile Asn Asp Thr Leu Glu Ser Phe Arg Glu Asn145 150 155 160 Leu Asn Asp Pro Arg Phe Asp Tyr Asn Lys Phe Phe Arg Lys Arg Met 165 170 175 Glu Ile Ile Tyr Asp Lys Gln Lys Asn Phe Ile Asn Tyr Tyr Lys Ala 180 185 190 Gln Arg Glu Glu Asn Pro Glu Leu Ile Ile Asp Asp Ile Val Lys Thr 195 200 205 Tyr Leu Ser Asn Glu Tyr Ser Lys Glu Ile Asp Glu Leu Asn Thr Tyr 210 215 220 Ile Glu Glu Ser Leu Asn Lys Ile Thr Gln Asn Ser Gly Asn Asp Val225 230 235 240 Arg Asn Phe Glu Glu Phe Lys Asn Gly Glu Ser Phe Asn Leu Tyr Glu 245 250 255 Gln Glu Leu Val Glu Arg Trp Asn Leu Ala Ala Ala Ser Asp Ile Leu 260 265 270 Arg Ile Ser Ala Leu Lys Glu Ile Gly Gly Met Tyr Leu Asp Val Asp 275 280 285 Met Leu Pro Gly Ile Gln Pro Asp Leu Phe Glu Ser Ile Glu Lys Pro 290 295 300 Ser Ser Val Thr Val Asp Phe Trp Glu Met Thr Lys Leu Glu Ala Ile305 310 315 320 Met Lys Tyr Lys Glu Tyr Ile Pro Glu Tyr Thr Ser Glu His Phe Asp 325 330 335 Met Leu Asp Glu Glu Val Gln Ser Ser Phe Glu Ser Val Leu Ala Ser 340 345 350 Lys Ser Asp Lys Ser Glu Ile Phe Ser Ser Leu Gly Asp Met Glu Ala 355 360 365 Ser Pro Leu Glu Val Lys Ile Ala Phe Asn Ser Lys Gly Ile Ile Asn 370 375 380 Gln Gly Leu Ile Ser Val Lys Asp Ser Tyr Cys Ser Asn Leu Ile Val385 390 395 400 Lys Gln Ile Glu Asn Arg Tyr Lys Ile Leu Asn Asn Ser Leu Asn Pro 405 410 415 Ala Ile Ser Glu Asp Asn Asp Phe Asn Thr Thr Thr Asn Thr Phe Ile 420 425 430 Asp Ser Ile Met Ala Glu Ala Asn Ala Asp Asn Gly Arg Phe Met Met 435 440 445 Glu Leu Gly Lys Tyr Leu Arg Val Gly Phe Phe Pro Asp Val Lys Thr 450 455 460 Thr Ile Asn Leu Ser Gly Pro Glu Ala Tyr Ala Ala Ala Tyr Gln Asp465 470 475 480 Leu Leu Met Phe Lys Glu Gly Ser Met Asn Ile His Leu Ile Glu Ala 485 490 495 Asp Leu Arg Asn Phe Glu Ile Ser Lys Thr Asn Ile Ser Gln Ser Thr 500 505 510 Glu Gln Glu Met Ala Ser Leu Trp Ser Phe Asp Asp Ala Arg Ala Lys 515 520 525 Ala Gln Phe Glu Glu Tyr Lys Arg Asn Tyr Phe Glu Gly Ser Leu Gly 530 535 540 Glu Asp Asp Asn Leu Asp Phe Ser Gln Asn Ile Val Val Asp Lys Glu545 550 555 560 Tyr Leu Leu Glu Lys Ile Ser Ser Leu Ala Arg Ser Ser Glu Arg Gly 565 570 575 Tyr Ile His Tyr Ile Val Gln Leu Gln Gly Asp Lys Ile Ser Tyr Glu 580 585 590 Ala Ala Cys Asn Leu Phe Ala Lys Thr Pro Tyr Asp Ser Val Leu Phe 595 600 605 Gln Lys Asn Ile Glu Asp Ser Glu Ile Ala Tyr Tyr Tyr Asn Pro Gly 610 615 620 Asp Gly Glu Ile Gln Glu Ile Asp Lys Tyr Lys Ile Pro Ser Ile Ile625 630 635 640 Ser Asp Arg Pro Lys Ile Lys Leu Thr Phe Ile Gly His Gly Lys Asp 645 650 655 Glu Phe Asn Thr Asp Ile Phe Ala Gly Phe Asp Val Asp Ser Leu Ser 660 665 670 Thr Glu Ile Glu Ala Ala Ile Asp Leu Ala Lys Glu Asp Ile Ser Pro 675 680 685 Lys Ser Ile Glu Ile Asn Leu Leu Gly Cys Asn Met Phe Ser Tyr Ser 690 695 700 Ile Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu Leu Leu Lys Val