Patent application title: ANTIBODIES AGAINST CLOSTRIDIUM DIFFICILE TOXINS AND USES THEREOF
Inventors:
IPC8 Class: AC07K1612FI
USPC Class:
1 1
Class name:
Publication date: 2021-09-02
Patent application number: 20210269511
Abstract:
Antibodies that specifically bind to toxins of C. difficile, antigen
binding portions thereof, and methods of making and using the antibodies
and antigen binding portions thereof are provided herein.Claims:
1. An isolated monoclonal antibody that binds to Clostridium difficile
(C. difficile) toxin A, or an antigen binding portion thereof, wherein
the antibody, or antigen binding portion thereof, comprises: (i) a heavy
chain variable region CDR1 comprising SEQ ID NO: 7; a heavy chain
variable region CDR2 comprising SEQ ID NO: 8; a heavy chain variable
region CDR3 comprising SEQ ID NO: 9; a light chain variable region CDR1
comprising SEQ ID NO: 16; a light chain variable region CDR2 comprising
SEQ ID NO: 17; and a light chain variable region CDR3 comprising SEQ ID
NO: 18; (ii) a heavy chain variable region CDR1 comprising SEQ ID NO: 10;
a heavy chain variable region CDR2 comprising SEQ ID NO: 11; a heavy
chain variable region CDR3 comprising SEQ ID NO: 12; a light chain
variable region CDR1 comprising SEQ ID NO: 19; a light chain variable
region CDR2 comprising SEQ ID NO: 20; and a light chain variable region
CDR3 comprising SEQ ID NO: 21; or (iii) a heavy chain variable region
CDR1 comprising SEQ ID NO: 13; a heavy chain variable region CDR2
comprising SEQ ID NO: 14; a heavy chain variable region CDR3 comprising
SEQ ID NO: 15; a light chain variable region CDR1 comprising SEQ ID NO:
22; a light chain variable region CDR2 comprising SEQ ID NO: 23; and a
light chain variable region CDR3 comprising SEQ ID NO: 24.
2. The isolated monoclonal antibody, or an antigen binding portion thereof, of claim 1, wherein the antibody, or antigen binding portion thereof, comprises a heavy chain variable region CDR1 comprising SEQ ID NO: 7, a heavy chain variable region CDR2 comprising SEQ ID NO: 8, a heavy chain variable region CDR3 comprising SEQ ID NO: 9, a light chain variable region CDR1 comprising SEQ ID NO: 16, a light chain variable region CDR2 comprising SEQ ID NO: 17, and a light chain variable region CDR3 comprising SEQ ID NO: 18.
3. The isolated monoclonal antibody, or an antigen binding portion thereof, of claim 1, wherein the antibody, or antigen binding portion thereof, comprises a heavy chain variable region CDR1 comprising SEQ ID NO: 10, a heavy chain variable region CDR2 comprising SEQ ID NO: 11, a heavy chain variable region CDR3 comprising SEQ ID NO: 12, a light chain variable region CDR1 comprising SEQ ID NO: 19, a light chain variable region CDR2 comprising SEQ ID NO: 20, and a light chain variable region CDR3 comprising SEQ ID NO: 21.
4. The isolated monoclonal antibody, or an antigen binding portion thereof, of claim 1, wherein the antibody, or antigen binding portion thereof, comprises a heavy chain variable region CDR1 comprising SEQ ID NO: 13, a heavy chain variable region CDR2 comprising SEQ ID NO: 14, a heavy chain variable region CDR3 comprising SEQ ID NO: 15, a light chain variable region CDR1 comprising SEQ ID NO: 22, a light chain variable region CDR2 comprising SEQ ID NO: 23, and a light chain variable region CDR3 comprising SEQ ID NO: 24.
5. An isolated monoclonal antibody that binds to Clostridium difficile (C. difficile) toxin A, or an antigen binding portion thereof, wherein the antibody, or antigen binding portion thereof, comprises a heavy chain variable region having an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 and/or a light chain variable region having an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.
6. The isolated monoclonal antibody, or an antigen binding portion thereof, of claim 5, wherein the antibody, or antigen binding portion thereof, comprises a heavy chain variable region having an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 1.
7. The isolated monoclonal antibody, or an antigen binding portion thereof, of claim 5, wherein the antibody, or antigen binding portion thereof, comprises a heavy chain variable region having an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 2.
8. The isolated monoclonal antibody, or an antigen binding portion thereof, of claim 5, wherein the antibody, or antigen binding portion thereof, comprises a heavy chain variable region having an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 3.
9. The isolated monoclonal antibody, or an antigen binding portion thereof, of claim 5, wherein the antibody, or antigen binding portion thereof, comprises a light chain variable region having an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 4.
10. The isolated monoclonal antibody, or an antigen binding portion thereof, of claim 5, wherein the antibody, or antigen binding portion thereof, comprises a light chain variable region having an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 5.
11. The isolated monoclonal antibody, or an antigen binding portion thereof, of claim 5, wherein the antibody, or antigen binding portion thereof, comprises a light chain variable region having an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 6.
12. The isolated monoclonal antibody, or an antigen binding portion thereof, of claim 5, wherein the antibody, or antigen binding portion thereof, comprises heavy and light chain variable regions comprising amino acid sequences at least 95% identical to the amino acid sequences set forth in SEQ ID NOs: 1 and 4, respectively.
13. The isolated monoclonal antibody, or an antigen binding portion thereof, of claim 5, wherein the antibody, or antigen binding portion thereof, comprises heavy and light chain variable regions comprising amino acid sequences at least 95% identical to the amino acid sequences set forth in SEQ ID NOs: 2 and 5, respectively.
14. The isolated monoclonal antibody, or an antigen binding portion thereof, of claim 5, wherein the antibody, or antigen binding portion thereof, comprises heavy and light chain variable regions comprising amino acid sequences at least 95% identical to the amino acid sequences set forth in SEQ ID NOs: 3 and 6, respectively.
15. The isolated monoclonal antibody, or antigen binding portion thereof, of claim 1, wherein the antibody is a human antibody, a humanized antibody or a chimeric antibody.
16. The antigen binding portion of claim 1, wherein the antigen binding portion is a Fab, Fab'2, ScFv, Fd, Fv or dAb.
17. The isolated monoclonal antibody of claim 1, wherein the antibody is an IgG1 or IgG3 isotype.
18. The isolated monoclonal antibody, or antigen binding portion thereof, of claim 1, wherein the K.sub.D of the antibody, or antigen binding portion thereof, is less than 20.times.10.sup.-6 M.
19. The isolated monoclonal antibody, or antigen binding portion thereof, of claim 1, wherein the antibody, or antigen binding portion thereof, neutralizes toxin A in vitro or in vivo.
Description:
RELATED INFORMATION
[0001] The application is a continuation of U.S. patent application Ser. No. 15/924,703, filed on Mar. 19, 2018, which is a continuation of U.S. patent application Ser. No. 14/926,706, filed on Oct. 29, 2015, which is a continuation of U.S. patent application Ser. No. 14/080,598, filed on Nov. 14, 2013, now U.S. Pat. No. 9,217,029, which is a divisional application of U.S. patent application Ser. No. 13/490,757, filed on Jun. 7, 2012, now U.S. Pat. No. 8,609,111, which is a continuation of U.S. patent application Ser. No. 12/533,501, filed on Jul. 31, 2009, now U.S. Pat. No. 8,236,311, which is a divisional application of U.S. patent application Ser. No. 11/051,453, filed on Feb. 4, 2005, now U.S. Pat. No. 7,625,559, which claims priority to U.S. provisional patent application No. 60/542,357, filed on Feb. 6, 2004, and U.S. provisional patent application No. 60/613,854, filed on Sep. 28, 2004, the entire contents both of which are hereby incorporated by reference.
[0002] The contents of any patents, patent applications, and references cited throughout this specification are hereby incorporated by reference in their entireties.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 16, 2020, is named MJI-001DV1CNDVCN3_Sequence_Listing.txt and is 99,987 bytes in size.
BACKGROUND OF THE INVENTION
[0004] Clostridium difficile (C. difficile) is a gram-positive bacterium that causes gastrointestinal disease in humans. C. difficile is the most common cause of infectious diarrhea in hospital patients, and is one of the most common nosocomial infections overall (Kelly et al., New Eng. J. Med., 330:257-62, 1994). In fact, disease associated with this pathogen may afflict as many as three million hospitalized patients per year in the United States (McFarland et al., New Eng. J. Med., 320:204-10, 1989; Johnson et al., Lancet, 336:97-100, 1990).
[0005] Treatment with antibiotics such as ampicillin, amoxicillin, cephalosporins, and clindamycin that disrupt normal intestinal flora can allow colonization of the gut with C. difficile and lead to C. difficile disease (Kelly and Lamont, Annu. Rev. Med., 49:375-90, 1998). The onset of C. difficile disease typically occurs four to nine days after antibiotic treatment begins, but can also occur after discontinuation of antibiotic therapy. C. difficile can produce symptoms ranging from mild to severe diarrhea and colitis, including pseudomembranous colitis (PMC), a severe form of colitis characterized by abdominal pain, watery diarrhea, and systemic illness (e.g., fever, nausea). Relapsing disease can occur in up to 20% of patients treated for a first episode of disease, and those who relapse are at a greater risk for additional relapses (Kelly and Lamont, Annu. Rev. Med., 49:375-90, 1998).
[0006] C. difficile disease is believed to be caused by the actions of two exotoxins, toxin A and toxin B, on gut epithelium. Both toxins are high molecular weight proteins (280-300 kDa) that catalyze covalent modification of Rho proteins, small GTP-binding proteins involved in actin polymerization, in host cells. Modification of Rho proteins by the toxins inactivates them, leading to depolymerization of actin filaments and cell death. Both toxins are lethal to mice when injected parenterally (Kelly and Lamont, Annu. Rev. Med., 49:375-90, 1998).
[0007] C. difficile disease can be diagnosed by assays that detect the presence or activity of toxin A or toxin B in stool samples, e.g., enzyme immunoassays. Cytotoxin assays can be used to detect toxin activity. To perform a cytotoxin assay, stool is filtered to remove bacteria, and the cytopathic effects of toxins on cultured cells are determined (Merz et al., J. Clin. Microbiol., 32:1142-47, 1994).
[0008] C. difficile treatment is complicated by the fact that antibiotics trigger C. difficile associated disease. Nevertheless, antibiotics are the primary treatment option at present. Antibiotics least likely to cause C. difficile associated disease such as vancomycin and metronidazole are frequently used. Vancomycin resistance evolving in other microorganisms is a cause for concern in using this antibiotic for treatment, as it is the only effective treatment for infection with other microorganisms (Gerding, Curr. Top. Microbiol. Immunol., 250:127-39, 2000). Probiotic approaches, in which a subject is administered non-pathogenic microorganisms that presumably compete for niches with the pathogenic bacteria, are also used. For example, treatment with a combination of vancomycin and Saccharomyces boulardii has been reported (McFarland et al., JAMA., 271(24):1913-8, 1994. Erratum in: JAMA, 272(7):518, 1994).
[0009] Vaccines have been developed that protect animals from lethal challenge in infectious models of disease (Torres et al., Infect. Immun. 63(12):4619-27, 1995). In addition, polyclonal antibodies have been shown to protect hamsters from disease when administered by injection or feeding (Giannasca et al., Infect. Immun. 67(2):527-38, 1999; Kink and Williams, Infect. Immun., 66(5):2018-25, 1998). Murine monoclonal antibodies have been isolated that bind to C. difficile toxins and neutralize their activities in vivo and in vitro (Corthier et al., Infect. Immun., 59(3):1192-5, 1991). There are some reports that human polyclonal antibodies containing toxin neutralizing antibodies can prevent C. difficile relapse (Salcedo et al., Gut., 41(3):366-70, 1997). Antibody response against toxin A has been correlated with disease outcome, indicating the efficacy of humoral responses in controlling infection. Individuals with robust toxin A ELISA responses had less severe disease compared to individuals with low toxin A antibody levels (Kyne et al., Lancet, 357(9251):189-93, 2001).
[0010] The individual role of toxin A and toxin B in disease pathogenesis, and the role of anti-toxin antibodies in protection from C. difficile disease are controversial and may depend on the host. In humans, the anti-toxin A antibody response has been correlated to disease outcome, suggesting a requirement for anti-toxin A response for protection. This observation is in contrast with reports of disease-causing C. difficile organisms that express only toxin B, implying that toxin B can contribute to disease in humans. These toxin A-negative strains can also cause disease in hamsters (Sambol et al., J. Infect. Dis., 183(12):1760-6, 2001).
SUMMARY OF THE INVENTION
[0011] This invention is based, in part, on the discovery that administration of antibodies against C. difficile toxin A to a subject can protect the subject from relapse of C. difficile-mediated disease in vivo. Administration of antibodies to one or both of toxin A and toxin B can prevent primary C. difficile-mediated disease. High affinity antibodies against C. difficile toxins can be produced, e.g., in mice, such as transgenic mice expressing human immunoglobulin gene segments. These antibodies can neutralize toxin cytotoxicity in vitro, and neutralize toxin enterotoxicity in vivo. Antibodies that recognize toxin A and/or toxin B can inhibit and protect from disease in vivo.
[0012] In one aspect, the invention features isolated human monoclonal antibodies or antigen binding portions thereof that specifically bind to an exotoxin of Clostridium difficile (C. difficile). In certain embodiments, the antibodies or antigen binding portions thereof specifically bind to C. difficile toxin A (toxin A). In other embodiments, the antibody or antigen binding portions thereof specifically bind to C. difficile toxin B (toxin B). In other embodiments, the antibodies or antigen binding portions thereof specifically bind to both toxin A and toxin B.
[0013] In certain embodiments, the antibodies or antigen binding portions thereof neutralize toxin A in vitro, inhibit binding of toxin A to mammalian cells, and/or inhibit C. difficile-mediated disease in vivo.
[0014] In various embodiments, the antibodies or antigen binding portions thereof have one or more of the following characteristics: when administered to a mouse, they protect the mouse against administration of a C. difficile toxin in an amount that would be fatal to a control mouse not administered the antibody; protect from or inhibit C. difficile-mediated colitis, antibiotic-associated colitis, or pseudomembranous colitis (PMC) in a subject; protect from or inhibit diarrhea in a subject; and/or inhibit relapse of C. difficile-mediated disease.
[0015] The antibodies or antigen binding portions thereof can specifically bind to an epitope within the N-terminal half of toxin A, e.g., an epitope between amino acids 1-1256 of toxin A. In other embodiments, the antibodies or antigen binding portions thereof specifically bind to an epitope within the C-terminal receptor binding domain of toxin A, e.g., an epitope between amino acids 1852-2710 of toxin A, or an epitope between amino acids 659-1852, e.g., an epitope within amino acid residues 900-1852, 900-1200, or 920-1033 of toxin A. In other embodiments, the antibodies or antigen binding portions thereof specifically bind an epitope within amino acids 1-600, 400-600, or 415-540 of toxin A. Other particular antibodies or antigen binding portions thereof, can specifically bind to an epitope within amino acid residues 1-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 900-1000, 1100-1200, 1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1800-1900, 1900-200, 2100-2200 or 2200-2300, 2300-2400, 2400-2500, 2500-2600, 2600-2710 of toxin A, or any interval, portion or range thereof.
[0016] In certain embodiments, the antibodies or antigen binding portions thereof specifically bind to toxin A with a K.sub.D of less than about 20.times.10.sup.-6 M. In a particular embodiment, the antibody, or antigen binding portion thereof, specifically binds to toxin A with a K.sub.D of less than about 10.times.10.sup.-7 M, less than about 10.times.10.sup.-8 M, less than about 10.times.10.sup.-9 M, or less than about 10.times.10.sup.-10 M. In other particular embodiments, the antibody, or antigen binding portion thereof, specifically binds to toxin A with a K.sub.D of less than about 50.times.10.sup.-10 M, less than about 20.times.10.sup.-10 M, less than about 15.times.10.sup.-10 M, less than about 8.times.10.sup.-10 M, or less than about 5.times.10.sup.-10 M.
[0017] In various other embodiments, the antibodies or antigen binding portions thereof include a variable heavy chain region including an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or more identical to a variable heavy chain region amino acid sequence of the antibody produced by clone 3D8 (SEQ ID NO:1), 1B11 (SEQ ID NO:2), or 3H2 (SEQ ID NO:3).
[0018] In certain embodiments, the antibodies or antigen binding portions thereof include a variable light chain region comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or more identical to a variable light chain region amino acid sequence of the antibody produced by clone 3D8 (SEQ ID NO:4), 1B11 (SEQ ID NO:5), or 3H2 (SEQ ID NO:6).
[0019] In certain embodiments, the antibodies or antigen binding portions thereof each include both a variable heavy chain region including an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or more identical to a variable heavy chain region amino acid sequence of the antibody produced by clone 3D8 (SEQ ID NO:1), 1B11 (SEQ ID NO:2), or 3H2 (SEQ ID NO:3), and a variable light chain region including an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or more identical to a variable light chain amino acid sequence of clone 3D8 (SEQ ID NO:4), 1B11 (SEQ ID NO:5), or 3H2 (SEQ ID NO:6).
[0020] In various embodiments, the antibodies or antigen binding portions thereof specifically bind to an epitope that overlaps with an epitope bound by an antibody produced by clone 3D8, 1B11, or 3H2 and/or compete for binding to toxin A with an antibody produced by clone 3D8, 1B11, or 3H2.
[0021] A variable heavy chain region of the antibodies or antigen binding portions thereof can include one or more complementarity determining regions (CDRs) that are at least 80%, 85%, 90%, 95%, or 99%, or more identical to a CDR of the antibody produced by clone 3D8 (SEQ ID NOs:7-9), 1B11 (SEQ ID NOs:10-12), or 3H2 (SEQ ID NOs:13-15) (also shown in Table 1).
[0022] A variable light chain region of the antibodies or antigen binding portions thereof can include one or more CDRs that are at least 80%, 85%, 90%, 95%, or 99%, or more identical to a CDR of a variable light chain region of the antibody produced by clone 3D8 (SEQ ID NOs:16-18), 1B11 (SEQ ID NOs:19-21), or 3H2 (SEQ ID NOs:22-24) (also shown in Table 2).
[0023] A variable heavy chain region of the antibodies or antigen binding portions thereof can include one or more complementarity determining regions (CDRs) that are at least 80%, 85%, 90%, 95%, or 99%, or more identical to a CDR of the antibody produced by clone 3D8 (SEQ ID NOs:7-9), 1B11 (SEQ ID NOs:10-12), or 3H2 (SEQ ID NOs:13-15), and a variable light chain region of the antibodies or antigen binding portions thereof can include one or more CDRs that are at least 80%, 85%, 90%, 95%, 99%, or more identical to a CDR of a variable light chain region of the antibody produced by clone 3D8 (SEQ ID NOs:16-18), 1B11 (SEQ ID NOs:19-21), or 3H2 (SEQ ID NOs:22-24).
[0024] A variable heavy chain region of the antibodies or antigen binding portions thereof can include three CDRs that are at least 80%, 85%, 90%, 95%, or 99%, or more identical to a CDR of a variable heavy chain region of the antibody produced by clone 3D8 (SEQ ID NOs:7-9), 1B11 (SEQ ID NOs:10-12), or 3H2 (SEQ ID NOs:13-15).
[0025] In some embodiments, a variable light chain region of the antibodies or antigen binding portions thereof includes three CDRs that are at least 80%, 85%, 90%, 95%, 99%, or more identical to a CDR of a variable light chain region of the antibody produced by clone 3D8 (SEQ ID NOs:16-18), 1B11 (SEQ ID NOs:19-21), or 3H2 (SEQ ID NOs:22-24).
[0026] In some embodiments, a variable light chain region of the antibodies or antigen binding portions thereof includes one or more CDRs that are at least 80%, 85%, 90%, 95%, or 99%, or more identical to a CDR of a variable light chain region of the antibody produced by clone 3D8 (SEQ ID NOs:16-18), 1B11 (SEQ ID NOs:19-21), or 3H2 (SEQ ID NOs:22-24), and a variable heavy chain region of the antibodies or antigen binding portions thereof includes three CDRs that are at least 80%, 85%, 90%, 95%, or 99%, or more identical to a CDR of a variable heavy chain region of the antibody produced by clone 3D8 (SEQ ID NOs:7-9), 1B11 (SEQ ID NOs:10-12), or 3H2 (SEQ ID NOs:13-15). The variable light chain region can include three CDRs that are at least 80%, 85%, 90%, 95%, or 99%, or more identical to a CDR of a variable light chain region of the antibody produced by clone 3D8 (SEQ ID NOs:16-18), 1B11 (SEQ ID NOs:19-21), or 3H2 (SEQ ID NOs:22-24).