Lys705 710 715 720 Asp Lys Ile Ser Glu Leu Met Pro Ser Ile Ser Gln Asp Ser Ile Ile 725 730 735 Val Ser Ala Asn Gln Tyr Glu Val Arg Ile Asn Ser Glu Gly Arg Arg 740 745 750 Glu Leu Leu Asp His Ser Gly Glu Trp Ile Asn Lys Glu Glu Ser Ile 755 760 765 Ile Lys Asp Ile Ser Ser Lys Glu Tyr Ile Ser Phe Asn Pro Lys Glu 770 775 780 Asn Lys Ile Thr Val Lys Ser Lys Asn Leu Pro Glu Leu Ser Thr Leu785 790 795 800 Leu Gln Glu Ile Arg Asn Asn Ser Asn Ser Ser Asp Ile Glu Leu Glu 805 810 815 Glu Lys Val Met Leu Thr Glu Cys Glu Ile Asn Val Ile Ser Asn Ile 820 825 830 Asp Thr Gln Ile Val Glu Glu Arg Ile Glu Glu Ala Lys Asn Leu Thr 835 840 845 Ser Asp Ser Ile Asn Tyr Ile Lys Asp Glu Phe Lys Leu Ile Glu Ser 850 855 860 Ile Ser Asp Ala Leu Cys Asp Leu Lys Gln Gln Asn Glu Leu Glu Asp865 870 875 880 Ser His Phe Ile Ser Phe Glu Asp Ile Ser Glu Thr Asp Glu Gly Phe 885 890 895 Ser Ile Arg Phe Ile Asn Lys Glu Thr Gly Glu Ser Ile Phe Val Glu 900 905 910 Thr Glu Lys Thr Ile Phe Ser Glu Tyr Ala Asn His Ile Thr Glu Glu 915 920 925 Ile Ser Lys Ile Lys Gly Thr Ile Phe Asp Thr Val Asn Gly Lys Leu 930 935 940 Val Lys Lys Val Asn Leu Asp Thr Thr His Glu Val Asn Thr Leu Asn945 950 955 960 Ala Ala Phe Phe Ile Gln Ser Leu Ile Glu Tyr Asn Ser Ser Lys Glu 965 970 975 Ser Leu Ser Asn Leu Ser Val Ala Met Lys Val Gln Val Tyr Ala Gln 980 985 990 Leu Phe Ser Thr Gly Leu Asn Thr Ile Thr Asp Ala Ala Lys Val Val 995 1000 1005 Glu Leu Val Ser Thr Ala Leu Asp Glu Thr Ile Asp Leu Leu Pro Thr 1010 1015 1020 Leu Ser Glu Gly Leu Pro Ile Ile Ala Thr Ile Ile Asp Gly Val Ser1025 1030 1035 1040 Leu Gly Ala Ala Ile Lys Glu Leu Ser Glu Thr Ser Asp Pro Leu Leu 1045 1050 1055 Arg Gln Glu Ile Glu Ala Lys Ile Gly Ile Met Ala Val Asn Leu Thr 1060 1065 1070 Thr Ala Thr Thr Ala Ile Ile Thr Ser Ser Leu Gly Ile Ala Ser Gly 1075 1080 1085 Phe Ser Ile Leu Leu Val Pro Leu Ala Gly Ile Ser Ala Gly Ile Pro 1090 1095 1100 Ser Leu Val Asn Asn Glu Leu Val Leu Arg Asp Lys Ala Thr Lys Val1105 1110 1115 1120 Val Asp Tyr Phe Lys His Val Ser Leu Val Glu Thr Glu Gly Val Phe 1125 1130 1135 Thr Leu Leu Asp Asp Lys Ile Met Met Pro Gln Asp Asp Leu Val Ile 1140 1145 1150 Ser Glu Ile Asp Phe Asn Asn Asn Ser Ile Val Leu Gly Lys Cys Glu 1155 1160 1165 Ile Trp Arg Met Glu Gly Gly Ser Gly His Thr Val Thr Asp Asp Ile 1170 1175 1180 Asp His Phe Phe Ser Ala Pro Ser Ile Thr Tyr Arg Glu Pro His Leu1185 1190 1195 1200 Ser Ile Tyr Asp Val Leu Glu Val Gln Lys Glu Glu Leu Asp Leu Ser 1205 1210 1215 Lys Asp Leu Met Val Leu Pro Asn Ala Pro Asn Arg Val Phe Ala Trp 1220 1225 1230 Glu Thr Gly Trp Thr Pro Gly Leu Arg Ser Leu Glu Asn Asp Gly Thr 1235 1240 1245 Lys Leu Leu Asp Arg Ile Arg Asp Asn Tyr Glu Gly Glu Phe Tyr Trp 1250 1255 1260 Arg Tyr Phe Ala Phe Ile Ala Asp Ala Leu Ile Thr Thr Leu Lys Pro1265 1270 1275 1280 Arg Tyr Glu Asp Thr Asn Ile Arg Ile Asn Leu Asp Ser Asn Thr Arg 1285 1290 1295 Ser Phe Ile Val Pro Ile Ile Thr Thr Glu Tyr Ile Arg Glu Lys Leu 1300 1305 1310 Ser Tyr Ser Phe Tyr Gly Ser Gly Gly Thr Tyr Ala Leu Ser Leu Ser 1315 1320 1325 Gln Tyr Asn Met Gly Ile Asn Ile Glu Leu Ser Glu Ser Asp Val Trp 1330 1335 1340 Ile Ile Asp Val Asp Asn Val Val Arg Asp Val Thr Ile Glu Ser Asp1345 1350 1355 1360 Lys Ile Lys Lys Gly Asp Leu Ile Glu Gly Ile Leu Ser Thr Leu Ser 1365 1370 1375 Ile Glu Glu Asn Lys Ile Ile Leu Asn Ser His Glu Ile Asn Phe Ser 1380 1385 1390 Gly Glu Val Asn Gly Ser Asn Gly Phe Val Ser Leu Thr Phe Ser Ile 1395 1400 1405 Leu Glu Gly Ile Asn Ala Ile Ile Glu Val Asp Leu Leu Ser Lys Ser 1410 1415 1420 Tyr Lys Leu Leu Ile Ser Gly Glu Leu Lys Ile Leu Met Leu Asn Ser1425 1430 1435 1440 Asn His Ile Gln Gln Lys Ile Asp Tyr Ile Gly Phe Asn Ser Glu Leu 1445 1450 1455 Gln Lys Asn Ile Pro Tyr Ser Phe Val Asp Ser Glu Gly Lys Glu Asn 1460 1465 1470 Gly Phe Ile Asn Gly Ser Thr Lys Glu Gly Leu Phe Val Ser Glu Leu 1475 1480 1485 Pro Asp Val Val Leu Ile Ser Lys Val Tyr Met Asp Asp Ser Lys Pro 1490 1495 1500 Ser Phe Gly Tyr Tyr Ser Asn Asn Leu Lys Asp Val Lys Val Ile Thr1505 1510 1515 1520 Lys Asp Asn Val Asn Ile Leu Thr Gly Tyr Tyr Leu Lys Asp Asp Ile 1525 1530 1535 Lys Ile Ser Leu Ser Leu Thr Leu Gln Asp Glu Lys Thr Ile Lys Leu 1540 1545 1550 Asn Ser Val His Leu Asp Glu Ser Gly Val Ala Glu Ile Leu Lys Phe 1555 1560 1565 Met Asn Arg Lys Gly Asn Thr Asn Thr Ser Asp Ser Leu Met Ser Phe 1570 1575 1580 Leu Glu Ser Met Asn Ile Lys Ser Ile Phe Val Asn Phe Leu Gln Ser1585 1590 1595 1600 Asn Ile Lys Phe Ile Leu Asp Ala Asn Phe Ile Ile Ser Gly Thr Thr 1605 1610 1615 Ser Ile Gly Gln Phe Glu Phe Ile Cys Asp Glu Asn Asp Asn Ile Gln 1620 1625 1630 Pro Tyr Phe Ile Lys Phe Asn Thr Leu Glu Thr Asn Tyr Thr Leu Tyr 1635 1640 1645 Val Gly Asn Arg Gln Asn Met Ile Val Glu Pro Asn Tyr Asp Leu Asp 1650 1655 1660 Asp Ser Gly Asp Ile Ser Ser Thr Val Ile Asn Phe Ser Gln Lys Tyr1665 1670 1675 1680 Leu Tyr Gly Ile Asp Ser Cys Val Asn Lys Val Val Ile Ser Pro Asn 1685 1690 1695 Ile Tyr Thr Asp Glu Ile Asn Ile Thr Pro Val Tyr Glu Thr Asn Asn 1700 1705 1710 Thr Tyr Pro Glu Val Ile Val Leu Asp Ala Asn Tyr Ile Asn Glu Lys 1715 1720 1725 Ile Asn Val Asn Ile Asn Asp Leu Ser Ile Arg Tyr Val Trp Ser Asn 1730 1735 1740 Asp Gly Asn Asp Phe Ile Leu Met Ser Thr Ser Glu Glu Asn Lys Val1745 1750 1755 1760 Ser Gln Val Lys Ile Arg Phe Val Asn Val Phe Lys Asp Lys Thr Leu 1765 1770 1775 Ala Asn Lys Leu Ser Phe Asn Phe Ser Asp Lys Gln Asp Val Pro Val 1780 1785 1790 Ser Glu Ile Ile Leu Ser Phe Thr Pro Ser Tyr Tyr Glu Asp Gly Leu 1795 1800 1805 Ile Gly Tyr Asp Leu Gly Leu Val Ser Leu Tyr Asn Glu Lys Phe Tyr 1810 1815 1820 Ile