[0027] In certain embodiments, a variable heavy chain region of the antibodies or antigen binding portions thereof includes three CDRs that are identical to a CDR of a variable heavy chain region of the antibody produced by clone 3D8 (SEQ ID NOs:7-9), 1B11 (SEQ ID NOs:10-12), or 3H2 (SEQ ID NOs:13-15), and a variable light chain region of the antibodies or antigen binding portions thereof includes three CDRs that are identical to a CDR of a variable light chain region of the antibody produced by clone 3D8 (SEQ ID NOs:16-18), 1B11 (SEQ ID NOs:19-21), or 3H2 (SEQ ID NOs:22-24), e.g., a variable light chain region and variable heavy chain region of the antibody or antigen binding portion thereof are identical to a variable light chain region and variable heavy chain region of the antibody produced by clone 3D8 (SEQ ID NO:1, SEQ ID NO:4), 1B11 (SEQ ID NO:2, SEQ ID NO:5), or 3H2 (SEQ ID NO:3, SEQ ID NO:6).
[0028] In some embodiments, the antibodies or antigen binding portions thereof neutralize toxin B in vitro, inhibit binding of toxin B to mammalian cells, and/or neutralize toxin B in vivo.
[0029] In some embodiments, the antibodies or antigen binding portions thereof specifically bind to an epitope in a C-terminal portion of toxin B (e.g., between amino acids 1777-2366 of toxin B). Other particular antibodies or antigen binding portions thereof, can specifically bind to an epitope within amino acid residues 1-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 900-1000, 1100-1200, 1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1800-1900, 1900-200, 2100-2200 or 2200-2366 of toxin B, or any interval, portion or range thereof.
[0030] In certain embodiments, the antibodies or antigen binding portions thereof specifically bind to toxin B with a K.sub.D of less than about 20.times.10.sup.-6 M. In a particular embodiment, the antibody, or antigen binding portion thereof, specifically binds to toxin B with a K.sub.D of less than about 10.times.10.sup.-7 M, less than about 10.times.10.sup.-8 M, less than about 10.times.10.sup.-9 M, or less than about 10.times.10.sup.-10 M. In other particular embodiments, the antibody, or antigen binding portion thereof, specifically binds to toxin B with a K.sub.D of less than about 50.times.10.sup.-10 M, less than about 20.times.10.sup.-10 M, less than about 15.times.10.sup.-10 M, less than about 8.times.10.sup.-10 M, or less than about 5.times.10.sup.-10 M.
[0031] In various other embodiments, the antibodies or antigen binding portions thereof include a variable heavy chain region including an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or more identical to a variable heavy chain region amino acid sequence of the antibody produced by clone 124-152 (i.e., the amino acid sequence shown in SEQ ID NO:54), 2A11, or 1 G10.
[0032] In certain embodiments, the antibodies or antigen binding portions thereof include a variable light chain region comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or more identical to a variable heavy chain region amino acid sequence of the antibody produced by clone 124-152 (i.e., the amino acid sequence shown in SEQ ID NO:58), 2A11, or 1 G10.
[0033] In certain embodiments, the antibodies or antigen binding portions thereof each include both a variable heavy chain region including an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99%, or more identical to a variable heavy chain region amino acid sequence of the antibody produced by clone 124-152 (i.e., the amino acid sequence shown in SEQ ID NO:54), 2A11, or 1 G10, and a variable light chain region including an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or more identical to a variable light chain amino acid sequence of the antibody produced by clone 124-152 (i.e., the amino acid sequence shown in SEQ ID NO:58), 2A11, or 1 G10.
[0034] In various embodiments, the antibodies or antigen binding portions thereof specifically bind to an epitope that overlaps with an epitope bound by an antibody produced by clone 124-152, 2A11, or 1 G10 and/or compete for binding to toxin B with an antibody produced by clone 124-152, 2A11, or 1 G10.
[0035] A variable heavy chain region of the antibodies or antigen binding portions thereof can include one or more complementarity determining regions (CDRs) that are at least 80%, 85%, 90%, 95%, or 99%, or more identical to a CDR of the antibody produced by clone 124-152 (SEQ ID NOs: 62, 64, or 66), 2A11, or 1 G10 (Table 3).
[0036] A variable light chain region of the antibodies or antigen binding portions thereof can include one or more complementarity determining regions (CDRs) that are at least 80%, 85%, 90%, 95%, or 99%, or more identical to a CDR of the antibody produced by clone 124-152 (SEQ ID NOs: 68, 70, or 72), 2A11, or 1 G10 (Table 4).
[0037] A variable heavy chain region of the antibodies or antigen binding portions thereof can include one or more complementarity determining regions (CDRs) that are at least 80%, 85%, 90%, 95%, or 99%, or more identical to a CDR of the antibody produced by clone 124-152 (SEQ ID NOs: 62, 64, or 66), 2A11, or 1 G10, and a variable light chain region of the antibodies or antigen binding portions thereof can include one or more CDRs that are at least 80%, 85%, 90%, 95%, 99%, or more identical to a CDR of a variable light chain region of the antibody produced by clone 124-152 (SEQ ID NOs: 68, 70, or 72), 2A11, or 1 G10.
[0038] A variable heavy chain region of the antibodies or antigen binding portions thereof can include three CDRs that are at least 80%, 85%, 90%, 95%, or 99%, or more identical to a CDR of a variable heavy chain region of the antibody produced by clone 124-152 (SEQ ID NOs: 62, 64, or 66), 2A11, or 1 G10.
[0039] In certain embodiments, the variable light chain region of the antibodies or antigen binding portions thereof includes three CDRs that are at least 80%, 85%, 90%, 95%, 99%, or more identical to a CDR of a variable light chain region of the antibody produced by clone 124-152 (SEQ ID NOs: 68, 70, or 72), 2A11, or 1 G10.
[0040] In other embodiments, the variable light chain region of the antibodies or antigen binding portions thereof includes one or more CDRs that are at least 80%, 85%, 90%, 95%, or 99%, or more identical to a CDR of a variable light chain region of the antibody produced by clone 124-152 (SEQ ID NOs: 68, 70, or 72), 2A11, or 1 G10, and a variable heavy chain region of the antibodies or antigen binding portions thereof includes three CDRs that are at least 80%, 85%, 90%, 95%, or 99%, or more identical to a CDR of a variable heavy chain region of the antibody produced by clone 124-152 (SEQ ID NOs: 62, 64, or 66), 2A11, or 1 G10. The variable light chain region can include three CDRs that are at least 80%, 85%, 90%, 95%, or 99%, or more identical to a CDR of a variable light chain region of the antibody produced by clone 124-152 (SEQ ID NOs: 68, 70, or 72), 2A11, or 1 G10.
[0041] In still other embodiments, the variable heavy chain region of the antibodies or antigen binding portions thereof includes three CDRs that are identical to a CDR of a variable heavy chain region of the antibody produced by clone 124-152 (SEQ ID NOs: 62, 64, or 66), 2A11, or 1 G10, and a variable light chain region of the antibodies or antigen binding portions thereof includes three CDRs that are identical to a CDR of a variable light chain region of the antibody produced by clone 124-152 (SEQ ID NOs: 68, 70, or 72), 2A11, or 1 G10, e.g., a variable light chain region and variable heavy chain region of the antibody or antigen binding portion thereof are identical to a variable light chain region and variable heavy chain region of the antibody produced by clone 124-152 (SEQ ID NOs: 62, 64, or 66), 2A11, or 1 G10.
[0042] The antibodies or antigen binding portions thereof can be full-length antibodies, can include an effector domain, e.g., an Fc domain, can be immunoglobulin gamma isotype antibodies, single-chain antibodies, or Fab fragments. The antibodies or antigen binding portions thereof can further include a pharmaceutically acceptable carrier and/or a label.
[0043] In various embodiments, compositions including the antibodies or antigen binding portions thereof are free of other human polypeptides (e.g., they contain less than 5% human polypeptides other than the antibodies or antigen binding portions thereof).
[0044] In yet another aspect, the invention features compositions including: (a) an isolated human monoclonal antibody or antigen binding portion thereof that specifically binds to an exotoxin of C. difficile; and (b) a polyclonal antibody or antigen binding portion thereof that specifically binds to an exotoxin of C. difficile.
[0045] In one embodiment, the human monoclonal antibody or antigen binding portion thereof specifically binds to C. difficile toxin A, and the polyclonal antibody or antigen binding portion thereof specifically binds to C. difficile toxin B. In one embodiment, the human monoclonal antibody or antigen binding portion thereof specifically binds to C. difficile toxin B, and the polyclonal antibody or antigen binding portion thereof specifically binds to C. difficile toxin A. The antibodies can include other features described herein.
[0046] In another aspect, the invention features isolated human monoclonal antibodies or antigen binding portions thereof that specifically bind to an exotoxin of Clostridium difficile (C. difficile), wherein the antibodies: (a) include a heavy chain variable region that is the product of or derived from a human VH 3-33 gene; and/or (b) include a light chain variable region that is the product of or derived from a human V.kappa. gene selected from the group consisting of V.kappa. L19, V.kappa. L6 and V.kappa. L15. The antibodies or antigen binding portions thereof can include other features described herein.
[0047] In another aspect, the invention features isolated human monoclonal antibodies or antigen binding portions thereof that specifically bind to an exotoxin of Clostridium difficile (C. difficile), wherein the antibodies: (a) include a heavy chain variable region that is the product of or derived from a human VH 5-51 gene; and/or (b) include a light chain variable region that is the product of or derived from a human V.kappa. A27 gene. The antibodies or antigen binding portions thereof also can include other features described herein.
[0048] In another aspect, the invention features isolated polypeptides that include an antigen binding portion of an antibody produced by hybridoma clone 3D8, 1B11, or 3H2 (also referred to herein as "3D8", "1B11", and "3H2").
[0049] In another aspect, the invention features isolated polypeptides that include an antigen binding portion of an antibody produced by hybridoma clone 124-152, 2A11, or 1 G10 (also referred to herein as "124-152", "2A11", and "1 G10").
[0050] In another aspect, the invention features isolated monoclonal antibodies or antigen binding portions thereof that specifically bind to an exotoxin of C. difficile, neutralize the toxin, inhibit, and/or protect from C. difficile-mediated disease. In one embodiment, the antibodies or antigen binding portions thereof are mammalian (e.g., human) antibodies or antigen binding portions thereof. The antibodies or antigen binding portions thereof can include other features described herein.
[0051] In another aspect, the invention features compositions including: (a) an isolated human monoclonal antibody or antigen binding portion thereof that specifically binds to C. difficile toxin A; and (b) an isolated human monoclonal antibody or antigen binding portion thereof that specifically binds to C. difficile toxin B.
[0052] In another aspect, the invention features isolated nucleic acids including a sequence encoding polypeptides at least 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NOs:1, 2, 3, 4, 5, or 6; e.g., wherein the nucleic acid sequence is at least 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NOs:38, 39, 40, 35, 36, or 37. The invention also features expression vectors including a nucleic acid encoding a polypeptide at least 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NOs:1, 2, 3, 4, 5, or 6; e.g., wherein the nucleic acid sequence is at least 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NOs:38, 39, 40, 35, 36, or 37, as well as host cells, e.g., bacterial cells, e.g., E. coli cells, including a nucleic acid encoding a polypeptide at least 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NOs:1, 2, 3, 4, 5, or 6; e.g., wherein the nucleic acid sequence is at least 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NOs:38, 39, 40, 35, 36, or 37.
[0053] In another aspect, the invention features isolated nucleic acids including a sequence encoding a polypeptide that is at least 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NOs: 54, 56, 58, or 60, for example, wherein the nucleic acid sequence is at least 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NOs: 55, 57, 59, or 61. The invention also features expression vectors including a nucleic acid encoding a polypeptide at least 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NOs: 54, 56, 58, or 60, for example, wherein the nucleic acid sequence is at least 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NOs: 55, 57, 59, or 61. The invention also provides host cells, e.g., bacterial cells, e.g., E. coli cells, that include a nucleic acid encoding a polypeptide that is at least 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NOs: 54, 56, 58, or 60, for example, wherein the nucleic acid sequence is at least 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NOs: 55, 57, 59, or 61.
[0054] The host cells can also be eukaryotic cells, e.g., yeast cells, mammalian cells, e.g., Chinese hamster ovary (CHO) cells, NSO cells, or myeloma cells.
[0055] In another aspect, the invention features kits including an isolated human monoclonal antibody or antigen binding portion thereof that specifically binds to an exotoxin of Clostridium difficile (C. difficile), e.g., an antibody or antigen binding portion thereof described herein. The kit can include instructions for use in preventing or treating C. difficile-mediated disease.
[0056] The kit can further include a polyclonal antibody or antigen binding portion thereof that specifically binds an exotoxin of C. difficile. In one embodiment, the human monoclonal antibody or antigen binding portion thereof specifically binds to C. difficile toxin A. In one embodiment, the polyclonal antibody or antigen binding portion thereof specifically binds to C. difficile toxin B.
[0057] In another aspect, the invention features kits including: (a) an isolated human monoclonal antibody that specifically binds to C. difficile toxin A; and (b) an isolated human monoclonal antibody that specifically binds to C. difficile toxin B.
[0058] The invention also features methods of treating C. difficile disease in a subject by administering to the subject an isolated human monoclonal antibody or antigen binding portion thereof that specifically binds to an exotoxin of Clostridium difficile (C. difficile) in an amount effective to inhibit C. difficile disease, e.g., C. difficile-mediated colitis, antibiotic-associated colitis, C. difficile-mediated pseudomembranous colitis (PMC), or diarrhea, or relapse of C. difficile-mediated disease. The antibody or antigen binding portion thereof can be administered, e.g., intravenously, intramuscularly, or subcutaneously, to the subject.
[0059] The antibody or antigen binding portion thereof can be administered alone or in combination with another therapeutic agent, e.g., a second human monoclonal antibody or antigen binding portion thereof. In one example, the antibody or antigen binding portion thereof specifically binds to C. difficile toxin A, and the second human monoclonal antibody or antigen binding portion thereof specifically binds to C. difficile toxin B. In another example, the second agent is an antibiotic, e.g., vancomycin or metronidazole. The second agent can be polyclonal gamma-globulin (e.g., human gamma-globulin).
[0060] In a particular embodiment, an antibody or antigen binding portion thereof is administered which includes a variable light chain region and a variable heavy chain region identical to the variable light chain region and variable heavy chain region of the antibody produced by clone 3D8 (i.e., including a variable light chain region sequence identical to SEQ ID NO:4 and a variable heavy chain region sequence identical to SEQ ID NO:1.
[0061] In another embodiment, this antibody or antigen binding portion thereof is administered in combination with an antibody or antigen binding portion thereof which includes a variable light chain region and a variable heavy chain region identical to the variable light chain region and variable heavy chain region of the antibody produced by clone 124-152 (i.e., including a variable light chain region sequence identical to SEQ ID NO:58 and a variable heavy chain region sequence identical to SEQ ID NO:54).
[0062] In yet another embodiment, an antibody or antigen binding portion produced by clone 3D8 (i.e., including a variable light chain region sequence identical to SEQ ID NO:4 and a variable heavy chain region sequence identical to SEQ ID NO:1), is administered in combination with an antibody or antigen binding portion thereof produced by clone 124-152 (i.e., including a variable light chain region sequence identical to SEQ ID NO:58 and a variable heavy chain region sequence identical to SEQ ID NO:54).
[0063] In another aspect, the invention features methods for making an antibody or antigen binding portion thereof that specifically binds to an exotoxin of C. difficile, by immunizing a transgenic non-human animal having a genome comprising a human heavy chain transgene and a human light chain transgene with a composition that includes an inactivated exotoxin, and isolating an antibody from the animal. The exotoxin can be inactivated, for example, by treatment with UDP-dialdehyde or by mutation (e.g., using recombinant methods). The method can further include evaluating binding of the antibody to the exotoxin.
[0064] The invention also features methods for making a human monoclonal antibody or antigen binding portion thereof by providing a nucleic acid encoding a human monoclonal antibody or antigen binding portion thereof that specifically binds to an exotoxin of C. difficile, and expressing the nucleic acid in a host cell.
[0065] In yet another aspect, the invention features a hybridoma or transfectoma including a nucleic acid encoding antigen binding portions (e.g., CDRs, or variable regions) of the antibody produced by clone 3D8, 1B11, or 3H2.
[0066] In yet another aspect, the invention features a hybridoma or transfectoma including a nucleic acid encoding antigen binding portions (e.g., CDRs, or variable regions) of the antibody produced by clone 124-152, 2A11, or 1 G10.
[0067] In addition, the invention features a method for making a hybridoma that expresses an antibody that specifically binds to an exotoxin of C. difficile by immunizing a transgenic non-human animal having a genome that includes a human heavy chain transgene and a human light chain transgene, with a composition that includes the exotoxin, wherein the toxin is inactivated; isolating splenocytes from the animal; generating hybridomas from the splenocytes; and selecting a hybridoma that produces an antibody that specifically binds to the exotoxin.
[0068] Treatment of humans with human monoclonal antibodies offers several advantages. For example, the antibodies are likely to be less immunogenic in humans than non-human antibodies. The therapy is rapid; toxin inactivation can occur as soon as the antibody reaches sites of infection and directly neutralizes the disease-causing toxin(s). Human antibodies localize to appropriate sites in humans more efficiently than non-human antibodies. Furthermore, the treatment is specific for C. difficile, and is unlikely to disrupt normal gut flora, unlike traditional antibiotic therapies.
[0069] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0070] FIG. 1 is a table listing the amino acid sequences of the VH and VL chains encoded by mRNA sequences from each clone. Lowercase letters represent amino acids in the leader peptide. CDRs are underlined. Clone 3D8, which expresses 6 unique light chain V regions, only expressed the group I amino acid sequence.
[0071] FIG. 2A is a representation of the amino acid and nucleic acid sequences of the VL chain expressed by clone 3D8. The V-segment and J-segment genes are listed above the amino acid and nucleic acid sequences. The CDRs are overlined.
[0072] FIG. 2B is a representation of the amino acid and nucleic acid sequences of the VH chain expressed by clone 3D8. The V-segment, D-segment and J-segment genes are listed above the amino acid and nucleic acid sequences. The CDRs are overlined.
[0073] FIG. 3A is a representation of the amino acid and nucleic acid sequences of the VL chain expressed by clone 1B11. The V-segment and J-segment genes are listed above the amino acid and nucleic acid sequences. The CDRs are overlined.
[0074] FIG. 3B is a representation of the amino acid and nucleic acid sequences of the VH chain expressed by clone 1B11. The V-segment, D-segment, and J-segment genes are listed above the amino acid and nucleic acid sequences. The CDRs are overlined.
[0075] FIG. 4A is a representation of the amino acid and nucleic acid sequences of the VL chain expressed by clone 33.3H2 (referred to herein as 3H2; 33.3H2 and 3H2 are used interchangeably herein). The V-segment and J-segment genes are listed above the amino acid and nucleic acid sequences. The CDRs are overlined.
[0076] FIG. 4B is a representation of the amino acid and nucleic acid sequences of the VH chain expressed by clone 33.3H2. The V-segment and J-segment genes are listed above the amino acid and nucleic acid sequences. The CDRs are overlined.
[0077] FIG. 5 is a graph depicting the results of ELISA assays, which measured binding of anti-toxin A monoclonal antibodies to toxin A.
[0078] FIG. 6A and FIG. 6B are a set of graphs depicting results of in vitro neutralization assays in the presence and absence of anti-toxin A monoclonal antibodies. FIG. 6A depicts results for assays performed with IMR-90 cells. FIG. 6B depicts results for assays performed with T-84 cells.
[0079] FIG. 7 is a schematic representation of the toxin A polypeptide, indicating fragments that were analyzed for epitope mapping studies.
[0080] FIG. 8A and FIG. 8B are schematic representations of toxin A fragments analyzed for epitope mapping studies.
[0081] FIG. 9 is a table listing the results of in vivo assays to determine mouse protection from lethal challenge with toxin A by anti-toxin A monoclonal antibodies.