Asn Asn Phe Gly Met Met Val Ser Gly Leu Ile Tyr Ile Asn Asp1825 1830 1835 1840 Ser Leu Tyr Tyr Phe Lys Pro Pro Val Asn Asn Leu Ile Thr Gly Phe 1845 1850 1855 Val Thr Val Gly Asp Asp Lys Tyr Tyr Phe Asn Pro Ile Asn Gly Gly 1860 1865 1870 Ala Ala Ser Ile Gly Glu Thr Ile Ile Asp Asp Lys Asn Tyr Tyr Phe 1875 1880 1885 Asn Gln Ser Gly Val Leu Gln Thr Gly Val Phe Ser Thr Glu Asp Gly 1890 1895 1900 Phe Lys Tyr Phe Ala Pro Ala Asn Thr Leu Asp Glu Asn Leu Glu Gly1905 1910 1915 1920 Glu Ala Ile Asp Phe Thr Gly Lys Leu Ile Ile Asp Glu Asn Ile Tyr 1925 1930 1935 Tyr Phe Asp Asp Asn Tyr Arg Gly Ala Val Glu Trp Lys Glu Leu Asp 1940 1945 1950 Gly Glu Met His Tyr Phe Ser Pro Glu Thr Gly Lys Ala Phe Lys Gly 1955 1960 1965 Leu Asn Gln Ile Gly Asp Tyr Lys Tyr Tyr Phe Asn Ser Asp Gly Val 1970 1975 1980 Met Gln Lys Gly Phe Val Ser Ile Asn Asp Asn Lys His Tyr Phe Asp1985 1990 1995 2000 Asp Ser Gly Val Met Lys Val Gly Tyr Thr Glu Ile Asp Gly Lys His 2005 2010 2015 Phe Tyr Phe Ala Glu Asn Gly Glu Met Gln Ile Gly Val Phe Asn Thr 2020 2025 2030 Glu Asp Gly Phe Lys Tyr Phe Ala His His Asn Glu Asp Leu Gly Asn 2035 2040 2045 Glu Glu Gly Glu Glu Ile Ser Tyr Ser Gly Ile Leu Asn Phe Asn Asn 2050 2055 2060 Lys Ile Tyr Tyr Phe Asp Asp Ser Phe Thr Ala Val Val Gly Trp Lys2065 2070 2075 2080 Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe Asp Glu Asp Thr Ala Glu 2085 2090 2095 Ala Tyr Ile Gly Leu Ser Leu Ile Asn Asp Gly Gln Tyr Tyr Phe Asn 2100 2105 2110 Asp Asp Gly Ile Met Gln Val Gly Phe Val Thr Ile Asn Asp Lys Val 2115 2120 2125 Phe Tyr Phe Ser Asp Ser Gly Ile Ile Glu Ser Gly Val Gln Asn Ile 2130 2135 2140 Asp Asp Asn Tyr Phe Tyr Ile Asp Asp Asn Gly Ile Val Gln Ile Gly2145 2150 2155 2160 Val Phe Asp Thr Ser Asp Gly Tyr Lys Tyr Phe Ala Pro Ala Asn Thr 2165 2170 2175 Val Asn Asp Asn Ile Tyr Gly Gln Ala Val Glu Tyr Ser Gly Leu Val 2180 2185 2190 Arg Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Thr Ile Glu 2195 2200 2205 Thr Gly Trp Ile Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr Tyr Phe 2210 2215 2220 Asn Pro Glu Thr Lys Lys Ala Cys Lys Gly Ile Asn Leu Ile Asp Asp2225 2230 2235 2240 Ile Lys Tyr Tyr Phe Asp Glu Lys Gly Ile Met Arg Thr Gly Leu Ile 2245 2250 2255 Ser Phe Glu Asn Asn Asn Tyr Tyr Phe Asn Glu Asn Gly Glu Met Gln 2260 2265 2270 Phe Gly Tyr Ile Asn Ile Glu Asp Lys Met Phe Tyr Phe Gly Glu Asp 2275 2280 2285 Gly Val Met Gln Ile Gly Val Phe Asn Thr Pro Asp Gly Phe Lys Tyr 2290 2295 2300 Phe Ala His Gln Asn Thr Leu Asp Glu Asn Phe Glu Gly Glu Ser Ile2305 2310 2315 2320 Asn Tyr Thr Gly Trp Leu Asp Leu Asp Glu Lys Arg Tyr Tyr Phe Thr 2325 2330 2335 Asp Glu Tyr Ile Ala Ala Thr Gly Ser Val Ile Ile Asp Gly Glu Glu 2340 2345 2350 Tyr Tyr Phe Asp Pro Asp Thr Ala Gln Leu Val Ile Ser Glu 2355 2360 2365