[0082] FIG. 10 is a graph depicting the results of mouse ileal loop fluid accumulation assays to measure efficacy of anti-toxin antibody neutralization in vivo.
[0083] FIG. 11A is a schematic diagram of the timeline of administration of various agents to hamsters in a hamster relapse model.
[0084] FIG. 11B is a graph depicting the results of the assays as the percentage of hamsters surviving clindamycin treatment followed by C. difficile challenge.
[0085] FIG. 12 is a graph depicting results of hamster relapse assays as the percentage of hamsters surviving clindamycin treatment followed by C. difficile challenge.
[0086] FIG. 13 is a graph depicting results of assays in which in vitro neutralization of toxin A and toxin B was measured in the presence and absence of polyclonal antisera from goats immunized with toxoid B. "G330" refers to samples in which sera from goat #330 were tested. "G331" refers to samples in which sera from goat #331 were tested.
[0087] FIG. 14 is a schematic diagram of the timeline of administration of various agents to hamsters in a hamster relapse model.
[0088] FIG. 15 is a graph depicting the results of hamster relapse assays as the percentage of hamsters surviving clindamycin treatment followed by C. difficile challenge. Hamsters were treated with vancomycin, vancomycin and 3D8, vancomycin and antisera from goat #331, or vancomycin, 3D8, and antisera from goat #331.
[0089] FIG. 16 is a graph depicting the results of hamster relapse assays as the percentage of healthy animals after clindamycin treatment followed by C. difficile challenge. "Goat 331" refers to antisera from goat #331.
[0090] FIG. 17 is a graph depicting the results of hamster relapse assays as the percentage of hamsters surviving clindamycin treatment followed by C. difficile challenge. Hamsters were immunized with a fragment of toxin B prior to clindamycin treatment. Hamsters were treated with vancomycin, vancomycin and 3D8, or received no treatment.
[0091] FIG. 18 is a graph depicting the results of hamster relapse assays as the percentage of healthy animals after clindamycin treatment followed by C. difficile challenge. Hamsters were immunized with a fragment of toxin B prior to clindamycin treatment.
[0092] FIG. 19 is a schematic diagram of the timeline of administration of various agents to hamsters in a C. difficile direct challenge model. "331" refers to antisera from goat #331. "Clinda" refers to treatment with clindamycin.
[0093] FIG. 20 is a graph depicting the results of direct challenge assays as the percentage of hamsters surviving direct C. difficile challenge.
[0094] FIG. 21 is a graph depicting the results of direct challenge assays as the percentage of healthy animals after direct challenge with C. difficile.
[0095] FIG. 22 is a representation of the amino acid sequence of C. difficile toxin A.
[0096] FIG. 23 is a representation of the amino acid sequence of C. difficile toxin B.
[0097] FIG. 24 is a graph depicting the results of primary challenge assays as the percentage of hamsters surviving direct C. difficile challenge.
[0098] FIG. 25 is a graph depicting the results of primary challenge assays as the percentage of hamsters surviving direct C. difficile challenge.
[0099] FIG. 26 is a graph depicting the results of primary challenge assays as the percentage of hamsters surviving direct C. difficile challenge.
[0100] FIG. 27 is a graph depicting results of assays in which in vitro neutralization of toxin A and toxin B was measured in the presence of monoclonal antibodies to toxin B or goat polyclonal sera against toxin B.
[0101] FIG. 28 is a representation of the amino acid and nucleic acid sequences of the VH chain expressed by clone 124-152. The V-segment, D-segment and J-segment genes are listed above the amino acid and nucleic acid sequences. The CDRs are overlined.
[0102] FIG. 29 is a representation of the amino acid and nucleic acid sequences of the VL chain expressed by clone 124-152. The V-segment and J-segment genes are listed above the amino acid and nucleic acid sequences. The CDRs are overlined.
[0103] FIG. 30 is a representation of the amino acid and related germline sequence of the VH chain expressed by clone 124-152. The V-segment, D-segment and J-segment genes are listed above the amino acid sequences. The CDRs are overlined.
[0104] FIG. 31 is a representation of the amino acid and related germline sequences of the VL chain expressed by clone 124-152. The V-segment and J-segment genes are listed above the amino acid sequences. The CDRs are overlined.
[0105] FIG. 32 is a schematic representation of the toxin B polypeptide, indicating fragments that were analyzed for epitope mapping studies.
[0106] Like reference symbols in the various drawings indicate like elements.
DETAILED DESCRIPTION OF THE INVENTION
[0107] In order to provide a clear understanding of the specification and claims, the following definitions are conveniently provided below.
Definitions
[0108] The term "toxin A" refers to the toxin A protein encoded by C. difficile. The amino acid sequence of C. difficile toxin A (SEQ ID NO:41) is provided in GenBank.RTM. under accession number A37052, version GI 98593 (see also FIG. 22). "Toxin B" refers to the toxin B protein encoded by C. difficile. The amino acid sequence of C. difficile toxin B (SEQ ID NO: 42) is provided in GenBank.RTM. under accession number S70172, version GI 7476000 (see also FIG. 23). "Protein" is used interchangeably with "polypeptide."
[0109] An "anti-C. difficile antibody" is an antibody that interacts with (e.g., binds to) a protein or other component produced by C. difficile bacteria. An "anti-toxin antibody" is an antibody that interacts with a toxin produced by C. difficile (e.g., toxin A or toxin B). An anti-toxin protein antibody may bind to an epitope, e.g., a conformational or a linear epitope, or to a fragment of the full-length toxin protein.
[0110] A "human antibody," is an antibody that has variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies described herein 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).
[0111] An anti-toxin antibody, or antigen binding portion thereof, can be administered alone or in combination with a second agent. The subject can be a patient infected with C. difficile, or having a symptom of C. difficile-associated disease ("CDAD"; e.g., diarrhea, colitis, abdominal pain) or a predisposition towards C. difficile-associated disease (e.g., undergoing treatment with antibiotics, or having experienced C. difficile-associated disease and at risk for relapse of the disease). The treatment can be to cure, heal, alleviate, relieve, alter, remedy, ameliorate, palliate, improve, or affect the infection and the disease associated with the infection, the symptoms of the disease, or the predisposition toward the disease.
[0112] An amount of an anti-toxin antibody effective to treat a CDAD, or a "therapeutically effective amount," is an amount of the antibody that is effective, upon single or multiple dose administration to a subject, in inhibiting CDAD in a subject. A therapeutically effective amount of the antibody or antibody fragment may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion is outweighed by the therapeutically beneficial effects. The ability of an antibody to inhibit a measurable parameter can be evaluated in an animal model system predictive of efficacy in humans. For example, the ability of an anti-toxin antibody to protect mice from lethal challenge with C. difficile can predict efficacy in humans. Other animal models predictive of efficacy are described herein, such as the intestinal ligation model described in the Examples. Alternatively, this property of an antibody or antibody composition can be evaluated by examining the ability of the compound to modulate, such modulation in vitro by assays known to the skilled practitioner. In vitro assays include binding assays, such as ELISA, and neutralization assays.
[0113] An amount of an anti-toxin antibody effective to prevent a disorder, or a "a prophylactically effective amount," of the antibody is an amount that is effective, upon single- or multiple-dose administration to the subject, in preventing or delaying the occurrence of the onset or recurrence of CDAD, or inhibiting a symptom thereof. However, if longer time intervals of protection are desired, increased doses can be administered.
[0114] The terms "agonize," "induce," "inhibit," "potentiate," "elevate," "increase," "decrease," or the like, e.g., which denote quantitative differences between two states, refer to a difference, e.g., a statistically or clinically significant difference, between the two states.
[0115] As used herein, "specific binding" or "specifically binds to" refers to the ability of an antibody to: (1) bind to a toxin of C. difficile with an affinity of at least 1.times.10.sup.7 M.sup.-1, and (2) bind to a toxin of C. difficile with an affinity that is at least two-fold greater than its affinity for a nonspecific antigen.
[0116] An "antibody" is a protein including at least one or two, heavy (H) chain variable regions (abbreviated herein as VHC), and at least one or two light (L) chain variable regions (abbreviated herein as VLC). The VHC and VLC 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). The extent of the framework region and CDRs has been precisely defined (see, Kabat, E. A., et al. Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991, and Chothia, C. et al., J. Mol. Biol. 196:901-917, 1987, which are incorporated herein by reference). Preferably, each VHC and VLC 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.
[0117] The VHC or VLC chain of the antibody can further include all or part of a heavy or light chain constant region. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are inter-connected by, e.g., disulfide bonds. The heavy chain constant region includes three domains, CH1, CH2 and CH3. The light chain constant region is comprised of one domain, CL. The variable region of the heavy and light chains contains a binding domain that interacts with an antigen. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The term "antibody" includes intact immunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof), wherein the light chains of the immunoglobulin may be of types kappa or lambda.
[0118] "Immunoglobulin" refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. The recognized human immunoglobulin genes include the kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Full-length immunoglobulin "light chains" (about 25 KD and 214 amino acids) are encoded by a variable region gene at the NH.sub.2-terminus (about 110 amino acids) and a kappa or lambda constant region gene at the COOH-terminus. Full-length immunoglobulin "heavy chains" (about 50 KD and 446 amino acids), are similarly encoded by a variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes, e.g., gamma (encoding about 330 amino acids). The term "immunoglobulin" includes an immunoglobulin having: CDRs from a human or non-human source. The framework of the immunoglobulin can be human, humanized, or non-human, e.g., a murine framework modified to decrease antigenicity in humans, or a synthetic framework, e.g., a consensus sequence.
[0119] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG.sub.1) that is encoded by heavy chain constant region genes.
[0120] The term "antigen binding portion" of an antibody (or simply "antibody portion," or "portion"), as used herein, refers to a portion of an antibody that specifically binds to a toxin of C. difficile (e.g., toxin A), e.g., a molecule in which one or more immunoglobulin chains is not full length, but which specifically binds to a toxin. Examples of binding portions encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VLC, VHC, CL and CH1 domains; (ii) a F(ab').sub.2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VHC and CH1 domains; (iv) a Fv fragment consisting of the VLC and VHC domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature 341:544-546, 1989), which consists of a VHC domain; and (vi) an isolated complementarity determining region (CDR) having sufficient framework to specifically bind, e.g., an antigen binding portion of a variable region. An antigen binding portion of a light chain variable region and an antigen binding portion of a heavy chain variable region, e.g., the two domains of the Fv fragment, VLC and VHC, can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VLC and VHC regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also encompassed within the term "antigen binding portion" of an antibody. These antibody portions are obtained using conventional techniques known to those with skill in the art, and the portions are screened for utility in the same manner as are intact antibodies.
[0121] The term "monospecific antibody" refers to an antibody that displays a single binding specificity and affinity for a particular target, e.g., epitope. This term includes a "monoclonal antibody" or "monoclonal antibody composition," which as used herein refer to a preparation of antibodies or portions thereof with a single molecular composition.
[0122] The term "recombinant" antibody, as used herein, refers to antibodies that are prepared, expressed, created, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial antibody library, antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes or antibodies prepared, expressed, created, or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant antibodies include humanized, CDR grafted, chimeric, in vitro generated (e.g., by phage display) antibodies, and may optionally include constant regions derived from human germline immunoglobulin sequences.
[0123] As used herein, the term "substantially identical" (or "substantially homologous") refers to a first amino acid or nucleotide sequence that contains a sufficient number of identical or equivalent (e.g., with a similar side chain, e.g., conserved amino acid substitutions) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have similar activities. In the case of antibodies, the second antibody has the same specificity and has at least 50% of the affinity of the first antibody.
[0124] Calculations of "homology" between two sequences are performed as follows. The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The length of a reference sequence aligned for comparison purposes is at least 50% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
[0125] The comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithm. The percent homology between two amino acid sequences is determined using the Needleman and Wunsch, J. Mol. Biol. 48:444-453, 1970, algorithm which has been incorporated into the GAP program in the GCG software package, using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
[0126] As used herein, the term "hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions" describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. 6.3.1-6.3.6, 1989, which is incorporated herein by reference. Aqueous and nonaqueous methods are described in that reference and either can be used. Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions: 6.times. sodium chloride/sodium citrate (SSC) at about 45.degree. C., followed by two washes in 0.2.times.SSC, 0.1% SDS at least at 50.degree. C. (the temperature of the washes can be increased to 55.degree. C. for low stringency conditions); 2) medium stringency hybridization conditions: 6.times.SSC at about 45.degree. C., followed by one or more washes in 0.2.times.SSC, 0.1% SDS at 60.degree. C.; 3) high stringency hybridization conditions: 6.times.SSC at about 45.degree. C., followed by one or more washes in 0.2.times.SSC, 0.1% SDS at 65.degree. C.; and 4) very high stringency hybridization conditions: 0.5 M sodium phosphate, 7% SDS at 65.degree. C., followed by one or more washes at 0.2.times.SSC, 1% SDS at 65.degree. C.
[0127] It is understood that the antibodies and antigen binding portions thereof described herein may have additional conservative or non-essential amino acid substitutions, which do not have a substantial effect on the polypeptide functions. Whether or not a particular substitution will be tolerated, i.e., will not adversely affect desired biological properties, such as binding activity, can be determined as described in Bowie et al., Science, 247:1306-1310, 1990. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
[0128] A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of a polypeptide, such as a binding agent, e.g., an antibody, without substantially altering a biological activity, whereas an "essential" amino acid residue results in such a change.
[0129] Unless otherwise defined, 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 methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Overview
[0130] C. difficile is a gram positive, toxin-producing bacterium that causes antibiotic-associated diarrhea and colitis in humans. Provided herein are methods and compositions for treatment and prevention of C. difficile-associated disease (CDAD). The compositions include antibodies that recognize proteins and other molecular components (e.g., lipids, carbohydrates, nucleic acids) of C. difficile bacteria, including antibodies that recognize toxins produced by C. difficile (e.g., toxin A and toxin B). In particular, human monoclonal antibodies are provided. In certain embodiments, these human monoclonal antibodies are produced in mice expressing human immunoglobulin gene segments (described below). Combinations of anti-toxin antibodies are also provided.
[0131] The new methods include administering antibodies (and antigen-binding portions thereof) that bind to a C. difficile toxin to a subject to inhibit CDAD in the subject. For example, human monoclonal anti-toxin A antibodies described herein can neutralize toxin A and inhibit relapse of C. difficile-mediated disease. In other examples, combinations of anti-toxin A antibodies (e.g., anti-toxin A monoclonal antibodies) and anti-toxin B antibodies can be administered to inhibit primary disease and reduce the incidence of disease relapse. The human monoclonal antibodies may localize to sites of disease (e.g., the gut) in vivo.
1. Generation of Antibodies
Immunogens
[0132] In general, animals are immunized with antigens expressed by C. difficile to produce antibodies. For producing anti-toxin antibodies, animals are immunized with inactivated toxins, or toxoids. Toxins can be inactivated, e.g., by treatment with formaldehyde, glutaraldehyde, peroxide, or oxygen treatment (see, e.g., Relyveld et al., Methods in Enzymology 93:24, 1983; Woodrow and Levine, eds., New Generation Vaccines, Marcel Dekker, Inc., New York, 1990). Mutant C. difficile toxins with reduced toxicity can be produced using recombinant methods (see, e.g., U.S. Pat. Nos. 5,085,862; 5,221,618; 5,244,657; 5,332,583; 5,358,868; and 5,433,945). For example, mutants containing deletions or point mutations in the toxin active site can be made. Recombinant fragments of the toxins can be used as immunogens. Another approach is to inactivate the toxin by treatment with UDP-dialdehyde (Genth et al., Inf. and Immun., 68(3):1094-1101, 2000). This method preserves the native structure of the toxin more readily than other treatments, and thus can elicit antibodies more reactive to the native toxin. This method is also described in Example 1, below.
[0133] Anti-toxin antibodies that bind and neutralize toxin A can interact with specific epitopes of toxin A. For example, an anti-toxin A antibody can bind an epitope in an N-terminal region of toxin A (e.g., between amino acids 1-1033 of toxin A), or a C-terminal region (e.g., between amino acids 1853-2710 of toxin A). In one example, an antibody that binds and neutralizes toxin A binds to an epitope within amino acids 1853-2710 of toxin A.
[0134] Similarly, anti-toxin B antibodies can recognize a specific epitope of toxin B, e.g., an N-terminal epitope, or a C-terminal epitope. In one example, an antibody that binds and neutralizes toxin B binds to an epitope within amino acids 1777-2366 of toxin B.
Generation of Human Monoclonal Antibodies in HuMAb Mice
[0135] Monoclonal antibodies can be produced in a manner not possible with polyclonal antibodies. Polyclonal antisera vary from animal to animal, whereas monoclonal preparations exhibit a uniform antigenic specificity. Murine animal systems are useful to generate monoclonal antibodies, and immunization protocols, techniques for isolating and fusing splenocytes, and methods and reagents for producing hybridomas are well known. Monoclonal antibodies can be produced by a variety of techniques, including conventional monoclonal antibody methodology, e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature, 256: 495, 1975. See generally, Harlow, E. and Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988.
[0136] Although these standard techniques are known, it is desirable to use humanized or human antibodies rather than murine antibodies to treat human subjects, because humans mount an immune response to antibodies from mice and other species. The immune response to murine antibodies is called a human anti-mouse antibody or HAMA response (Schroff, R. et al., Cancer Res., 45, 879-885, 1985) and is a condition that causes serum sickness in humans and results in rapid clearance of the murine antibodies from an individual's circulation. The immune response in humans has been shown to be against both the variable and the constant regions of murine immunoglobulins. Human monoclonal antibodies are safer for administration to humans than antibodies derived from other animals and human polyclonal antibodies.
[0137] One useful type of animal in which to generate human monoclonal antibodies is a transgenic mouse that expresses human immunoglobulin genes rather than its own mouse immunoglobulin genes. Such transgenic mice, e.g., "HuMAb.TM." mice, contain human immunoglobulin gene miniloci that encode unrearranged human heavy (.mu. and .gamma.) and .kappa. light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous .mu. and .kappa. chain loci (see e.g., Lonberg, N. et al., Nature 368(6474): 856-859, 1994, and U.S. Pat. No. 5,770,429). Accordingly, the mice exhibit reduced expression of mouse IgM or .kappa., and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgGic monoclonal antibodies (Lonberg, N. et al., supra; reviewed in Lonberg, N. Handbook of Experimental Pharmacology 113:49-101, 1994; Lonberg, N. and Huszar, D., Intern. Rev. Immunol., 13: 65-93, 1995, and Harding, F. and Lonberg, N., Ann. N.Y. Acad. Sci., 764:536-546, 1995).
[0138] The preparation of such transgenic mice is described in further detail in Taylor, L. et al., Nucleic Acids Research, 20:6287-6295, 1992; Chen, J. et al., International Immunology 5: 647-656, 1993; Tuaillon et al., Proc. Natl. Acad. Sci., USA 90:3720-3724, 1993; Choi et al., Nature Genetics, 4:117-123, 1993; Chen, J. et al., EMBO J., 12: 821-830, 1993; Tuaillon et al., J. Immunol., 152:2912-2920, 1994; Taylor, L. et al., International Immunology, 6: 579-591, 1994; and Fishwild, D. et al., Nature Biotechnology, 14: 845-851, 1996. See further, U.S. Pat. Nos. 5,545,806; 5,569,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650, 5,814,318, 5,874,299 and 5,877,397, all by Lonberg and Kay, and PCT Publication Nos. WO 01/14424, WO 98/24884, WO 94/25585, WO 93/1227, and WO 92/03918.
[0139] To generate fully human monoclonal antibodies to an antigen, HuMAb mice can be immunized with an immunogen, as described by Lonberg, N. et al. Nature, 368(6474): 856-859, 1994; Fishwild, D. et al., Nature Biotechnology, 14: 845-851, 1996 and WO 98/24884. Preferably, the mice will be 6-16 weeks of age upon the first immunization. For example, a purified preparation of inactivated toxin A can be used to immunize the HuMAb mice intraperitoneally. To generate antibodies against C. difficile proteins, lipids, and/or carbohydrate molecules, mice can be immunized with killed or nonviable C. difficile organisms.