3818PRTArtificial SequenceSynthetic 381Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 3828PRTArtificial SequenceSynthetic 382Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 38317PRTArtificial SequenceSynthetic 383Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15 Xaa38412PRTArtificial SequenceSynthetic 384Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 3853PRTArtificial SequenceSynthetic 385Xaa Xaa Xaa1 3869PRTArtificial SequenceSynthetic 386Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 3878PRTArtificial SequenceSynthetic 387Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 3888PRTArtificial SequenceSynthetic 388Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 38924PRTArtificial SequenceSynthetic 389Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 3907PRTArtificial SequenceSynthetic 390Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 3913PRTArtificial SequenceSynthetic 391Xaa Xaa Xaa1 39210PRTArtificial SequenceSynthetic 392Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 39310PRTArtificial SequenceSynthetic 393Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 3949PRTArtificial SequenceSynthetic 394Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 39516PRTArtificial SequenceSynthetic 395Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15 3967PRTArtificial SequenceSynthetic 396Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 3973PRTArtificial SequenceSynthetic 397Xaa Xaa Xaa1 3989PRTArtificial SequenceSynthetic 398Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5

Claims

1. An isolated antibody that specifically binds to Clostridium difficile toxin A or to toxin B, or that binds to, or cross reacts with, both toxin A and B, wherein: a) the isolated antibody or antigen-binding fragment thereof that specifically binds toxin A of Clostridium difficile comprises the three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 98, 114, 130, 146 and 162; and the three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 106, 122, 138, 154 and 170; b) the isolated antibody or antigen-binding fragment thereof that specifically binds toxin B of Clostridium difficile comprises the HCDR1, HCDR2 and HCDR3 contained within a HCVR amino acid sequence selected from the group consisting of SEQ ID NOs: 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338 and 354; and the LCDR1, LCDR2 and LCDR3 contained within a LCVR amino acid sequence selected from the group consisting of SEQ ID NOs: 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346 and 362; and c) the isolated antibody or antigen-binding fragment that binds to, or cross reacts with both toxin A and toxin B of Clostridium difficile comprises the HCDR1, HCDR2 and HCDR3 contained within a HCVR amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 34, 50, 66 and 82; and the LCDR1, LCDR2 and LCDR3 contained within a LCVR amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 42, 58, 74 and 90.

2. The isolated antibody or antigen-binding fragment thereof of claim 1 that specifically binds toxin A of Clostridium difficile, wherein the antibody, or antigen-binding fragment thereof comprises: (a) a HCVR having an amino acid sequence of SEQ ID NO: 146; and (b) a LCVR having an amino acid sequence of SEQ ID NO: 154.

3. The isolated antibody or antigen-binding fragment thereof of claim 1 that specifically binds toxin A of Clostridium difficile, wherein the antibody, or antigen-binding fragment thereof comprises a HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2/10, 98/106, 114/122, 130/138, 146/154 and 162/170.