[0140] HuMAb transgenic mice respond best when initially immunized intraperitoneally (IP) with antigen in complete Freund's adjuvant, followed by IP immunizations every other week (up to a total of 6) with antigen in incomplete Freund's adjuvant. The immune response can be monitored over the course of the immunization protocol with plasma samples being obtained by retroorbital bleeds. The plasma can be screened, for example by ELISA or flow cytometry, and mice with sufficient titers of anti-toxin human immunoglobulin can be used for fusions. Mice can be boosted intravenously with antigen 3 days before sacrifice and removal of the spleen. It is expected that 2-3 fusions for each antigen may need to be performed. Several mice are typically immunized for each antigen.
[0141] The mouse splenocytes can be isolated and fused with PEG to a mouse myeloma cell line based upon standard protocols. The resulting hybridomas are then screened for the production of antigen-specific antibodies. For example, single cell suspensions of splenic lymphocytes from immunized mice are fused to one-sixth the number of P3X63-Ag8.653 nonsecreting mouse myeloma cells (ATCC, CRL 1580) with 50% PEG. Cells are plated at approximately 2.times.10.sup.5 in flat bottom microtiter plate, followed by a two week incubation in selective medium containing 20% fetal Clone Serum, 18% "653" conditioned media, 5% origen (IGEN), 4 mM L-glutamine, 1 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin, 50 mg/ml gentamycin and 1.times.HAT (Sigma; the HAT is added 24 hours after the fusion). After two weeks, cells are cultured in medium in which the HAT is replaced with HT. Supernatants from individual wells are then screened by ELISA for human anti-toxin cell monoclonal IgM and IgG antibodies. The antibody secreting hybridomas are replated, screened again, and if still positive for human IgG, anti-toxin monoclonal antibodies, can be subcloned at least twice by limiting dilution. The stable subclones are then cultured in vitro to generate small amounts of antibody in tissue culture medium for characterization.
[0142] In one embodiment, the transgenic animal used to generate human antibodies to the toxin contains at least one, typically 2-10, and sometimes 25-50 or more copies of the transgene described in Example 12 of WO 98/24884 (e.g., pHC1 or pHC2) bred with an animal containing a single copy of a light chain transgene described in Examples 5, 6, 8, or 14 of WO 98/24884, and the offspring bred with the J.sub.H deleted animal described in Example 10 of WO 98/24884, the contents of which are hereby expressly incorporated by reference. Animals are bred to homozygosity for each of these three traits. Such animals have the following genotype: a single copy (per haploid set of chromosomes) of a human heavy chain unrearranged mini-locus (described in Example 12 of WO 98/24884), a single copy (per haploid set of chromosomes) of a rearranged human K light chain construct (described in Example 14 of WO 98/24884), and a deletion at each endogenous mouse heavy chain locus that removes all of the functional J.sub.H segments (described in Example 10 of WO 98/24884). Such animals are bred with mice that are homozygous for the deletion of the J.sub.H segments (Examples 10 of WO 98/24884) to produce offspring that are homozygous for the J.sub.H deletion and hemizygous for the human heavy and light chain constructs. The resultant animals are injected with antigens and used for production of human monoclonal antibodies against these antigens.
[0143] B cells isolated from such an animal are monospecific with regard to the human heavy and light chains because they contain only a single copy of each gene. Furthermore, they will be monospecific with regard to human or mouse heavy chains because both endogenous mouse heavy chain gene copies are nonfunctional by virtue of the deletion spanning the J.sub.H region introduced as described in Examples 9 and 12 of WO 98/24884. Furthermore, a substantial fraction of the B cells will be monospecific with regards to the human or mouse light chains, because expression of the single copy of the rearranged human kappa light chain gene will allelically and isotypically exclude the rearrangement of the endogenous mouse kappa and lambda chain genes in a significant fraction of B-cells.
[0144] In one embodiment, the transgenic mouse will exhibit immunoglobulin production with a significant repertoire, ideally substantially similar to that of a native mouse. Thus, for example, in embodiments where the endogenous Ig genes have been inactivated, the total immunoglobulin levels will range from about 0.1 to 10 mg/ml of serum, e.g., 0.5 to 5 mg/ml, or at least about 1.0 mg/ml. When a transgene capable of effecting a switch to IgG from IgM has been introduced into the transgenic mouse, the adult mouse ratio of serum IgG to IgM is preferably about 10:1. The IgG to IgM ratio will be much lower in the immature mouse. In general, greater than about 10%, e.g., about 40 to 80% of the spleen and lymph node B cells will express exclusively human IgG protein.
[0145] The repertoire in the transgenic mouse will ideally approximate that shown in a non-transgenic mouse, usually at least about 10% as high, preferably 25 to 50% or more as high. Generally, at least about a thousand different immunoglobulins (ideally IgG), preferably 10.sup.4 to 10.sup.6 or more, will be produced, depending primarily on the number of different V, J, and D regions introduced into the mouse genome. Typically, the immunoglobulins will exhibit an affinity for preselected antigens of at least about 10.sup.7M.sup.-1, 10.sup.9M.sup.-1, 10.sup.10M.sup.-1, 10.sup.11M.sup.-1, 10.sup.12M.sup.-1, or greater, e.g., up to 10.sup.13M.sup.-1 or greater.
[0146] HuMAb mice can produce B cells that undergo class-switching via intratransgene switch recombination (cis-switching) and express immunoglobulins reactive with the toxin. The immunoglobulins can be human sequence antibodies, wherein the heavy and light chain polypeptides are encoded by human transgene sequences, which may include sequences derived by somatic mutation and V region recombinatorial joints, as well as germline-encoded sequences. These human sequence immunoglobulins can be referred to as being substantially identical to a polypeptide sequence encoded by a human VL or VH gene segment and a human JL or JL segment, even though other non-germline sequences may be present as a result of somatic mutation and differential V-J and V-D-J recombination joints. With respect to such human sequence antibodies, the variable regions of each chain are typically at least 80 percent encoded by human germline V, J, and, in the case of heavy chains, D, gene segments. Frequently at least 85 percent of the variable regions are encoded by human germline sequences present on the transgene. Often 90 or 95 percent or more of the variable region sequences are encoded by human germline sequences present on the transgene. However, since non-germline sequences are introduced by somatic mutation and VJ and VDJ joining, the human sequence antibodies will frequently have some variable region sequences (and less frequently constant region sequences) that are not encoded by human V, D, or J gene segments as found in the human transgene(s) in the germline of the mice. Typically, such non-germline sequences (or individual nucleotide positions) will cluster in or near CDRs, or in regions where somatic mutations are known to cluster.
[0147] The human sequence antibodies that bind to the toxin can result from isotype switching, such that human antibodies comprising a human sequence gamma chain (such as gamma 1, gamma 2, or gamma 3) and a human sequence light chain (such as K) are produced. Such isotype-switched human sequence antibodies often contain one or more somatic mutation(s), typically in the variable region and often in or within about 10 residues of a CDR) as a result of affinity maturation and selection of B cells by antigen, particularly subsequent to secondary (or subsequent) antigen challenge. These high affinity human sequence antibodies have binding affinities of at least about 1.times.10.sup.9 M.sup.-1, typically at least 5.times.10.sup.9 M.sup.-1, frequently more than 1.times.10.sup.10 M.sup.-1, and sometimes 5.times.10.sup.10 M.sup.-1 to 1.times.10.sup.11 M.sup.-1 or greater.
[0148] Anti-toxin antibodies can also be raised in other mammals, including non-transgenic mice, humans, rabbits, and goats.
Anti-Toxin a Antibodies
[0149] Human monoclonal antibodies that specifically bind to toxin A include antibodies produced by the 3D8, 1B11, and 3H2 clones described herein. Antibodies with variable heavy chain and variable light chain regions that are at least 80%, or more, identical to the variable heavy and light chain regions of 3D8, 1B11, and 3H2 can also bind to toxin A. In related embodiments, anti-toxin A antibodies include, for example, the complementarity determining regions (CDR) of variable heavy chains and/or variable light chains of 3D8, 1B11, or 3H2. The CDRs of the variable heavy chain regions from these clones are shown in Table 1, below.
TABLE-US-00001 TABLE 1 Variable Heavy Chain CDR Amino Acid Sequences Clone Chain CDR Amino Acid Sequence SEQ ID NO: 3D8 H CDR1 NYGMH 7 1B11 H CDR1 SYGMH 10 3H2 H CDR1 KYGMH 13 3D8 H CDR2 LIWYDGSNEDYTDSVKG 8 1B11 H CDR2 VIWASGNKKYYIESVEG 11 3H2 H CDR2 VIWYDGTNKYYADSMKG 14 3D8 H CDR3 WGMVRGVIDVFDI 9 1B11 H CDR3 ANFDY 12 3H2 H CDR3 DPPTANY 15
[0150] The CDRs of the variable light chain regions from these clones are shown in table 2, below.
TABLE-US-00002 TABLE 2 Variable Light Chain CDR Amino Acid Sequences Clone Chain CDR Amino Acid Sequence SEQ ID NO: 3D8 L CDR1 RASQGISSWLA 16 1B11 L CDR1 RASQSVSSYLA 19 3H2 L CDR1 RASQGISSWLA 22 3D8 L CDR2 AASSLQS 17 1B11 L CDR2 DASNRAT 20 3H2 L CDR2 AASSLQS 23 3D8 L CDR3 QQANSFPWT 18 1B11 L CDR3 QQRSNWSQFT 21 3H2 L CDR3 QQYKSYPVT 24
[0151] CDRs are the portions of immunoglobulins that determine specificity for a particular antigen. In certain embodiments, CDRs corresponding to the CDRs in tables 1 and 2 having sequence variations (e.g., conservative substitutions) may bind to toxin A. For example, CDRs, in which 1, 2 3, 4, or 5 residues, or less than 20% of total residues in the CDR, are substituted or deleted can be present in an antibody (or antigen binding portion thereof) that binds toxin A.
[0152] Similarly, anti-toxin antibodies can have CDRs containing a consensus sequence, as sequence motifs conserved amongst multiple antibodies can be important for binding activity. For example, CDR1 of a variable light chain region of the antibodies or antigen binding portions thereof can include the amino acid sequence R-A-S-Q-X-X-S-S-X-L-A (SEQ ID NO: 25), CDR2 of a variable light chain region of the antibodies or antigen binding portions thereof can include the amino acid sequence A-S-X-X-X-S/T (SEQ ID NO:26), and/or CDR3 of a variable light chain region of the antibodies or antigen binding portions thereof can include the amino acid sequence Q-Q-X-X-S/N-X-P/S (SEQ ID NO:27), wherein X is any amino acid.
[0153] In some embodiments, CDR1 of a variable heavy chain region of the antibodies or antigen binding portions thereof includes the amino acid sequence Y-G-M-H (SEQ ID NO:28), and/or CDR2 of a variable heavy chain region of the antibodies or antigen binding portions thereof includes the amino acid sequence I-W-X-X-G-X-X-X-Y-X-X-S-X-X-G (SEQ ID NO:29), wherein X is any amino acid.
[0154] Human anti-toxin antibodies can include variable regions that are the product of, or derived from, specific human immunoglobulin genes. For example, the antibodies can include a variable heavy chain region that is the product of, or derived from a human VH3-33 gene. Numerous sequences for antibodies derived from this gene are available in GenBank.RTM. (see, e.g., Acc. No: AJ555951, GI No:29836865; Acc. No:AJ556080, GI No.:29837087; Acc. No.: AJ556038, GI No.:29837012, and other human VH3-33 rearranged gene segments provided in GenBank.RTM.). The antibodies can also, or alternatively, include a light chain variable region that is the product of, or derived from a human V.kappa. L19 gene (see, e.g., GenBank.RTM. Acc. No. AJ556049, GI No:29837033 for a partial sequence of a rearranged human V.kappa. L19 gene segment). As known in the art, and described in this section, above, variable immunoglobulin regions of recombined antibodies are derived by a process of recombination in vivo in which variability is introduced to genomic segments encoding the regions. Accordingly, variable regions derived from a human VH-33 or V.kappa. L19 gene can include nucleotides that are different that those in the gene found in non-lymphoid tissues. These nucleotide differences are typically concentrated in the CDRs.
Anti-Toxin B Antibodies
[0155] Human monoclonal antibodies that specifically bind to toxin B include antibodies produced by the 124-152, 2A11, and 1 G10 clones described herein. Antibodies with variable heavy chain and variable light chain regions that are at least 80%, or more, identical to the variable heavy and light chain regions of -152, 2A11, and 1 G10 can also bind to toxin B. In related embodiments, anti-toxin B antibodies include, for example, the complementarity determining regions (CDR) of variable heavy chains and/or variable light chains of -152, 2A11, or 1 G10. The CDRs of the variable heavy chain regions from these clones are shown in Table 3, below.
TABLE-US-00003 TABLE 3 Variable Heavy Chain CDR Amino Acid Sequences SEQ ID NO: SEQ ID NO: Clone Chain CDR Amino Acid Sequence (a.a.) (n.t.) 124-152 H CDR1 SYWIG 62 63 124-152 H CDR2 IFYPGDSSTRYSPSFQG 64 65 124-152 H CDR3 RRNWGNAFDI 66 67
[0156] The CDRs of the variable light chain regions from these clones are shown in Table 4, below.
TABLE-US-00004 TABLE 4 Variable Light Chain CDR Amino Acid Sequences SEQ ID NO: SEQ ID NO: Clone Chain CDR Amino Acid Sequence (a.a.) (n.t.) 124-152 L CDR1 RASQSVSSSYLAW 68 69 124-152 L CDR2 GASSRAT 70 71 124-152 L CDR3 QQYGSSTWT 72 73
[0157] CDRs are the portions of immunoglobulins that determine specificity for a particular antigen. In certain embodiments, CDRs corresponding to the CDRs in Tables 3 and 4 having sequence variations (e.g., conservative substitutions) may bind to toxin B. For example, CDRs, in which 1, 2, 3, 4, or 5 residues, or less than 20% of total residues in the CDR, are substituted or deleted can be present in an antibody (or antigen binding portion thereof) that binds toxin B.
[0158] Human anti-toxin B antibodies can include variable regions that are the product of, or derived from, specific human immunoglobulin genes (see FIGS. 28-31). For example, the antibodies can include a variable heavy chain region that is the product of, or derived from a human VH 5-51 gene. The antibodies can also, or alternatively, include a light chain variable region that is the product of, or derived from a human V.kappa. A27 gene and/or JK1 gene. As known in the art, and described in this section, above, variable immunoglobulin regions of recombined antibodies are derived by a process of recombination in vivo in which variability is introduced to genomic segments encoding the regions. Accordingly, variable regions derived from a human VH-5-51 or V.kappa. A27/JK1 gene can include nucleotides that are different that those in the gene found in non-lymphoid tissues. These nucleotide differences are typically concentrated in the CDRs.
2. Production and Modification of Antibodies
[0159] Many different forms of anti-toxin antibodies can be useful in the inhibition of CDAD. The antibodies can be of the various isotypes, including: IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA1, IgA2, IgD, or IgE. Preferably, the antibody is an IgG isotype, e.g., IgG1. The antibody molecules can be full-length (e.g., an IgG1 or IgG4 antibody) or can include only an antigen-binding fragment (e.g., a Fab, F(ab').sub.2, Fv or a single chain Fv fragment). These include monoclonal antibodies (e.g., human monoclonal antibodies), recombinant antibodies, chimeric antibodies, and humanized antibodies, as well as antigen-binding portions of the foregoing.
[0160] Anti-toxin antibodies or portions thereof useful in the present invention can also be recombinant antibodies produced by host cells transformed with DNA encoding immunoglobulin light and heavy chains of a desired antibody. Recombinant antibodies may be produced by known genetic engineering techniques. For example, recombinant antibodies can be produced by cloning a nucleotide sequence, e.g., a cDNA or genomic DNA, encoding the immunoglobulin light and heavy chains of the desired antibody. The nucleotide sequence encoding those polypeptides is then inserted into an expression vector so that both genes are operatively linked to their own transcriptional and translational expression control sequences. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. Typically, both genes are inserted into the same expression vector. Prokaryotic or eukaryotic host cells may be used.
[0161] Expression in eukaryotic host cells is preferred because such cells are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody. However, any antibody produced that is inactive due to improper folding can be renatured according to well known methods (Kim and Baldwin, Ann. Rev. Biochem., 51:459-89, 1982). It is possible that the host cells will produce portions of intact antibodies, such as light chain dimers or heavy chain dimers, which also are antibody homologs according to the present invention.
[0162] The antibodies described herein also can be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods as is well known in the art (Morrison, S., Science, 229:1202, 1985). For example, in one embodiment, the gene(s) of interest, e.g., human antibody genes, can be ligated into an expression vector such as a eukaryotic expression plasmid such as used in a GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338 841, or in other expression systems well known in the art. The purified plasmid with the cloned antibody genes can be introduced in eukaryotic host cells such as CHO-cells or NSO-cells or alternatively other eukaryotic cells like a plant derived cells, fungi or yeast cells. The method used to introduce these genes can be any method described in the art, such as electroporation, lipofectine, lipofectamine or ballistic transfection, in which cells are bombarded with microparticles carrying the DNA of interest (Rodin, et al. Immunol. Lett., 74(3):197-200, 2000). After introducing these antibody genes in the host cells, cells expressing the antibody can be identified and selected. These cells represent the transfectomas which can then be amplified for their expression level and upscaled to produce antibodies. Recombinant antibodies can be isolated and purified from these culture supernatants and/or cells using standard techniques.
[0163] It will be understood that variations on the above procedures are useful in the present invention. For example, it may be desired to transform a host cell with DNA encoding either the light chain or the heavy chain (but not both) of an antibody. Recombinant DNA technology may also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding, e.g., the constant region may be modified by, for example, deleting specific amino acids. The molecules expressed from such truncated DNA molecules are useful in the methods described herein. In addition, bifunctional antibodies can be produced in which one heavy and one light chain bind to a toxin, and the other heavy and light chain are specific for an antigen other than the toxin, or another epitope of the toxin.
[0164] Chimeric antibodies can be produced by recombinant DNA techniques known in the art. For example, a gene encoding the Fc constant region of a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the murine Fc, and the equivalent portion of a gene encoding a human Fc constant region is substituted (see Robinson et al., International Patent Publication PCT/US86/02269; Akira, et al., European Patent Application 184, 187; Taniguchi, M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., International Application WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988 Science, 240:1041-1043); Liu et al. (1987) PNAS, 84:3439-3443; Liu et al., 1987, J. Immunol., 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al., 1987, Canc. Res., 47:999-1005; Wood et al. (1985) Nature, 314:446-449; and Shaw et al., 1988, J. Natl. Cancer Inst., 80:1553-1559). Chimeric antibodies can also be created by recombinant DNA techniques where DNA encoding murine V regions can be ligated to DNA encoding the human constant regions.
[0165] An antibody or an immunoglobulin chain can be humanized by methods known in the art. For example, once murine antibodies are obtained, variable regions can be sequenced. The location of the CDRs and framework residues can be determined (see, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol., 196:901-917). The light and heavy chain variable regions can, optionally, be ligated to corresponding constant regions. Indeed, it is understood that any of the antibodies described herein, including fully human antibodies, can be altered (e.g., by mutation, substitution) to contain a substitute constant region, e.g., Fc region, or portion(s) thereof to achieve, for example, a desired antibody structure, function (e.g., effector function), subtype, allotype, subclass, or the like. Anti-toxin antibodies can be sequenced using art-recognized techniques. CDR-grafted antibody molecules or immunoglobulins can be produced by CDR-grafting or CDR substitution, wherein one, two, or all CDRs of an immunoglobulin chain can be replaced. See e.g., U.S. Pat. No. 5,225,539; Jones et al., 1986, Nature, 321:552-525; Verhoeyan et al., 1988, Science, 239:1534; Beidler et al., 1988, J. Immunol., 141:4053-4060; and Winter, U.S. Pat. No. 5,225,539.
[0166] Winter describes a CDR-grafting method that may be used to prepare the antibodies of the present invention (UK Patent Application GB 2188638A, filed on Mar. 26, 1987; Winter U.S. Pat. No. 5,225,539), the contents of which is expressly incorporated by reference. For example, all of the CDRs of a particular antibody may be replaced with at least a portion of a human CDR (e.g., a CDR from clone 3D8, as shown in Tables 1 and 2, and/or clone 124-152, as shown in Tables 3 and 4, above) or only some of the CDRs may be replaced. It is only necessary to replace the number of CDRs required for binding of the antibody to a predetermined antigen (e.g., an exotoxin of C. difficile).
[0167] Humanized antibodies can be generated by replacing sequences of the Fv variable region that are not directly involved in antigen binding with equivalent sequences from human Fv variable regions. General methods for generating humanized antibodies are provided by Morrison, S. L., 1985, Science, 229:1202-1207, by Oi et al., 1986, BioTechniques, 4:214, and by Queen et al. U.S. Pat. Nos. 5,585,089, 5,693,761 and U.S. Pat. No. 5,693,762. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable regions from at least one of a heavy or light chain. Sources of such nucleic acid are well known to those skilled in the art and, for example, may be obtained from a hybridoma producing an antibody against a predetermined target, as described above. The recombinant DNA encoding the humanized antibody, or fragment thereof, can then be cloned into an appropriate expression vector. Other techniques for humanizing antibodies are described in Padlan et al. EP 519596 A1, published on Dec. 23, 1992.
[0168] Also within the scope of the invention are antibodies in which specific amino acids have been substituted, deleted, or added. In particular, preferred antibodies have amino acid substitutions in the framework region, such as to improve binding to the antigen. For example, a selected, small number of acceptor framework residues of the immunoglobulin chain can be replaced by the corresponding donor amino acids. Preferred locations of the substitutions include amino acid residues adjacent to the CDR, or which are capable of interacting with a CDR (see e.g., U.S. Pat. No. 5,585,089). Criteria for selecting amino acids from the donor are described in U.S. Pat. No. 5,585,089 (e.g., columns 12-16), the contents of which are hereby incorporated by reference. The acceptor framework can be a mature human antibody framework sequence or a consensus sequence.
[0169] A "consensus sequence" is a sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related sequences (See e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family of proteins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence. A "consensus framework" of an immunoglobulin refers to a framework region in the consensus immunoglobulin sequence.
[0170] An anti-toxin antibody, or antigen-binding portion thereof, can be derivatized or linked to another functional molecule (e.g., another peptide or protein). For example, an antibody can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody, a detectable agent, a cytotoxic agent, a pharmaceutical agent, and/or a protein or peptide that can mediate association with another molecule (such as a streptavidin core region or a polyhistidine tag).
[0171] One type of derivatized protein is produced by crosslinking two or more proteins (of the same type or of different types). Suitable crosslinkers include those that are heterobifunctional, having two distinct reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, Ill.
[0172] Useful detectable agents with which a protein can be derivatized (or labeled) include fluorescent compounds, various enzymes, prosthetic groups, luminescent materials, bioluminescent materials, and radioactive materials. Exemplary fluorescent detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, and, phycoerythrin. A protein or antibody can also be derivatized with detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, .beta.-galactosidase, acetylcholinesterase, glucose oxidase and the like. When a protein is derivatized with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a detectable reaction product. For example, when the detectable agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is detectable. A protein can also be derivatized with a prosthetic group (e.g., streptavidin/biotin and avidin/biotin). For example, an antibody can be derivatized with biotin, and detected through indirect measurement of avidin or streptavidin binding.
[0173] Labeled proteins and antibodies can be used, for example, diagnostically and/or experimentally in a number of contexts, including (i) to isolate a predetermined antigen by standard techniques, such as affinity chromatography or immunoprecipitation; and (ii) to detect a predetermined antigen (e.g., a toxin, e.g., in a cellular lysate or a patient sample) in order to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen.
[0174] An anti-toxin antibody or antigen-binding fragment thereof may be conjugated to another molecular entity, such as a label.
3. Screening Methods
[0175] Anti-toxin antibodies can be characterized for binding to the toxin by a variety of known techniques. Antibodies are typically characterized by ELISA first. Briefly, microtiter plates can be coated with the toxin or toxoid antigen in PBS, and then blocked with irrelevant proteins such as bovine serum albumin (BSA) diluted in PBS. Dilutions of plasma from toxin-immunized mice are added to each well and incubated for 1-2 hours at 37.degree. C. The plates are washed with PBS/Tween 20 and then incubated with a goat-anti-human IgG Fc-specific polyclonal reagent conjugated to alkaline phosphatase for 1 hour at 37.degree. C. After washing, the plates are developed with ABTS substrate, and analyzed at OD of 405. Preferably, mice which develop the highest titers will be used for fusions.
[0176] An ELISA assay as described above can be used to screen for antibodies and, thus, hybridomas that produce antibodies that show positive reactivity with the toxin. Hybridomas that produce antibodies that bind, preferably with high affinity, to the toxin can than be subcloned and further characterized. One clone from each hybridoma, which retains the reactivity of the parent cells (by ELISA), can then be chosen for making a cell bank, and for antibody purification.
[0177] To purify the anti-toxin antibodies, selected hybridomas can be grown in roller bottles, two-liter spinner-flasks or other culture systems. Supernatants can be filtered and concentrated before affinity chromatography with protein A-Sepharose (Pharmacia, Piscataway, N.J.) to purify the protein. After buffer exchange to PBS, the concentration can be determined by spectrophotometric methods.
[0178] To determine if the selected monoclonal antibodies bind to unique epitopes, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, Ill.). Biotinylated MAb binding can be detected with a streptavidin labeled probe. Anti-toxin antibodies can be further tested for reactivity with the toxin by Western blotting.
[0179] Other assays to measure activity of the anti-toxin antibodies include neutralization assays. In vitro neutralization assays can measure the ability of an antibody to inhibit a cytopathic effect on cells in culture (see Example 3, below). In vivo assays to measure toxin neutralization are described in Examples 5, 6, and 7, below.
4. Pharmaceutical Compositions and Kits
[0180] In another aspect, the present invention provides compositions, e.g., pharmaceutically acceptable compositions, which include an antibody molecule described herein or antigen binding portion thereof, formulated together with a pharmaceutically acceptable carrier.
[0181] "Pharmaceutically acceptable carriers" include any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible. The carriers can be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or epidermal administration (e.g., by injection or infusion).
[0182] The compositions of this invention may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Useful compositions are in the form of injectable or infusible solutions. A useful mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). For example, the antibody or antigen binding portion thereof can be administered by intravenous infusion or injection. In another embodiment, the antibody or antigen binding portion thereof is administered by intramuscular or subcutaneous injection.
[0183] The phrases "parenteral administration" and "administered parenterally" as used herein mean modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.
[0184] Therapeutic compositions typically should be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high antibody concentration. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., antibody or antibody portion) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the useful methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
[0185] The antibodies and antibody portions described herein can be administered by a variety of methods known in the art, and for many therapeutic applications. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.
[0186] In certain embodiments, an antibody, or antibody portion thereof may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound (and other ingredients, if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound of the invention by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. Therapeutic compositions can be administered with medical devices known in the art.
[0187] Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
[0188] An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of an antibody or antibody portion of the invention is 0.1-60 mg/kg, e.g., 0.5-25 mg/kg, 1-2 mg/kg, or 0.75-10 mg/kg. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.
[0189] Also within the scope of the invention are kits including an anti-toxin antibody or antigen binding portion thereof. The kits can include one or more other elements including: instructions for use; other reagents, e.g., a label, a therapeutic agent, or an agent useful for chelating, or otherwise coupling, an antibody to a label or therapeutic agent, or other materials for preparing the antibody for administration; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.
[0190] Various combinations of antibodies can be packaged together. For example, a kit can include antibodies that bind to toxin A (e.g., antibodies that include the variable heavy and light chain regions of 3D8) and antibodies that bind to toxin B (e.g., human monoclonal anti-toxin B antibodies, e.g., 124-152, 2A11, and/or 1 G10, or polyclonal antisera reactive with toxin B). The antibodies can be mixed together, or packaged separately within the kit.
[0191] Instructions for use can include instructions for therapeutic application including suggested dosages and/or modes of administration, e.g., in a patient with a symptom of CDAD. Other instructions can include instructions on coupling of the antibody to a chelator, a label or a therapeutic agent, or for purification of a conjugated antibody, e.g., from unreacted conjugation components.
[0192] The kit can include a detectable label, a therapeutic agent, and/or a reagent useful for chelating or otherwise coupling a label or therapeutic agent to the antibody. Coupling agents include agents such as N-hydroxysuccinimide (NHS). In such cases the kit can include one or more of a reaction vessel to carry out the reaction or a separation device, e.g., a chromatographic column, for use in separating the finished product from starting materials or reaction intermediates.
[0193] The kit can further contain at least one additional reagent, such as a diagnostic or therapeutic agent, e.g., a diagnostic or therapeutic agent as described herein, and/or one or more additional anti-toxin or anti-C. difficile antibodies (or portions thereof), formulated as appropriate, in one or more separate pharmaceutical preparations.
[0194] Other kits can include optimized nucleic acids encoding anti-toxin antibodies, and instructions for expression of the nucleic acids.
5. Therapeutic Methods and Compositions
[0195] The new proteins and antibodies have in vitro and in vivo therapeutic, prophylactic, and diagnostic utilities. For example, these antibodies can be administered to cells in culture, e.g., in vitro or ex vivo, or to a subject, e.g., in vivo, to treat, inhibit, prevent relapse, and/or diagnose C. difficile and disease associated with C. difficile.
[0196] As used herein, the term "subject" is intended to include human and non-human animals. The term "non-human animals" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, chickens, mice, dogs, cats, pigs, cows, and horses.
[0197] The proteins and antibodies can be used on cells in culture, e.g., in vitro or ex vivo. For example, cells can be cultured in vitro in culture medium and the contacting step can be effected by adding the anti-toxin antibody or fragment thereof, to the culture medium. The methods can be performed on virions or cells present in a subject, as part of an in vivo (e.g., therapeutic or prophylactic) protocol. For in vivo embodiments, the contacting step is effected in a subject and includes administering an anti-toxin antibody or portion thereof to the subject under conditions effective to permit binding of the antibody, or portion, to any toxin expressed by bacteria in the subject, e.g., in the gut.
[0198] Methods of administering antibody molecules are described herein. Suitable dosages of the molecules used will depend on the age and weight of the subject and the particular drug used. The antibody molecules can be used as competitive agents for ligand binding to inhibit or reduce an undesirable interaction, e.g., to inhibit binding of toxins to the gastrointestinal epithelium.
[0199] The anti-toxin antibodies (or antigen binding portions thereof) can be administered in combination with other anti-C. difficile antibodies (e.g., other monoclonal antibodies, polyclonal gamma-globulin). Combinations of antibodies that can be used include an anti-toxin A antibody or antigen binding portion thereof and an anti-toxin B antibody or antigen binding portion thereof. The anti-toxin A antibody can be 3D8, an antibody that includes the variable regions of 3D8, or an antibody with variable regions at least 90% identical to the variable regions of 3D8. The anti-toxin B antibody can be 124-152, 2A11, 1 G10, or an antibody with variable regions at least 90% identical to the variable regions of the foregoing, e.g., 124-152. Combinations of anti-toxin A (e.g., 3D8) and anti-toxin B antibodies (e.g., 124-152) can provide potent inhibition of CDAD.
[0200] It is understood that any of the agents of the invention, for example, anti-toxin A or anti-toxin B antibodies, or fragments thereof, can be combined, for example in different ratios or amounts, for improved therapeutic effect. Indeed, the agents of the invention can be formulated as a mixture, or chemically or genetically linked using art recognized techniques thereby resulting in covalently linked antibodies (or covalently linked antibody fragments), having both anti-toxin A and anti-toxin B binding properties. The combined formulation may be guided by a determination of one or more parameters such as the affinity, avidity, or biological efficacy of the agent alone or in combination with another agent. The agents of the invention can also be administered in combination with other agents that enhance access, half-life, or stability of the therapeutic agent in targeting, clearing, and/or sequestering C. difficile or an antigen thereof.
[0201] Such combination therapies are preferably additive and even synergistic in their therapeutic activity, e.g., in the inhibition, prevention (e.g., of relapse), and/or treatment of C. difficile-related diseases or disorders (see, e.g., Example 16 which shows the efficacy of single and combined antibody therapies). Administering such combination therapies can decrease the dosage of the therapeutic agent (e.g., antibody or antibody fragment mixture, or cross-linked or genetically fused bispecific antibody or antibody fragment) needed to achieve the desired effect.
[0202] Immunogenic compositions that contain an immunogenically effective amount of a toxin, or fragments thereof, are described herein, and can be used in generating anti-toxin antibodies. Immunogenic epitopes in a toxin sequence can be identified according to methods known in the art, and proteins, or fragments containing those epitopes can be delivered by various means, in a vaccine composition. Suitable compositions can include, for example, lipopeptides (e.g., Vitiello et al., J. Clin. Invest. 95:341 (1995)), peptide compositions encapsulated in poly(DL-lactide-co-glycolide) ("PLG") microspheres (see, e.g., Eldridge et al., Molec. Immunol. 28:287-94 (1991); Alonso et al., Vaccine 12:299-306 (1994); Jones et al., Vaccine 13:675-81 (1995)), peptide compositions contained in immune stimulating complexes (ISCOMS) (see, e.g., Takahashi et al., Nature 344:873-75 (1990); Hu et al., Clin. Exp. Immunol. 113:235-43 (1998)), and multiple antigen peptide systems (MAPs) (see, e.g., Tam, Proc. Natl. Acad. Sci. U.S.A. 85:5409-13 (1988); Tam, J. Immunol. Methods 196:17-32 (1996)).
[0203] Useful carriers that can be used with immunogenic compositions of the invention are well known, and include, for example, thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly L-lysine, poly L-glutamic acid, influenza, hepatitis B virus core protein, and the like. The compositions can contain a physiologically tolerable (i.e., acceptable) diluent such as water, or saline, typically phosphate buffered saline. The compositions and vaccines also typically include an adjuvant. Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are examples of materials well known in the art. Additionally, CTL responses can be primed by conjugating toxins (or fragments, inactive derivatives or analogs thereof) to lipids, such as tripalmitoyl-S-glycerylcysteinyl-seryl-serine (P.sub.3CSS).
[0204] The anti-toxin antibodies can be administered in combination with other agents, such as compositions to treat CDAD. For example, therapeutics that can be administered in combination with anti-toxin antibodies include antibiotics used to treat CDAD, such as vancomycin, metronidazole, or bacitracin. The antibodies can be used in combination with probiotic agents such as Saccharomyces boulardii. The antibodies can also be administered in combinations with a C. difficile vaccine, e.g., a toxoid vaccine.
6. Other Methods
[0205] An anti-toxin antibody (e.g., monoclonal antibody) can be used to isolate toxins by standard techniques, such as affinity chromatography or immunoprecipitation. Moreover, an anti-toxin antibody can be used to detect the toxin (e.g., in a stool sample), e.g., to screen samples for the presence of C. difficile. Anti-toxin antibodies can be used diagnostically to monitor levels of the toxin in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen.
EXEMPLIFICATION
[0206] Throughout the examples, the following materials and methods were used unless otherwise stated.
Materials and Methods
[0207] In general, the practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, recombinant DNA technology, immunology (especially, e.g., antibody technology), and standard techniques in polypeptide preparation. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: Cold Spring Harbor Laboratory Press (1989); Antibody Engineering Protocols (Methods in Molecular Biology), 510, Paul, S., Humana Pr (1996); Antibody Engineering: A Practical Approach (Practical Approach Series, 169), McCafferty, Ed., Irl Pr (1996); Antibodies: A Laboratory Manual, Harlow et al., C.S.H.L. Press, Pub. (1999); and Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons (1992).
EXAMPLES
[0208] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
Example 1. Generation of Anti-Toxin a Monoclonal Antibodies
[0209] C. difficile toxin A was obtained either from Techlab, Inc. (Blacksburg, Va.), or by recombinant production. The toxin was purified and inactivated prior to immunization. Inactivation was performed by treatment with reactive UDP-dialdehyde, which results in alkylation of catalytic residues while preserving native toxin structure. For the detailed protocol, see Genth et al., Inf and Immun. 68(3):1094-1101, 2000. Briefly, purified toxin A was incubated with UDP-2',3'-dialdehyde (0.1-1.0 mM) in buffer for 18 hours at 37.degree. C., filtered through a 100 kDa-cutoff filter to remove unreacted UDP-2',3'-dialdehyde, and washed with buffer. Inactivated toxin A (toxoid A) was used for immunization.
[0210] HCo7 transgenic mice, generated as described above in the section entitled "Generation of Human Monoclonal Antibodies in HuMAb Mice" and supplied by Medarex, Milpitas, Calif., were immunized intraperitoneally 6-12 times each with 10 .mu.g of toxoid in RIBI adjuvant. In the HCo7 transgenic mice, the endogenous mouse kappa light chain gene has been homozygously disrupted as described in Chen et al. (1993) EMBO J. 12:811-820 and the endogenous mouse heavy chain gene has been homozygously disrupted as described in Example 1 of PCT Publication WO 01/09187. The HCo7 transgenic mice carry a human kappa light chain transgene, KCo5, as described in Fishwild et al. (1996) Nature Biotechnology 14:845-851, and the HCo7 human heavy chain transgene as described in U.S. Pat. Nos. 5,545,806; 5,625,825; and 5,545,807. Serum was collected from each mouse and tested for reactivity to toxin A by ELISA and neutralization of cytotoxicity on IMR-90 cells. Mice that tested positive for toxin A-reactive and neutralizing antiserum were injected with 5-10 .mu.g toxoid A through the tail vein. Mice were sacrificed and spleens were isolated for fusion to hybridomas approximately 3 days after tail vein injection was performed.
[0211] Clonal hybridomas were generated and screened by ELISA. Percentages of kappa/gamma light chain positive, antigen-specific, and neutralizing clones identified by screening clones generated from four separate hybridoma fusions are listed in Table 5.
TABLE-US-00005 TABLE 5 % kappa/gamma Fusion positive % antigen specific % neutralizing 1 5.7 (94/1632) 3.4 (56/1632) 0.7 (12/1632) 2 0.2 (1/384) 0 (0/384) 0 (0/384) 3 1.8 (14/768) 0.39 (3/768) 4 4.4 (43/960) 1.7 (17/960)
[0212] Three hybridoma clones were selected for further analysis: 3D8, 1B11, and 33.3H2. CDNAs from each clone were amplified by RT-PCR from mRNA, cloned, and sequenced. One heavy chain V region consensus sequence was found for each clone. All three clones utilized a VH region derived from the same germline V region gene (VH 3-33), but utilized different J sequences. The amino acid sequences of the VH and VL regions from each clone are shown in FIG. 1 (SEQ ID NOs: 1-6). The complementarity determining regions (CDRs) are overlined in the Figure.
[0213] Sequence analysis of the kappa V (VK light chain) genes revealed that HuMAb 1B11 and 33.3H2 each express one consensus kappa chain V sequence. The 1B11 hybridoma expressed a V.kappa. light chain derived from the V.kappa. L6 germline gene, whereas the 33.3H2 hybridoma expresses a V.kappa. light chain derived from the V.kappa. L15 germline gene. Upon analysis of the V.kappa. clones from HuMAb 3D8, 6 (I-VI) light chains were expressed at the mRNA level (FIG. 1). To determine which of the light chains were expressed at the protein level, mass spectroscopy and N-terminal sequencing of the purified 3D8 antibody were performed. When light chains were isolated from cellular protein and analyzed by mass spectroscopy, a single light chain was seen with a mass of 23,569 Daltons. This corresponded to the light chain with the group I amino acid sequence depicted in FIG. 1, which is derived from the V.kappa. L19 germline gene. N-terminal sequencing of the light chain confirmed this result. FIGS. 2A, 3A, and 4A depict the nucleotide and the amino acid sequences of the V.kappa. of each 3D8 (group I; SEQ ID NOs: 4, and 30-34), 1B11 (SEQ ID NO: 5), and 33.3H2 (SEQ ID NO:6) respectively. The CDRs are overlined and the germline V.kappa. and J.kappa. are shown.
[0214] Thus, the 3D8 antibody comprises a heavy chain variable region that is the product of or derived from a human VH 3-33 gene and a light chain variable region that is the product of or derived from a human V.kappa. L19 gene. The 1B11 antibody comprises a heavy chain variable region that is the product of or derived from a human VH 3-33 gene and a light chain variable region that this the product of or derived from a human V.kappa. L6 gene. The 33.3H2 antibody comprises a heavy chain variable region that is the product of or derived from a human VH 3-33 gene and a light chain variable region that this the product of or derived from a human V.kappa. L15 gene.