4. The isolated antibody of claim 1, or an antigen-binding fragment thereof that specifically binds toxin A of Clostridium difficile, wherein the antibody comprises: (a) a HCDR1 domain having an amino acid sequence selected from SEQ ID NOs: 4, 100, 116, 132, 148 and 164; (b) a HCDR2 domain having an amino acid sequence selected from SEQ ID NOs: 6, 102, 118, 134, 150 and 166; (c) a HCDR3 domain having an amino acid sequence selected from SEQ ID NOs: 8, 104, 120, 136, 152 and 168; (d) a LCDR1 domain having an amino acid sequence selected from SEQ ID NOs: 12, 108, 124, 140, 156, and 172; (e) a LCDR2 domain having an amino acid sequence selected from SEQ ID NOs: 14, 110, 126, 142, 158 and 174; and (f) a LCDR3 domain having an amino acid sequence selected from SEQ ID NOs: 16, 112, 128, 144, 160 and 176.

5. The isolated antibody of claim 4, or an antigen-binding fragment thereof that specifically binds toxin A of Clostridium difficile, wherein the antibody comprises: (a) a HCDR1 domain having an amino acid sequence of SEQ ID NO: 148; (b) a HCDR2 domain having an amino acid sequence of SEQ ID NO: 150; (c) a HCDR3 domain having an amino acid sequence of SEQ ID NO: 152; (d) a LCDR1 domain having an amino acid sequence of SEQ ID NO: 156; (e) a LCDR2 domain having an amino acid sequence of SEQ ID NO: 158; and (f) a LCDR3 domain having an amino acid sequence of SEQ ID NO: 160.

6. The isolated antibody of claim 1, or an antigen-binding fragment thereof that specifically binds Clostridium difficile toxin B, wherein the antibody comprises: (a) a HCVR having the amino acid sequence of SEQ ID NO: 274; and (b) a LCVR having the amino acid sequence of SEQ ID NO: 282.

7. The isolated antibody of claim 1, or an antigen-binding fragment thereof that specifically binds Clostridium difficile toxin B, wherein the isolated antibody or antigen-binding fragment thereof comprises: a HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 178/186, 194/202, 210/218, 226/234, 242/250, 258/266, 274/282, 290/298, 306/314, 322/330, 338/346 and 354/362.

8. The isolated antibody of claim 1, or an antigen-binding fragment thereof that specifically binds Clostridium difficile toxin B, wherein the antibody comprises: (a) a HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 180, 196, 212, 228, 244, 260, 276, 292, 308, 324, 340 and 356; (b) a HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 182, 198, 214, 230, 246, 262, 278, 294, 310, 326, 342 and 358; (c) a HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 184, 200, 216, 232, 248, 264, 280, 296, 312, 328, 344 and 360; (d) a LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 188, 204, 220, 236, 252, 268, 284, 300, 316, 332, 348 and 364; (e) a LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 190, 206, 222, 238, 254, 270, 286, 302, 318, 334, 350 and 366; and (f) a LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 192, 208, 224, 240, 256, 272, 288, 304, 320, 336, 352 and 368.

9. The isolated antibody of claim 8, or an antigen-binding fragment thereof that specifically binds Clostridium difficile toxin B, wherein the antibody comprises: (a) a HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 276, (b) a HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 278; (c) a HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 280; (d) a LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 284; (e) a LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 286; and (f) a LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 288.

10. The isolated antibody of claim 1, or an antigen-binding fragment thereof that binds to, or cross reacts with both toxin A and toxin B of Clostridium difficile, wherein the antibody comprises (a) a HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 34; and (b) a LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 26 and 42.

11. The isolated antibody of claim 1, or an antigen-binding fragment thereof that binds to, or cross reacts with both toxin A and toxin B of Clostridium difficile, wherein the antibody or antigen-binding fragment thereof comprises a HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 18/26, 34/42, 50/58, 66/74 and 82/90.

12. The isolated antibody of claim 11, or an antigen-binding fragment thereof that binds to or cross reacts with both toxin A and toxin B of Clostridium difficile, wherein the antibody or antigen-binding fragment thereof comprises a HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 18/26 and 34/42.

13. The isolated antibody of claim 1, or an antigen-binding fragment thereof that binds to, or cross reacts with both toxin A and toxin B of Clostridium difficile, wherein the antibody comprises: (a) a HCDR1 domain having an amino acid sequence selected from SEQ ID NOs: 20, 36, 52, 68, and 84; (b) a HCDR2 domain having an amino acid sequence selected from SEQ ID NOs: 22, 38, 54, 70 and 86; (c) a HCDR3 domain having an amino acid sequence selected from SEQ ID NOs: 24, 40, 56, 72 and 88; (d) a LCDR1 domain having an amino acid sequence selected from SEQ ID NOs: 28, 44, 60, 76 and 92; (e) a LCDR2 domain having an amino acid sequence selected from SEQ ID NOs: 30, 46, 62, 78 and 94; and (f) a LCDR3 domain having an amino acid sequence selected from SEQ ID NOs: 32, 48, 64, 80 and 96.