[0215] The antibodies 3D8 and 1B11 express human IgG1 constant regions, and antibody 33.3H2 expresses human IgG3 constant regions. The antibodies described in Examples 2-7 were isolated from these hybridomas, and thus express the variable sequences shown in FIG. 1 along with human constant regions. DNA encoding the antigen binding portion of each clone was cloned into a vector to be expressed as a human antibody for administration to humans.
Example 2. Binding Activity of Anti-Toxin a Antibodies
[0216] Binding of each antibody to toxin A was determined by ELISA using standard techniques. The results of this assay are depicted in FIG. 5. Antibodies produced by 3D8, 1B11, and 33.3H2 were compared to a fourth human monoclonal antibody with toxin A binding activity, 8E6. FIG. 5 shows that the antibodies bind toxin A with comparable affinities.
[0217] The affinity of the 3D8 and 1B11 antibodies for toxin A was also measured with Biacore.RTM. instrument, which detects biomolecular binding interactions with surface plasmon resonance technology. Each antibody was added to protein A-coated sensor chips, and toxin A was allowed to flow over the chip to measure binding. 3D8 had a K.sub.D of 14.6.times.10.sup.-10M. 1B11 had a K.sub.D of 7.38.times.10.sup.-10M. Thus, the antibodies bind with high affinity to toxin A. These binding constants indicate that the antibodies have affinities suitable for use in human therapy.
Example 3. Toxin Neutralization by Anti-Toxin a Antibodies
[0218] Antibodies expressed by 1B11, 3D8, and 33.3H2 hybridomas were tested for toxin A neutralization activity in vitro. Cells were incubated in the presence of varying concentrations of toxin A, which causes cells to round up and lose adherence to cell culture dishes. Cytopathic effect (CPE) was determined by visual inspection of cells. A CPE score from 0-4 was determined, based on the results of the visual inspection (4=100% cytotoxicity, 0=0% toxicity). The results of these assays are depicted in FIGS. 6A and 6B. Neutralization of toxicity against a human lung fibroblast cell line, IMR-90, and a human gut epithelial cell line, T-84, was determined. FIG. 6A shows that all of the antibodies had neutralizing capacity towards IMR-90 cells. The relative neutralizing activity of toxin A cytotoxicity on IMR-90 cells was 1B11>3H2>3D8. Interestingly, the relative neutralizing activity was 3D8.gtoreq.1B11>3H2 against T-84 cells, which are human colonic epithelial cells (FIG. 6A). T-84 cells are believed to be more sensitive to toxin A than other cell types. T-84 cells may provide a more relevant target cell to determine toxin A cytotoxicity.
Example 4. Epitope Mapping of Anti-Toxin A Antibodies
[0219] The epitope of toxin A bound by each monoclonal antibody was determined by western blotting. Recombinant E. coli clones were constructed which express four fragments of toxin A representing the enzymatic domain (i.e., amino acids 1-659 of toxin A), the receptor binding domain (i.e., amino acids 1853-2710 of toxin A), and the two regions in between (i.e., amino acids 660-1255 and 1256-1852 of toxin A). The appropriate segments of the toxin A gene were PCR-amplified from genomic DNA prepared from C. difficile strain ATCC 43255. The fragments were cloned using a pET vector and transformed into BL21 DE3 cells for expression. The vector provides inducible expression and affinity domains for purification (i.e., a His-tag) and detection (i.e., a V5 epitope tag). Expression was induced with IPTG and fragments were purified by affinity chromatography. Binding to four different fragments of toxin A was measured: fragment 1 corresponded to amino acids 1-659; fragment 2 corresponded to amino acids 660-1255; fragment 3 corresponded to amino acids 1256-1852; and fragment 4 corresponded to amino acids 1853-2710 (FIG. 7). 1B11 reacted with fragments 1 and 2. 33.3H2 reacted with fragment 2. 3D8 and another human monoclonal antibody, 6B4, reacted with fragment 4 (the receptor binding domain). A polyclonal antiserum from rabbits immunized with toxoid A reacted with all four fragments.
[0220] The 1B11 and 33.3H2 epitopes were mapped in further detail. To map the 1B11 epitope, subfragments of fragment 1 (amino acids 1-659) corresponding to amino acids 1-540, 1-415, 1-290, and 1-165, were generated (FIG. 8A). 1B11 bound to fragment 1 and to the fragment containing amino acids 1-540. 1B11 did not bind to the other subfragments. Therefore, the epitope bound by 1B11 maps between amino acids 415-540 of toxin A.
[0221] To map the 33.3H2 epitope, subfragments of fragment 2 (amino acids 660-1255) corresponding to amino acids 660-1146, 660-1033, 660-920, and 660-807, were generated (FIG. 8B). 33.3H2 bound to the fragments corresponding to amino acids 660-1255, 660-1146, and 660-1033. 33.3H2 did not bind to the other subfragments. Therefore, the epitope bound by 33.3H2 maps between amino acids 920-1033 of toxin A.
Example 5. Protection of Mice from Lethal Toxin a Challenge by Administration of Anti-Toxin A Antibodies
[0222] Each antibody was tested for the ability to protect mice from challenge with a lethal dose of toxin A. Swiss Webster female mice, each weighing 10-20 grams, were injected intraperitoneally with up to 250 .mu.g of 3D8, 1B11, or 33.3H2, or a control antibody (anti-respiratory syncytial virus antibody, Medlmmune) prior to challenge with toxin A. Approximately 24 hours after injection, mice were challenged with a dose of toxin A greater than 10 times the lethal dose (LD.sub.50), typically 100 ng. Animals were observed for signs of toxicity for the next 7 days. The results of these experiments are summarized in FIG. 9. The data is expressed as percentage survival. Numbers in parenthesis refer to antibody dose, if a dose other than 250 .mu.g was given. FIG. 9 shows that each of the antibodies was able to protect mice from lethal toxin A challenge to some extent. The percentage of mice surviving when treated with 3D8 ranged from 10-100 percent. The percentage of mice surviving when treated with 33.3H2 ranged from 20-100 percent. The percentage of mice surviving when treated with 1B11 ranged from 0-60 percent. The relative ability of these monoclonals to protect mice was 3H2.gtoreq.3D8>1B11.
Example 6. Neutralization of Toxin A Enterotoxicity in Ligated Mouse Intestinal Loops with Anti-Toxin A Antibodies
[0223] 3D8 and 33.3H2 antibodies were tested for neutralization of toxin A enterotoxicity in a mouse ileal loop model. This model measures toxin A-induced fluid accumulation in mouse intestine. To perform these experiments, each mouse was starved for 16 hours, anesthetized, and the ileum next to the cecum was exposed. A loop of 3 to 5 centimeters was doubly ligated at each end and injected with 10 .mu.g of toxin A. The ileal loop was returned to the abdominal cavity, the wound was closed, and the animal was allowed to recover. Four hours after surgery, the animal was euthanized and the loop was removed from the animal. The length of each segment was remeasured, and the intraluminal fluid was extracted. The volume of the fluid and the volume-to-length (V:L) ratio in milliliters per centimeter was calculated for each loop. Test mice were injected with antibody parenterally 1-2 days before surgery. The results of these experiments are depicted in FIG. 10. Injection with toxin A increased the weight to length ratio of intestinal fluid by 50%. Both 3D8 and 33.3H2 prevented this increase in fluid accumulation. Mice administered either antibody had a weight to length ratio comparable to mice that did not receive any toxin A injection. Therefore, 3D8 and 33.3H2 protect from intestinal fluid accumulation in vivo.
[0224] These results indicate that the anti-toxin A monoclonal antibodies protect from toxin A-mediated enterotoxicity in vivo. The mouse ligated loop data shows that these monoclonal antibodies can protect from mucosal damage when administered systemically.
Example 7. Protection of Hamsters from C. difficile Relapse with Anti-Toxin A Antibodies
[0225] 3D8 was tested in a hamster relapse model. Hamsters are sensitive to the toxic effects of C. difficile toxins, and typically die within 2-3 days of receiving a single dose of clindamycin in the presence of C. difficile. To test the efficacy of 3D8 in hamsters, a relapse model was used. In this model, hamsters were given a dose of clindamycin and a dose of C. difficile B1 spores one day later. One set of control hamsters received no additional antibiotic or antibody. A second set of control hamsters were treated with 10 mg/kg/day vancomycin. Vancomycin is an antibiotic used in the treatment of C. difficile disease. As shown in FIG. 11A, a test set of hamsters received 10 mg/kg/day vancomycin and 2 mg/kg/day of a rabbit polyclonal antiserum raised against toxin A each day for seven days after C. difficile exposure, as indicated by the arrows in the figure. A second test set of hamsters received 10 mg/kg/day vancomycin and 50 mg/kg/day 3D8 at the same time intervals. Hamster survival was plotted versus time and is shown in FIG. 11B.
[0226] FIG. 11B shows that all of the hamsters that received only clindamycin and C. difficile (diamonds) died within two days of challenge with the bacteria. Twelve percent (2/17) of hamsters treated with vancomycin (squares) survived challenge with bacteria; eighty-eight percent (15/17) died within eight days. Forty-one percent (7/17) of hamsters treated with vancomycin and 3D8 (crosses) survived challenge; fifty-nine (10/17) percent died within seven days. Sixty-four percent (7/11) of hamsters treated with vancomycin and polyclonal rabbit serum (triangles) survived the challenge with bacteria; thirty-six percent (4/11) died within nine days. These data are also depicted in FIG. 12 as the percentage of total survivors in each treatment group. As shown in the figure, the percentage of survivors was highest (sixty-four percent) in the group receiving vancomycin and polyclonal rabbit serum. The group receiving 3D8 and vancomycin had the second highest rate of survival (forty-one percent). Only twelve percent of vancomycin-treated hamsters survived. Those with no treatment all died. These data show that polyclonal and monoclonal anti-toxin antibodies protect from relapse of C. difficile disease in vivo when administered after infection.
Example 8. Production of Anti-Toxin A Antibodies for Administration in Humans
[0227] Nucleic acid sequences encoding the variable heavy chain and light chains of the 3D8 antibody were cloned into a pIE-UgammalF vector using standard recombinant DNA methodology. The vector was amplified in E. coli, purified, and transfected into CHO-dg44 cells. Transfected cells were plated at 4.times.10.sup.5 cells per well in a 96-well dish and selected for vector transfection with G418. One clone, designated 1D3, was originally selected by G418 resistance, then assayed along with other transfectomas for production of IgG. 1D3 had a higher level of IgG production relative to other transfectants during several rounds of expansion. The expression of the 3D8 antibody was amplified by growth in the presence of increasing concentrations of methotrexate. A culture capable of growth in 175 nM methotrexate was chosen for cloning single cells for further development. Plating the culture in 96 well plates at low density allowed generation of cultures arising from a single cell or clones. The cultures were screened for production of human IgG, and the cell that produced the highest level of IgG was selected for further use. The methotrexate-amplified clone was expanded to produce a cell bank including multiple frozen vials of cells.
[0228] To prepare antibodies from transfected cells, cells from a clone isolated in the previous steps are cultured and expanded as inoculum for a bioreactor. The bioreactor typically holds a 500 liter volume of culture medium. The cells are cultured in the bioreactor until cell viability drops, which indicates a maximal antibody concentration has been produced in the culture. The cells are removed by filtration. The filtrate is applied to a protein A column. Antibodies bind to the column, and are eluted with a low pH wash. Next, the antibodies are applied to a Q-Sepharose column to remove residual contaminants, such as CHO cell proteins, DNA, and other contaminants (e.g., viral contaminants, if present). Antibodies are eluted from the Q-Sepharose column, nano-filtered, concentrated, and washed in a buffer such as PBS. The preparation is then aseptically aliquoted into vials for administration.
Example 9. Preparation and Characterization of Polyclonal Anti-Toxin B Antibodies
[0229] Two Nubian goats (#330 and #331) were injected intramuscularly with 50 .mu.g UDP dialdehyde-inactivated toxin B (Techlab) and complete Freund's adjuvant. Booster doses of 25 .mu.g toxoid B with Freund's incomplete adjuvant were given intramuscularly at two-week intervals. Test bleeds were obtained after 4 immunizations. ELISA reactivity and neutralization of cytotoxicity against both toxin A and toxin B were assayed to measure the specificity and cross reactivity of the sera.
[0230] Both animals responded well to toxin B and to a lesser extent to toxin A as measured by ELISA. Sera from goat #331 had less toxin A cross-reactivity and was chosen for the majority of the subsequent experiments. Neutralization of cytotoxicity to IMR-90 cells was determined as described in Example 3. The results of cytotoxicity neutralization are depicted in FIG. 13, which shows that sera from both animals exhibited good toxin B neutralizing antibody titers and very low, but detectable, toxin A neutralizing antibody titers. The ability of the goat sera to protect mice from a lethal intraperitoneal challenge with toxin B (100 ng) was also confirmed (data not shown).
Example 10. Protection of Hamsters from C. difficile Relapse with Anti-Toxin a and Anti-Toxin B Antibodies
[0231] Groups of hamsters (n=20) were challenged with clindamycin and C. difficile, and then treated with vancomycin as described in the hamster model of relapse in Example 7. Antibodies (either 3D8, serum from goat #331, or 3D8 and serum from goat #331) were given twice daily after vancomycin treatment (FIG. 14). Animals were monitored for survival (FIG. 15) or illness (FIG. 16). Antibody doses were 1 ml twice daily for serum from goat #331 and 3 mg for 3D8 given twice daily. Animals receiving vancomycin only (i.e., no antibody treatment) served as a negative controls. As observed previously, 3D8 and vancomycin treatment alone demonstrated a partial protective effect, in which 10 out of 20 animals were protected from lethality (FIG. 15). Fifty percent of animals in this group remained healthy (FIG. 16). Six out of 20 animals receiving vancomycin treatment alone were protected (FIG. 15). Thirty percent remained healthy (FIG. 16). Partial protection (9/20 animals protected) was also observed when the goat serum was used alone (FIG. 15). Forty percent remained healthy. Protection was increased to nearly 100% when both goat serum and 3D8 were given together (18/20) and disease onset was delayed (FIG. 15). Ninety percent of these animals remained healthy (FIG. 16). Clearly, protection from illness followed a pattern similar to protection from lethality. These data demonstrate that 3D8 can be fully protective in the hamster disease model when toxin B is also neutralized.
Example 11. Protection of Hamsters from C. difficile Relapse in Hamsters Immunized with Toxin B
[0232] Hamsters were immunized intraperitoneally with 10 .mu.g of the COOH-terminal fragment of toxin B (corresponding to amino acids 1777-2366 of toxin B) expressed in E. coli and using RIBI as adjuvant. Animals received 7 doses of toxin B antigen. Neutralizing antibody responses were observed in the animals that were tested. Groups of immunized hamsters were challenged with clindamycin and C. difficile then treated with vancomycin as described in the hamster model of relapse in Example 7. Antibody (3D8, 3 mg/dose) was given twice daily after vancomycin treatment to 19 animals and compared to a negative control group (n=20) that received no treatment (FIGS. 17 and 18). Six animals were challenged without vancomycin treatment to ensure that hamsters immunized with toxin B antigen were susceptible to C. difficile infection. Animals were monitored for survival (FIG. 17) or illness (FIG. 18). FIG. 17 shows that immunized animals that were not given 3D8 relapsed at a similar rate to that observed previously (65% relapse). Toxin B-immunized animals receiving 3D8 were more fully protected from relapse than observed previously (10% relapse, as compared to approximately 50% relapse in animals not previously immunized with toxin B in other experiments).
[0233] FIG. 18 shows that some of the immunized animals receiving 3D8 became ill but recovered from their diarrhea. Thirty five percent of immunized animals receiving vancomycin alone remained healthy. In experiments in which toxin B reactive sera were not present in animals, virtually all animals that had diarrhea later died. These data provide further evidence that 3D8 can be fully protective in the hamster disease model when toxin B is also neutralized. Neutralization of toxin B in addition to toxin A was required for optimal protection from C. difficile disease in this model.
Example 12. Protection of Hamsters from Primary C. Difficile Challenge Using 3D8 in Hamsters Treated with Goat Anti-Toxin B Sera
[0234] Prevention of relapse of C. difficile disease in the hamsters was easier to demonstrate than protection from direct challenge (i.e., challenge without vancomycin administration). Experiments with rabbit sera demonstrated only weak protection from direct challenge and 3D8 had no detectable affect on direct challenge. Since 3D8 was more protective in a background of toxin B neutralizing antibodies, it was determined whether the combined administration of 3D8 and anti-toxin B antisera could prevent disease due to direct challenge. Groups of 5 hamsters were challenged after receiving once daily doses of 3D8 (3 mg), combined 3D8 (3 mg) and goat #331 (1 ml) sera, or no antibodies for the 3 days prior to challenge as depicted in FIG. 19. The data in FIG. 20 shows that animals receiving no antibodies or either 3D8 or goat sera alone all died with 48 hours of C. difficile challenge. Most animals (80%) receiving both 3D8 and goat sera survived and the affected animals survived for 10 days after challenge. FIG. 21 shows that animals treated with 3D8 and goat sera became ill but recovered. These data provide further evidence that 3D8 can be fully protective in the hamster disease model when toxin B is also neutralized. Neutralization of toxin B in addition to toxin A was required for optimal protection from C. difficile disease in this model.
[0235] The successful protection of hamsters directly challenged with C. difficile offers several advantages to the screening of new toxin B candidates. Smaller numbers of animals can be used since 100% of untreated animals die. Antibodies, such as monoclonal antibodies (e.g., human monoclonal antibodies) can be screened directly in hamsters because the procedure requires 100 mg or less of the test antibody. Other modes of testing, such as the relapse model, require the effort of producing gram quantities due to the low attack rate in the relapse model, which necessitates testing larger numbers of animals. Direct challenge experiments are also shorter in duration with a definitive read out within 3-4 days of C. difficile challenge compared to 7-10 in the relapse model. In addition, the elimination of vancomycin treatment from the screening method reduces the number of times animals are handled.
Example 13. Generation of Anti-Toxin B Monoclonal Antibodies
[0236] C. difficile toxin B was obtained either from Techlab, Inc. (Blacksburg, Va.), or by recombinant production. The toxin was purified and inactivated prior to immunization. Inactivation was performed by treatment with reactive UDP-dialdehyde, which results in alkylation of catalytic residues while preserving native toxin structure. Briefly, purified toxin B was incubated with UDP-2',3'-dialdehyde (0.1-1.0 mM) in buffer for 18 hours at 37.degree. C., filtered through a 100 kDa-cutoff filter to remove unreacted UDP-2',3'-dialdehyde, and washed with buffer. Inactivated toxin B (toxoid B) or recombinant toxin B fragments were used as immunogens. A toxin B receptor binding domain (amino acid residues 1777-2366) was expressed in E. coli as a fusion protein containing an immunotag (hexahistadine) for affinity purification using nickel chelate affinity chromatography (designated fragment 4; see Example 11).
[0237] Hco12 transgenic mice, generated as described above in the section entitled "Generation of Human Monoclonal Antibodies in HuMAb Mice" and supplied by Medarex, Milpitas, Calif., were immunized intraperitoneally 6-12 times each with 10 .mu.g of toxoid in RIBI adjuvant. In the Hco12 transgenic mice, the endogenous mouse kappa light chain gene has been homozygously disrupted as described in Chen et al. (1993) EMBO J. 12:811-820 and the endogenous mouse heavy chain gene has been homozygously disrupted as described in Example 1 of PCT Publication WO 01/09187. The Hco12 transgenic mice carry a human kappa light chain transgene, KCo5, as described in Fishwild et al. (1996) Nature Biotechnology 14:845-851, and the Hco12 human heavy chain transgene as described in U.S. Pat. Nos. 5,545,806; 5,625,825; and 5,545,807. Serum was collected from each mouse and tested for reactivity to toxin B by ELISA and neutralization of cytotoxicity on IMR-90 cells. Mice that tested positive for toxin B-reactive and neutralizing antiserum were injected with 5-10 .mu.g toxoid B or fragment 4 through the tail vein. Mice were sacrificed and spleens were isolated for fusion to hybridomas approximately 3 days after tail vein injection was performed.
[0238] Clonal hybridomas were generated and screened by ELISA. Three hybridoma clones were selected for further analysis: 124-152; 2A11; and 1 G10. In particular, cDNAs from the 124-152 clone were amplified by RT-PCR from mRNA, cloned, and sequenced. The heavy chain V region was determined to be derived from the germline sequence VH 5-51, the D region derived from the germline sequence 7-27, and the J sequence from the germline region JH3b. The light chain (kappa) regions were determined to be derived from A27 and the J region from JK1. The isotype of the 124-152 clone was determined to be IgG1. The amino acid sequences of the VH and VL regions of the 124-152 clone are shown in FIGS. 27-28. The complementarity determining regions (CDRs) are indicated in the Figures. The related germline sequences of the VH and VL regions are shown in FIGS. 30-31.