14. The isolated antibody of claim 13, or an antigen-binding fragment thereof that binds to, or cross reacts with both toxin A and toxin B of Clostridium difficile, wherein the isolated antibody or antigen-binding fragment comprises: (a) a HCDR1 domain having an amino acid sequence selected from SEQ ID NOs: 20, and 36; (b) a HCDR2 domain having an amino acid sequence selected from SEQ ID NOs: 22, and 38; (c) a HCDR3 domain having an amino acid sequence selected from SEQ ID NOs: 24, and 40; (d) a LCDR1 domain having an amino acid sequence selected from SEQ ID NOs: 28, and 44; (e) a LCDR2 domain having an amino acid sequence selected from SEQ ID NOs: 30, and 46; and (f) a LCDR3 domain having an amino acid sequence selected from SEQ ID NOs: 32, and 48.

15. A pharmaceutical composition comprising one or more antibodies of claim 1 and a pharmaceutically acceptable carrier or diluent.

16. The pharmaceutical composition of claim 15, wherein the composition comprises at least one antibody, or an antigen-binding fragment thereof that binds specifically to toxin A of Clostridium difficile and at least one antibody, or an antigen-binding fragment thereof that binds specifically to toxin B of Clostridium difficile, wherein: a) the antibody or antigen-binding fragment thereof that binds specifically to toxin A comprises the three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) contained within any one of the heavy chain variable region (HCVR) amino acid sequences selected from the group consisting of SEQ ID NOs: 2, 98, 114, 130, 146 and 162; and the three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) contained within any one of the light chain variable region (LCVR) amino acid sequences selected from the group consisting of SEQ ID NOs: 10, 106, 122, 138, 154 and 170; and wherein b) the antibody or antigen-binding fragment thereof that binds specifically to toxin B comprises the three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within any one of the HCVR amino acid sequences selected from the group consisting of SEQ ID NOs: 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338 and 354; and the three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within any one of the LCVR amino acid sequences selected from the group consisting of SEQ ID NOs: 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346 and 362.

17. The pharmaceutical composition of claim 16, wherein the antibody or an antigen-binding fragment thereof that specifically binds toxin A of Clostridium difficile comprises a HCVR/LCVR amino acid sequence pair of SEQ ID NO: 146/154, and wherein the antibody or an antigen-binding fragment thereof that specifically binds toxin B of Clostridium difficile comprises a HCVR/LCVR amino acid sequence pair of SEQ ID NO: 274/282.

18. The pharmaceutical composition of claim 16, comprising: a) an isolated first antibody, or antigen-binding fragment thereof that specifically binds toxin A of Clostridium difficile, comprising a HCDR1 having the amino acid sequence of SEQ ID NO: 148, a HCDR2 having the amino acid sequence of SEQ ID NO: 150, a HCDR3 having the amino acid sequence of SEQ ID NO: 152, a LCDR1 having the amino acid sequence of SEQ ID NO: 156, a LCDR2 having the amino acid sequence of SEQ ID NO: 158, a LCDR3 having the amino acid sequence of SEQ ID NO: 160; b) an isolated second antibody, or antigen-binding fragment thereof that specifically binds toxin B of Clostridium difficile, comprising a HCDR1 having the amino acid sequence of SEQ ID NO: 276, a HCDR2 having the amino acid sequence of SEQ ID NO: 278, a HCDR3 having the amino acid sequence of SEQ ID NO: 280, a LCDR1 having the amino acid sequence of SEQ ID NO: 284, a LCDR2 having the amino acid sequence of SEQ ID NO: 286, a LCDR3 having the amino acid sequence of SEQ ID NO: 288; and c) a pharmaceutically acceptable carrier or diluent.

19. The pharmaceutical composition of claim 15, wherein the antibodies contained within the composition are effective at neutralizing toxins A and B from a hypervirulent strain of Clostridium difficile.

20. The pharmaceutical composition of claim 19, wherein the hypervirulent strain of Clostridium difficile is a BI/NAP1/027 strain.

21. The pharmaceutical composition of claim 20, wherein the BI/NAP1/027 strain is selected from VA5, VA17, 6336 and 6443.

22. A method for treating a patient suffering from a Clostridium difficile-associated condition or disease, or for treating at least one symptom or complication associated with the condition or disease, or for preventing the development of a Clostridium difficile-associated condition or disease in a patient at risk thereof, the method comprising administering to the patient an effective amount of the pharmaceutical composition of claim 15, wherein the Clostridium difficile-associated condition or disease is either prevented, or lessened in severity and/or duration, or at least one symptom or complication associated with the condition or disease is prevented, or ameliorated, or that the frequency and/or duration of, or the severity of recurrences, or relapses with Clostridium difficile is reduced.

23. The method of claim 22, wherein the at least one symptom or complication associated with the Clostridium difficile-associated condition or disease is selected from the group consisting of anorexia, abdominal pain, abdominal bloating, diarrhea with or without bleeding, dehydration, malnutrition, pseudomembranous colitis, complete or segmental colonic resection, fever and systemic infection (sepsis), death, relapse of the Clostridium difficile condition or disease, and rejection of a transplanted tissue or organ.

24. The method of claim 22, wherein the patient at risk of developing a Clostridium difficile-associated condition or disease is selected from the group consisting of an elderly patient 65 years old), a patient who is immunocompromised due to underlying illness or due to administration of immunosuppressive therapeutics, a patient who has some underlying medical condition that may pre-dispose them to acquiring a Clostridium difficile infection, a patient hospitalized for an extended period of time (at least one week), a patient who has been treated for an extended period of time(.gtoreq.14 days) with broad spectrum antibiotics, a cancer patient, a transplant patient, and a patient on therapy with agents such as but not limited to a proton pump inhibitor, or histamine H2 receptor inhibitor that are used for treatment of gastrointestinal diseases or conditions to reduce or treat gastric acidity, gastroesophageal reflux disease (GERD), stomach and small intestine ulcers, or heartburn.

25. The method of claim 24, wherein the cancer patient is undergoing treatment with an anti-cancer drug, or undergoing radiotherapy to treat a cancer.

26. The method of claim 24, wherein the transplant patient is a patient receiving a hematopoietic stem cell transplant, or a solid tissue or organ transplant.

27. The method of claim 26, wherein the transplant patient is being treated with an immunosuppressive drug, or any transplant rejection drug, or who is undergoing treatment with a drug regimen to prevent tissue or organ graft rejection following the transplant.

28. The method of claim 22, wherein the pharmaceutical composition is administered to the patient in combination with a second therapeutic agent.

29. The method of claim 28, wherein the second therapeutic agent is selected from the group consisting of a toxoid, a Clostridium difficile vaccine, an antibiotic, another different antibody to Clostridium difficile toxin A and/or B, and any other palliative therapy useful for ameliorating at least one symptom associated with a Clostridium difficile-associated condition or disease.

30. The method of claim 29, wherein the at least one symptom or complication associated with the Clostridium difficile-associated condition or disease is selected from the group consisting of anorexia, abdominal pain, abdominal bloating, diarrhea with or without bleeding, dehydration, malnutrition, pseudomembranous colitis, complete or segmental colonic resection, fever and systemic infection (sepsis), death, relapse of the Clostridium difficile condition or disease, and rejection of a transplanted tissue or organ.

31. An isolated antibody that interacts with, or binds to, an epitope within amino acid residues 468-863 of the carboxy terminal receptor binding domain of toxin A produced by Clostridium difficile, or an antigen binding fragment thereof, wherein the carboxy terminal receptor binding domain of toxin A comprises the amino acid sequence of SEQ ID NO: 375.

32. An isolated antibody that interacts with or binds to an epitope within the carboxy terminal receptor binding domain of toxin A produced by Clostridium difficile, or an antigen binding fragment thereof, wherein the epitope is selected from the group consisting of residues 468-488 of SEQ ID NO: 375, residues 510-530 of SEQ ID NO: 375, residues 602-610 of SEQ ID NO: 375, residues 644-703 of SEQ ID NO: 375, residues 724-794 of SEQ ID NO: 375, residues 799-814 of SEQ ID NO: 375 and residues 858-863 of SEQ ID NO: 375.

33. The isolated antibody of claim 32, comprising the HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 146/154.

34. A pharmaceutical composition comprising the isolated antibody of claim 32 and a second isolated antibody that interacts with, or binds to toxin B of Clostridium difficile and a pharmaceutically acceptable carrier or diluent.

35. The pharmaceutical composition of claim 34, wherein the second antibody that interacts with or binds to toxin B of Clostridium difficile comprises the HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 274/282.