[0239] The antibodies 124-152; 2A11; and 1 G10 were isolated from corresponding hybridomas and tested for their binding characteristics (infra). DNA encoding the 124-152 clone was cloned into a vector to be expressed as a human antibody for administration to humans.
Example 14. Binding Activity of Anti-Toxin B Antibodies
[0240] Binding of each antibody to toxin B was determined by Biacore using standard techniques. The results of this assay are depicted in Table 6. Antibodies produced by 124-152; 2A11; and 1 G10 were compared to appropriate controls.
[0241] In particular, the affinity of the 124-152; 2A11; and 1 G10 antibodies for toxin B was measured with Biacore.RTM. instrument, which detects biomolecular binding interactions with surface plasmon resonance technology. Each antibody was added to protein A-coated sensor chips, and toxin B was allowed to flow over the chip to measure binding. 124-152 had a K.sub.D of 1.64.times.10.sup.-10M; 2A11 had a K.sub.D of 0.24.times.10.sup.-10M; and 1 G10 had a K.sub.D of 2.98.times.10.sup.-10M. Thus, the antibodies bind with high affinity to toxin B. These binding constants indicate that the antibodies have affinities suitable for use in vivo application, for example, human therapy.
TABLE-US-00006 TABLE 6 K.sub.D .times. 10.sup.-10 k.sub.a .times. 10.sup.5 k.sub.d .times. 10.sup.-5 Sample ID (M) (1/Ms) (1/s) 2A11 0.24 21 5.07 124.152 1.64 34.5 56.4 51.1G10 2.98 1.31 3.89
Example 15. Toxin Neutralization by Anti-Toxin B Antibodies
[0242] Antibodies expressed by 124-152; 2A11; and 1 G10 hybridomas were tested for toxin B neutralization activity in vitro. Cells were incubated in the presence of varying concentrations of a monoclonal antibody specific to toxin B which would prevent cells from rounding up after exposure to toxin B. Cytopathic effect (CPE) was determined by visual inspection of cells. A CPE score from 0-4 was determined, based on the results of the visual inspection (4=100% cytotoxicity, 0=0% toxicity). The results of these assays are depicted in FIG. 27. Neutralization of toxicity against a human lung fibroblast cell line, IMR-90. FIG. 27 shows that all of the antibodies had neutralizing capacity towards IMR-90 cells. The relative neutralizing activity of toxin A cytotoxicity on IMR-90 cells was 124-152>1 G10>2A11.
Example 16. Protection of Hamsters from Primary C. Difficile Challenge Using Anti-Toxin B Antibodies
[0243] Protection from direct challenge of an inoculum of C. difficile (clindamycin on day -1 and C. difficile spores on day O (1/100,000 dilution) was performed over a period of 4 to 10 days in the presence or absence of anti-toxin B antibodies. Groups of 5 hamsters were challenged after receiving once daily doses of 3D8 (20 mg total over 4 days), combined 3D8 (Id.) and goat #331 (3 ml) sera, 3D8 in combination with anti-toxin B antibodies 124-152 (18 mg total over 4 days), 2A11 (20 mg total over 4 days), or 1 G10 (20 mg total over 4 days) or no antibodies for 3 days prior to challenge as depicted in FIG. 24. The data in FIG. 24 shows that animals receiving no antibodies or either 3D8 or goat sera alone all died within 72 hours of C. difficile challenge whereas animals receiving 3D8 and an anti-toxin B antibody, and preferably in combination with 124-152, had a 40% survival rate (FIG. 24). A 10 day study similar to the foregoing (but using a more dilute C. difficile inoculum) was performed with increasing amounts of the anti-toxin B antibody 124-152 (0.56 mg, 1.7 mg, or 5.0 mg given at days -3, -2, -1, and 0). Animals receiving both 3D8 and goat sera survived and most animals (60%-70%) survived for 10 days after challenge if given 3D8 in combination with 124-152. Even the lowest dosage of the anti-toxin B antibody 124-152 (0.56 mg in combination with 3D8) was highly effective (70% survival; see FIG. 25). Results show that 124-152 and 3D8, alone, are less effective then when used in combination where a more than additive, indeed, synergistic therapeutic result is achieved (FIGS. 24-26). These data provide further evidence that the anti-toxin B antibody is highly effective, especially in combination with the anti-toxin A antibody 3D8. Neutralization of toxin B in addition to toxin A was determined to provide for protection from C. difficile disease in this model.
Example 17. Epitope Mapping of Anti-Toxin B Antibodies
[0244] The epitope of toxin B bound by each monoclonal antibody was determined by western blotting. Recombinant E. coli clones were constructed which express fragments of toxin B representing different domains of toxin B. The appropriate segments of the toxin B gene were PCR-amplified from DNA prepared from an appropriate C. difficile strain. The fragments were cloned into an expression vector and expressed in E. coli. Human monoclonal antibody 152 was used to probe toxin B fragment in western blots in order to map the binding epitope. Toxin B protein fragments were isolated from E. coli containing a portion of the toxin B genes and separated using SDS-PAGE. After electrophoresis, the toxin B fragments were transferred to nitrocellulose and probed with monoclonal antibody 152 followed by alkaline phosphatase conjugated goat anti human to detect MAb 152 binding. HuMab 152 was determined to bind to the --COOH fragment portion of toxin B between amino acids 1777 and 2366 (see, for example, FIG. 32).
Other Embodiments
[0245] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
Sequence CWU
1
1
831122PRTHomo sapiens 1Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln
Pro Gly Arg1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Asn Tyr 20
25 30Gly Met His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Leu Ile Trp Tyr Asp Gly Ser Asn Glu Asp Tyr Thr Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala
Arg Trp Gly Met Val Arg Gly Val Ile Asp Val Phe Asp Ile Trp
100 105 110Gly Gln Gly Thr Val Val Thr
Val Ser Ser 115 1202114PRTHomo sapiens 2Gln Met
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5
10 15Ser Leu Arg Leu Ser Cys Glu Ala
Ser Gly Phe Ser Phe Asn Ser Tyr 20 25
30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ser Val Ile Trp Ala
Ser Gly Asn Lys Lys Tyr Tyr Ile Glu Ser Val 50 55
60Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Ala Asn Phe Asp Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val 100 105
110Ser Ser3116PRTHomo sapiens 3Gln Val Gln Leu Val Glu Ser
Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Asn Lys Tyr 20 25 30Gly Met
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ala Val Ile Trp Tyr Asp Gly Thr Asn Lys
Tyr Tyr Ala Asp Ser Met 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Met Leu Tyr65
70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95Ala Arg Asp Pro Pro Thr Ala Asn Tyr Trp Gly Gln
Gly Thr Leu Val 100 105 110Thr
Val Ser Ser 1154107PRTHomo sapiens 4Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Val Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile
Ser Ser Trp 20 25 30Leu Ala
Trp Tyr Gln His Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Ala Asn Ser Phe Pro Trp 85
90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 1055108PRTHomo sapiens 5Glu Ile Val Leu Thr Gln
Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5
10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
Val Ser Ser Tyr 20 25 30Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35
40 45Tyr Asp Ala Ser Asn Arg Ala Thr Gly
Ile Pro Ala Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65
70 75 80Glu Asp Phe Ala Val
Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Ser Gln 85
90 95Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile
Lys 100 1056107PRTHomo sapiens 6Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5
10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Ser Ser Trp 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile
35 40 45Tyr Ala Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Tyr Lys Ser Tyr Pro Val 85
90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys 100 10575PRTHomo sapiens 7Asn Tyr Gly Met
His1 5817PRTHomo sapiens 8Leu Ile Trp Tyr Asp Gly Ser Asn
Glu Asp Tyr Thr Asp Ser Val Lys1 5 10
15Gly913PRTHomo sapiens 9Trp Gly Met Val Arg Gly Val Ile Asp
Val Phe Asp Ile1 5 10105PRTHomo sapiens
10Ser Tyr Gly Met His1 51117PRTHomo sapiens 11Val Ile Trp
Ala Ser Gly Asn Lys Lys Tyr Tyr Ile Glu Ser Val Glu1 5
10 15Gly125PRTHomo sapiens 12Ala Asn Phe
Asp Tyr1 5135PRTHomo sapiens 13Lys Tyr Gly Met His1
51417PRTHomo sapiens 14Val Ile Trp Tyr Asp Gly Thr Asn Lys Tyr Tyr
Ala Asp Ser Met Lys1 5 10
15Gly157PRTHomo sapiens 15Asp Pro Pro Thr Ala Asn Tyr1
51611PRTHomo sapiens 16Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala1
5 10177PRTHomo sapiens 17Ala Ala Ser Ser Leu
Gln Ser1 5189PRTHomo sapiens 18Gln Gln Ala Asn Ser Phe Pro
Trp Thr1 51911PRTHomo sapiens 19Arg Ala Ser Gln Ser Val Ser
Ser Tyr Leu Ala1 5 10207PRTHomo sapiens
20Asp Ala Ser Asn Arg Ala Thr1 52110PRTHomo sapiens 21Gln
Gln Arg Ser Asn Trp Ser Gln Phe Thr1 5
102211PRTHomo sapiens 22Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala1
5 10237PRTHomo sapiens 23Ala Ala Ser Ser Leu
Gln Ser1 5249PRTHomo sapiens 24Gln Gln Tyr Lys Ser Tyr Pro
Val Thr1 52511PRTHomo sapiensMOD_RES(5)..(6)Any amino
acidMOD_RES(9)..(9)Any amino acid 25Arg Ala Ser Gln Xaa Xaa Ser Ser Xaa
Leu Ala1 5 10266PRTHomo
sapiensMOD_RES(3)..(5)Any amino acidMOD_RES(6)..(6)Ser or Thr 26Ala Ser
Xaa Xaa Xaa Xaa1 5277PRTHomo sapiensMOD_RES(3)..(4)Any
amino acidMOD_RES(5)..(5)Ser or AsnMOD_RES(6)..(6)Any amino
acidMOD_RES(7)..(7)Pro or Ser 27Gln Gln Xaa Xaa Xaa Xaa Xaa1
5284PRTHomo sapiens 28Tyr Gly Met His12915PRTHomo
sapiensMOD_RES(3)..(4)Any amino acidMOD_RES(6)..(8)Any amino
acidMOD_RES(10)..(11)Any amino acidMOD_RES(13)..(14)Any amino acid 29Ile
Trp Xaa Xaa Gly Xaa Xaa Xaa Tyr Xaa Xaa Ser Xaa Xaa Gly1 5
10 1530107PRTHomo sapiens 30Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5
10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Ser Ser Trp 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile
35 40 45Tyr Ala Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Trp 85
90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 10531107PRTHomo sapiens 31Val Ile Trp
Met Thr Gln Ser Pro Ser Leu Leu Ser Ala Ser Thr Gly1 5
10 15Asp Arg Val Thr Ile Ser Cys Arg Met
Ser Gln Gly Ile Ser Ser Tyr 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile
35 40 45Tyr Ala Ala Ser Thr Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Trp 85
90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 10532107PRTHomo sapiens 32Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly1 5
10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Ser Ser Trp 20 25
30Leu Ala Trp Tyr Gln His Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45Tyr Ala Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Trp 85
90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 10533107PRTHomo sapiens 33Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly1 5
10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Ser Ser Trp 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile
35 40 45Tyr Ala Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Trp 85
90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 10534107PRTHomo sapiens 34Asp Ile Gln
Met Thr Gln Ser Leu Ser Ser Leu Ser Ala Ser Val Gly1 5
10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Ser Ser Trp 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile
35 40 45Tyr Ala Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Trp 85
90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 10535321DNAHomo sapiensCDS(1)..(321)
35gac atc cag atg acc cag tct cca tct tcc gtg tct gca tct gta gga
48Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly1
5 10 15gac aga gtc acc atc act
tgt cgg gcg agt cag ggt att agc agc tgg 96Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25
30tta gcc tgg tat cag cat aaa cca ggg aaa gcc cct aag
ctc ctg atc 144Leu Ala Trp Tyr Gln His Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45tat gct gca
tcc agt ttg caa agt ggg gtc cca tca agg ttc agc ggc 192Tyr Ala Ala
Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60agt gga tct ggg aca gat ttc act ctc acc atc agc
agc ctg cag cct 240Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75
80gaa gat ttt gca act tac tat tgt caa cag gct aat agt ttc cct tgg
288Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Trp
85 90 95acg ttc ggc caa ggg acc
aag gtg gaa atc aaa 321Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys 100 10536324DNAHomo
sapiensCDS(1)..(324) 36gaa att gtg ttg aca cag tct cca gcc acc ctg tct
ttg tct cca ggg 48Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser
Leu Ser Pro Gly1 5 10
15gaa aga gcc acc ctc tcc tgc agg gcc agt cag agt gtt agc agc tac
96Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30tta gcc tgg tac caa cag aaa
cct ggc cag gct ccc agg ctc ctc atc 144Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40
45tat gat gca tcc aac agg gcc act ggc atc cca gcc agg ttc agt
ggc 192Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser
Gly 50 55 60agt ggg tct ggg aca gac
ttc act ctc acc atc agc agc cta gag cct 240Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70
75 80gaa gat ttt gca gtt tat tac tgt cag cag cgt
agc aac tgg tct caa 288Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg
Ser Asn Trp Ser Gln 85 90
95ttc act ttc ggc cct ggg acc aaa gtg gat atc aaa
324Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 100
10537321DNAHomo sapiensCDS(1)..(321) 37gac atc cag atg acc cag
tct cca tcc tca ctg tct gca tct gta gga 48Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5
10 15gac aga gtc acc atc act tgt cgg gcg agt cag ggt
att agc agc tgg 96Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly
Ile Ser Ser Trp 20 25 30tta
gcc tgg tat cag cag aaa cca gag aaa gcc cct aag tcc ctg atc 144Leu
Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35
40 45tat gct gca tcc agt ttg caa agt ggg
gtc cca tca agg ttc agc ggc 192Tyr Ala Ala Ser Ser Leu Gln Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55
60agt gga tct ggg aca gat ttc act ctc acc atc agc agc ctg cag cct
240Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80gaa gat ttt gca act
tat tac tgc caa cag tat aag agt tac ccg gtc 288Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Tyr Lys Ser Tyr Pro Val 85
90 95act ttc ggc gga ggg acc aag gtg gag atc aaa
321Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 10538366DNAHomo sapiensCDS(1)..(366) 38cag gtg
cag ctg gtg gag tct ggg gga ggc gtg gtc cag cct ggc agg 48Gln Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5
10 15tcc ctg aga ctc tcc tgt gcg gcg
tct gga ttc agc ttc agt aac tat 96Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Ser Phe Ser Asn Tyr 20 25
30ggc atg cac tgg gtc cgc cag gct cca ggc aag ggg ctg gag tgg
gtg 144Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45gca ctt ata tgg tat
gat gga agt aat gag gac tat aca gac tcc gtg 192Ala Leu Ile Trp Tyr
Asp Gly Ser Asn Glu Asp Tyr Thr Asp Ser Val 50 55
60aag ggc cga ttc acc atc tcc aga gac aat tcc aag aac acg
ctg tat 240Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80ctg
caa atg aac agc ctg aga gcc gag gac acg gct gtg tat tac tgt 288Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95gcg aga tgg ggg atg gtt cgg
gga gtt atc gat gtt ttt gat atc tgg 336Ala Arg Trp Gly Met Val Arg
Gly Val Ile Asp Val Phe Asp Ile Trp 100 105
110ggc caa ggg aca gtg gtc acc gtc tct tca
366Gly Gln Gly Thr Val Val Thr Val Ser Ser 115
12039342DNAHomo sapiensCDS(1)..(342) 39cag atg cag ctg gtg gag tct
ggg ggc ggc gtg gtc cag cct ggg agg 48Gln Met Gln Leu Val Glu Ser
Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10
15tcc ctg aga ctc tcc tgt gaa gcg tct gga ttc tcc ttc
aat agc tat 96Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Ser Phe
Asn Ser Tyr 20 25 30ggc atg
cac tgg gtc cgc cag gct cca ggc aag ggg ctg gag tgg gtg 144Gly Met
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45tca gtc ata tgg gcc agt gga aat aag aaa
tat tat ata gaa tcc gtg 192Ser Val Ile Trp Ala Ser Gly Asn Lys Lys
Tyr Tyr Ile Glu Ser Val 50 55 60gag
ggc cga ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat 240Glu
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65
70 75 80ctg caa atg aac agc ctg
aga gcc gag gac acg gct gtg tat tac tgt 288Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95gcg aga gcc aat ttt gac tac tgg ggc cag gga acc
ctg gtc acc gtc 336Ala Arg Ala Asn Phe Asp Tyr Trp Gly Gln Gly Thr
Leu Val Thr Val 100 105 110tcc
tca 342Ser
Ser40348DNAHomo sapiensCDS(1)..(348) 40cag gtg cag ctg gtg gag tct ggg
gga ggc gtg gtc cag cct ggg agg 48Gln Val Gln Leu Val Glu Ser Gly
Gly Gly Val Val Gln Pro Gly Arg1 5 10
15tcc ctg aga ctc tcc tgt gca gcg tct gga ttc acc ttc aat
aaa tat 96Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn
Lys Tyr 20 25 30ggc atg cac
tgg gtc cgc cag gct cca ggc aag ggg ctg gag tgg gtg 144Gly Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45gca gtt ata tgg tat gat gga act aat aaa tac
tat gca gac tcc atg 192Ala Val Ile Trp Tyr Asp Gly Thr Asn Lys Tyr
Tyr Ala Asp Ser Met 50 55 60aag ggc
cga ttc acc atc tcc aga gac aat tcc aag aat atg ctg tat 240Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Met Leu Tyr65
70 75 80ctg caa atg aac agc cta aga
gcc gag gac acg gct gtg tat tac tgt 288Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95gcg aga gat ccc ccc act gct aac tac tgg ggc cag gga
acc ctg gtc 336Ala Arg Asp Pro Pro Thr Ala Asn Tyr Trp Gly Gln Gly
Thr Leu Val 100 105 110acc gtc
tcc tca 348Thr Val
Ser Ser 115412710PRTClostridium difficile 41Met Ser Leu Ile Ser
Lys Glu Glu Leu Ile Lys Leu Ala Tyr Ser Ile1 5
10 15Arg Pro Arg Glu Asn Glu Tyr Lys Thr Ile Leu
Thr Asn Leu Asp Glu 20 25
30Tyr Asn Lys Leu Thr Thr Asn Asn Asn Glu Asn Lys Tyr Leu Gln Leu
35 40 45Lys Lys Leu Asn Glu Ser Ile Asp
Val Phe Met Asn Lys Tyr Lys Thr 50 55
60Ser Ser Arg Asn Arg Ala Leu Ser Asn Leu Lys Lys Asp Ile Leu Lys65
70 75 80Glu Val Ile Leu Ile
Lys Asn Ser Asn Thr Ser Pro Val Glu Lys Asn 85
90 95Leu His Phe Val Trp Ile Gly Gly Glu Val Ser
Asp Ile Ala Leu Glu 100 105
110Tyr Ile Lys Gln Trp Ala Asp Ile Asn Ala Glu Tyr Asn Ile Lys Leu
115 120 125Trp Tyr Asp Ser Glu Ala Phe
Leu Val Asn Thr Leu Lys Lys Ala Ile 130 135
140Val Glu Ser Ser Thr Thr Glu Ala Leu Gln Leu Leu Glu Glu Glu
Ile145 150 155 160Gln Asn
Pro Gln Phe Asp Asn Met Lys Phe Tyr Lys Lys Arg Met Glu
165 170 175Phe Ile Tyr Asp Arg Gln Lys
Arg Phe Ile Asn Tyr Tyr Lys Ser Gln 180 185
190Ile Asn Lys Pro Thr Val Pro Thr Ile Asp Asp Ile Ile Lys
Ser His 195 200 205Leu Val Ser Glu
Tyr Asn Arg Asp Glu Thr Val Leu Glu Ser Tyr Arg 210
215 220Thr Asn Ser Leu Arg Lys Ile Asn Ser Asn His Gly
Ile Asp Ile Arg225 230 235
240Ala Asn Ser Leu Phe Thr Glu Gln Glu Leu Leu Asn Ile Tyr Ser Gln
245 250 255Glu Leu Leu Asn Arg
Gly Asn Leu Ala Ala Ala Ser Asp Ile Val Arg 260
265 270Leu Leu Ala Leu Lys Asn Phe Gly Gly Val Tyr Leu
Asp Val Asp Met 275 280 285Leu Pro
Gly Ile His Ser Asp Leu Phe Lys Thr Ile Ser Arg Pro Ser 290
295 300Ser Ile Gly Leu Asp Arg Trp Glu Met Ile Lys
Leu Glu Ala Ile Met305 310 315
320Lys Tyr Lys Lys Tyr Ile Asn Asn Tyr Thr Ser Glu Asn Phe Asp Lys
325 330 335Leu Asp Gln Gln
Leu Lys Asp Asn Phe Lys Leu Ile Ile Glu Ser Lys 340
345 350Ser Glu Lys Ser Glu Ile Phe Ser Lys Leu Glu
Asn Leu Asn Val Ser 355 360 365Asp
Leu Glu Ile Lys Ile Ala Phe Ala Leu Gly Ser Val Ile Asn Gln 370
375 380Ala Leu Ile Ser Lys Gln Gly Ser Tyr Leu
Thr Asn Leu Val Ile Glu385 390 395
400Gln Val Lys Asn Arg Tyr Gln Phe Leu Asn Gln His Leu Asn Pro
Ala 405 410 415Ile Glu Ser
Asp Asn Asn Phe Thr Asp Thr Thr Lys Ile Phe His Asp 420
425 430Ser Leu Phe Asn Ser Ala Thr Ala Glu Asn
Ser Met Phe Leu Thr Lys 435 440
445Ile Ala Pro Tyr Leu Gln Val Gly Phe Met Pro Glu Ala Arg Ser Thr 450
455 460Ile Ser Leu Ser Gly Pro Gly Ala
Tyr Ala Ser Ala Tyr Tyr Asp Phe465 470
475 480Ile Asn Leu Gln Glu Asn Thr Ile Glu Lys Thr Leu
Lys Ala Ser Asp 485 490
495Leu Ile Glu Phe Lys Phe Pro Glu Asn Asn Leu Ser Gln Leu Thr Glu
500 505 510Gln Glu Ile Asn Ser Leu
Trp Ser Phe Asp Gln Ala Ser Ala Lys Tyr 515 520
525Gln Phe Glu Lys Tyr Val Arg Asp Tyr Thr Gly Gly Ser Leu
Ser Glu 530 535 540Asp Asn Gly Val Asp
Phe Asn Lys Asn Thr Ala Leu Asp Lys Asn Tyr545 550
555 560Leu Leu Asn Asn Lys Ile Pro Ser Asn Asn
Val Glu Glu Ala Gly Ser 565 570
575Lys Asn Tyr Val His Tyr Ile Ile Gln Leu Gln Gly Asp Asp Ile Ser
580 585 590Tyr Glu Ala Thr Cys
Asn Leu Phe Ser Lys Asn Pro Lys Asn Ser Ile 595
600 605Ile Ile Gln Arg Asn Met Asn Glu Ser Ala Lys Ser
Tyr Phe Leu Ser 610 615 620Asp Asp Gly
Glu Ser Ile Leu Glu Leu Asn Lys Tyr Arg Ile Pro Glu625
630 635 640Arg Leu Lys Asn Lys Glu Lys
Val Lys Val Thr Phe Ile Gly His Gly 645
650 655Lys Asp Glu Phe Asn Thr Ser Glu Phe Ala Arg Leu
Ser Val Asp Ser 660 665 670Leu
Ser Asn Glu Ile Ser Ser Phe Leu Asp Thr Ile Lys Leu Asp Ile 675
680 685Ser Pro Lys Asn Val Glu Val Asn Leu
Leu Gly Cys Asn Met Phe Ser 690 695
700Tyr Asp Phe Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu Leu Leu Ser705
710 715 720Ile Met Asp Lys
Ile Thr Ser Thr Leu Pro Asp Val Asn Lys Asn Ser 725
730 735Ile Thr Ile Gly Ala Asn Gln Tyr Glu Val
Arg Ile Asn Ser Glu Gly 740 745
750Arg Lys Glu Leu Leu Ala His Ser Gly Lys Trp Ile Asn Lys Glu Glu
755 760 765Ala Ile Met Ser Asp Leu Ser
Ser Lys Glu Tyr Ile Phe Phe Asp Ser 770 775
780Ile Asp Asn Lys Leu Lys Ala Lys Ser Lys Asn Ile Pro Gly Leu
Ala785 790 795 800Ser Ile
Ser Glu Asp Ile Lys Thr Leu Leu Leu Asp Ala Ser Val Ser
805 810 815Pro Asp Thr Lys Phe Ile Leu
Asn Asn Leu Lys Leu Asn Ile Glu Ser 820 825
830Ser Ile Gly Asp Tyr Ile Tyr Tyr Glu Lys Leu Glu Pro Val
Lys Asn 835 840 845Ile Ile His Asn
Ser Ile Asp Asp Leu Ile Asp Glu Phe Asn Leu Leu 850
855 860Glu Asn Val Ser Asp Glu Leu Tyr Glu Leu Lys Lys
Leu Asn Asn Leu865 870 875
880Asp Glu Lys Tyr Leu Ile Ser Phe Glu Asp Ile Ser Lys Asn Asn Ser
885 890 895Thr Tyr Ser Val Arg
Phe Ile Asn Lys Ser Asn Gly Glu Ser Val Tyr 900
905 910Val Glu Thr Glu Lys Glu Ile Phe Ser Lys Tyr Ser
Glu His Ile Thr 915 920 925Lys Glu
Ile Ser Thr Ile Lys Asn Ser Ile Ile Thr Asp Val Asn Gly 930
935 940Asn Leu Leu Asp Asn Ile Gln Leu Asp His Thr
Ser Gln Val Asn Thr945 950 955
960Leu Asn Ala Ala Phe Phe Ile Gln Ser Leu Ile Asp Tyr Ser Ser Asn
965 970 975Lys Asp Val Leu
Asn Asp Leu Ser Thr Ser Val Lys Val Gln Leu Tyr 980
985 990Ala Gln Leu Phe Ser Thr Gly Leu Asn Thr Ile
Tyr Asp Ser Ile Gln 995 1000
1005Leu Val Asn Leu Ile Ser Asn Ala Val Asn Asp Thr Ile Asn Val
1010 1015 1020Leu Pro Thr Ile Thr Glu
Gly Ile Pro Ile Val Ser Thr Ile Leu 1025 1030
1035Asp Gly Ile Asn Leu Gly Ala Ala Ile Lys Glu Leu Leu Asp
Glu 1040 1045 1050His Asp Pro Leu Leu
Lys Lys Glu Leu Glu Ala Lys Val Gly Val 1055 1060
1065Leu Ala Ile Asn Met Ser Leu Ser Ile Ala Ala Thr Val
Ala Ser 1070 1075 1080Ile Val Gly Ile
Gly Ala Glu Val Thr Ile Phe Leu Leu Pro Ile 1085
1090 1095Ala Gly Ile Ser Ala Gly Ile Pro Ser Leu Val
Asn Asn Glu Leu 1100 1105 1110Ile Leu
His Asp Lys Ala Thr Ser Val Val Asn Tyr Phe Asn His 1115
1120 1125Leu Ser Glu Ser Lys Lys Tyr Gly Pro Leu
Lys Thr Glu Asp Asp 1130 1135 1140Lys
Ile Leu Val Pro Ile Asp Asp Leu Val Ile Ser Glu Ile Asp 1145
1150 1155Phe Asn Asn Asn Ser Ile Lys Leu Gly
Thr Cys Asn Ile Leu Ala 1160 1165
1170Met Glu Gly Gly Ser Gly His Thr Val Thr Gly Asn Ile Asp His
1175 1180 1185Phe Phe Ser Ser Pro Ser
Ile Ser Ser His Ile Pro Ser Leu Ser 1190 1195
1200Ile Tyr Ser Ala Ile Gly Ile Glu Thr Glu Asn Leu Asp Phe
Ser 1205 1210 1215Lys Lys Ile Met Met
Leu Pro Asn Ala Pro Ser Arg Val Phe Trp 1220 1225
1230Trp Glu Thr Gly Ala Val Pro Gly Leu Arg Ser Leu Glu
Asn Asp 1235 1240 1245Gly Thr Arg Leu
Leu Asp Ser Ile Arg Asp Leu Tyr Pro Gly Lys 1250
1255 1260Phe Tyr Trp Arg Phe Tyr Ala Phe Phe Asp Tyr
Ala Ile Thr Thr 1265 1270 1275Leu Lys
Pro Val Tyr Glu Asp Thr Asn Ile Lys Ile Lys Leu Asp 1280
1285 1290Lys Asp Thr Arg Asn Phe Ile Met Pro Thr
Ile Thr Thr Asn Glu 1295 1300 1305Ile
Arg Asn Lys Leu Ser Tyr Ser Phe Asp Gly Ala Gly Gly Thr 1310
1315 1320Tyr Ser Leu Leu Leu Ser Ser Tyr Pro
Ile Ser Thr Asn Ile Asn 1325 1330
1335Leu Ser Lys Asp Asp Leu Trp Ile Phe Asn Ile Asp Asn Glu Val
1340 1345 1350Arg Glu Ile Ser Ile Glu
Asn Gly Thr Ile Lys Lys Gly Lys Leu 1355 1360
1365Ile Lys Asp Val Leu Ser Lys Ile Asp Ile Asn Lys Asn Lys
Leu 1370 1375 1380Ile Ile Gly Asn Gln
Thr Ile Asp Phe Ser Gly Asp Ile Asp Asn 1385 1390
1395Lys Asp Arg Tyr Ile Phe Leu Thr Cys Glu Leu Asp Asp
Lys Ile 1400 1405 1410Ser Leu Ile Ile
Glu Ile Asn Leu Val Ala Lys Ser Tyr Ser Leu 1415
1420 1425Leu Leu Ser Gly Asp Lys Asn Tyr Leu Ile Ser
Asn Leu Ser Asn 1430 1435 1440Thr Ile
Glu Lys Ile Asn Thr Leu Gly Leu Asp Ser Lys Asn Ile 1445
1450 1455Ala Tyr Asn Tyr Thr Asp Glu Ser Asn Asn
Lys Tyr Phe Gly Ala 1460 1465 1470Ile
Ser Lys Thr Ser Gln Lys Ser Ile Ile His Tyr Lys Lys Asp 1475
1480 1485Ser Lys Asn Ile Leu Glu Phe Tyr Asn
Asp Ser Thr Leu Glu Phe 1490 1495
1500Asn Ser Lys Asp Phe Ile Ala Glu Asp Ile Asn Val Phe Met Lys
1505 1510 1515Asp Asp Ile Asn Thr Ile
Thr Gly Lys Tyr Tyr Val Asp Asn Asn 1520 1525
1530Thr Asp Lys Ser Ile Asp Phe Ser Ile Ser Leu Val Ser Lys
Asn 1535 1540 1545Gln Val Lys Val Asn
Gly Leu Tyr Leu Asn Glu Ser Val Tyr Ser 1550 1555
1560Ser Tyr Leu Asp Phe Val Lys Asn Ser Asp Gly His His
Asn Thr 1565 1570 1575Ser Asn Phe Met
Asn Leu Phe Leu Asp Asn Ile Ser Phe Trp Lys 1580
1585 1590Leu Phe Gly Phe Glu Asn Ile Asn Phe Val Ile
Asp Lys Tyr Phe 1595 1600 1605Thr Leu
Val Gly Lys Thr Asn Leu Gly Tyr Val Glu Phe Ile Cys 1610
1615 1620Asp Asn Asn Lys Asn Ile Asp Ile Tyr Phe
Gly Glu Trp Lys Thr 1625 1630 1635Ser
Ser Ser Lys Ser Thr Ile Phe Ser Gly Asn Gly Arg Asn Val 1640
1645 1650Val Val Glu Pro Ile Tyr Asn Pro Asp
Thr Gly Glu Asp Ile Ser 1655 1660
1665Thr Ser Leu Asp Phe Ser Tyr Glu Pro Leu Tyr Gly Ile Asp Arg
1670 1675 1680Tyr Ile Asn Lys Val Leu
Ile Ala Pro Asp Leu Tyr Thr Ser Leu 1685 1690
1695Ile Asn Ile Asn Thr Asn Tyr Tyr Ser Asn Glu Tyr Tyr Pro
Glu 1700 1705 1710Ile Ile Val Leu Asn
Pro Asn Thr Phe His Lys Lys Val Asn Ile 1715 1720
1725Asn Leu Asp Ser Ser Ser Phe Glu Tyr Lys Trp Ser Thr
Glu Gly 1730 1735 1740Ser Asp Phe Ile
Leu Val Arg Tyr Leu Glu Glu Ser Asn Lys Lys 1745
1750 1755Ile Leu Gln Lys Ile Arg Ile Lys Gly Ile Leu
Ser Asn Thr Gln 1760 1765 1770Ser Phe
Asn Lys Met Ser Ile Asp Phe Lys Asp Ile Lys Lys Leu 1775
1780 1785Ser Leu Gly Tyr Ile Met Ser Asn Phe Lys
Ser Phe Asn Ser Glu 1790 1795 1800Asn
Glu Leu Asp Arg Asp His Leu Gly Phe Lys Ile Ile Asp Asn 1805
1810 1815Lys Thr Tyr Tyr Tyr Asp Glu Asp Ser
Lys Leu Val Lys Gly Leu 1820 1825
1830Ile Asn Ile Asn Asn Ser Leu Phe Tyr Phe Asp Pro Ile Glu Phe
1835 1840 1845Asn Leu Val Thr Gly Trp
Gln Thr Ile Asn Gly Lys Lys Tyr Tyr 1850 1855
1860Phe Asp Ile Asn Thr Gly Ala Ala Leu Thr Ser Tyr Lys Ile
Ile 1865 1870 1875Asn Gly Lys His Phe
Tyr Phe Asn Asn Asp Gly Val Met Gln Leu 1880 1885
1890Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr Phe Ala
Pro Ala 1895 1900 1905Asn Thr Gln Asn
Asn Asn Ile Glu Gly Gln Ala Ile Val Tyr Gln 1910
1915 1920Ser Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr
Tyr Phe Asp Asn 1925 1930 1935Asn Ser
Lys Ala Val Thr Gly Trp Arg Ile Ile Asn Asn Glu Lys 1940
1945 1950Tyr Tyr Phe Asn Pro Asn Asn Ala Ile Ala
Ala Val Gly Leu Gln 1955 1960 1965Val
Ile Asp Asn Asn Lys Tyr Tyr Phe Asn Pro Asp Thr Ala Ile 1970
1975 1980Ile Ser Lys Gly Trp Gln Thr Val Asn
Gly Ser Arg Tyr Tyr Phe 1985 1990
1995Asp Thr Asp Thr Ala Ile Ala Phe Asn Gly Tyr Lys Thr Ile Asp
2000 2005 2010Gly Lys His Phe Tyr Phe
Asp Ser Asp Cys Val Val Lys Ile Gly 2015 2020
2025Val Phe Ser Thr Ser Asn Gly Phe Glu Tyr Phe Ala Pro Ala
Asn 2030 2035 2040Thr Tyr Asn Asn Asn
Ile Glu Gly Gln Ala Ile Val Tyr Gln Ser 2045 2050
2055Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Asp
Asn Asn 2060 2065 2070Ser Lys Ala Val
Thr Gly Trp Gln Thr Ile Asp Ser Lys Lys Tyr 2075
2080 2085Tyr Phe Asn Thr Asn Thr Ala Glu Ala Ala Thr
Gly Trp Gln Thr 2090 2095 2100Ile Asp
Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ala Glu Ala 2105
2110 2115Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys
Lys Tyr Tyr Phe Asn 2120 2125 2130Thr
Asn Thr Ala Ile Ala Ser Thr Gly Tyr Thr Ile Ile Asn Gly 2135
2140 2145Lys His Phe Tyr Phe Asn Thr Asp Gly
Ile Met Gln Ile Gly Val 2150 2155
2160Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr
2165 2170 2175Asp Ala Asn Asn Ile Glu
Gly Gln Ala Ile Leu Tyr Gln Asn Glu 2180 2185
2190Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp
Ser 2195 2200 2205Lys Ala Val Thr Gly
Trp Arg Ile Ile Asn Asn Lys Lys Tyr Tyr 2210 2215
2220Phe Asn Pro Asn Asn Ala Ile Ala Ala Ile His Leu Cys
Thr Ile 2225 2230 2235Asn Asn Asp Lys
Tyr Tyr Phe Ser Tyr Asp Gly Ile Leu Gln Asn 2240
2245 2250Gly Tyr Ile Thr Ile Glu Arg Asn Asn Phe Tyr
Phe Asp Ala Asn 2255 2260 2265Asn Glu
Ser Lys Met Val Thr Gly Val Phe Lys Gly Pro Asn Gly 2270
2275 2280Phe Glu Tyr Phe Ala Pro Ala Asn Thr His
Asn Asn Asn Ile Glu 2285 2290 2295Gly
Gln Ala Ile Val Tyr Gln Asn Lys Phe Leu Thr Leu Asn Gly 2300
2305 2310Lys Lys Tyr Tyr Phe Asp Asn Asp Ser
Lys Ala Val Thr Gly Trp 2315 2320
2325Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn Thr Ala
2330 2335 2340Glu Ala Ala Thr Gly Trp
Gln Thr Ile Asp Gly Lys Lys Tyr Tyr 2345 2350
2355Phe Asn Leu Asn Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr
Ile 2360 2365 2370Asp Gly Lys Lys Tyr
Tyr Phe Asn Thr Asn Thr Phe Ile Ala Ser 2375 2380
2385Thr Gly Tyr Thr Ser Ile Asn Gly Lys His Phe Tyr Phe
Asn Thr 2390 2395 2400Asp Gly Ile Met
Gln Ile Gly Val Phe Lys Gly Pro Asn Gly Phe 2405
2410 2415Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn
Asn Ile Glu Gly 2420 2425 2430Gln Ala
Ile Leu Tyr Gln Asn Lys Phe Leu Thr Leu Asn Gly Lys 2435
2440 2445Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Ala
Val Thr Gly Leu Arg 2450 2455 2460Thr
Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ala Val 2465
2470 2475Ala Val Thr Gly Trp Gln Thr Ile Asn
Gly Lys Lys Tyr Tyr Phe 2480 2485
2490Asn Thr Asn Thr Ser Ile Ala Ser Thr Gly Tyr Thr Ile Ile Ser
2495 2500 2505Gly Lys His Phe Tyr Phe
Asn Thr Asp Gly Ile Met Gln Ile Gly 2510 2515
2520Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr Phe Ala Pro Ala
Asn 2525 2530 2535Thr Asp Ala Asn Asn
Ile Glu Gly Gln Ala Ile Arg Tyr Gln Asn 2540 2545
2550Arg Phe Leu Tyr Leu His Asp Asn Ile Tyr Tyr Phe Gly
Asn Asn 2555 2560 2565Ser Lys Ala Ala
Thr Gly Trp Val Thr Ile Asp Gly Asn Arg Tyr 2570
2575 2580Tyr Phe Glu Pro Asn Thr Ala Met Gly Ala Asn
Gly Tyr Lys Thr 2585 2590 2595Ile Asp
Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gln Ile 2600
2605 2610Gly Val Phe Lys Gly Ser Asn Gly Phe Glu
Tyr Phe Ala Pro Ala 2615 2620 2625Asn
Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Arg Tyr Gln 2630
2635 2640Asn Arg Phe Leu His Leu Leu Gly Lys
Ile Tyr Tyr Phe Gly Asn 2645 2650
2655Asn Ser Lys Ala Val Thr Gly Trp Gln Thr Ile Asn Gly Lys Val
2660 2665 2670Tyr Tyr Phe Met Pro Asp
Thr Ala Met Ala Ala Ala Gly Gly Leu 2675 2680
2685Phe Glu Ile Asp Gly Val Ile Tyr Phe Phe Gly Val Asp Gly
Val 2690 2695 2700Lys Ala Pro Gly Ile
Tyr Gly 2705 2710422367PRTClostridium difficile 42Met
Ser Leu Val Asn Arg Lys Gln Leu Glu Lys Met Ala Asn Val Arg1
5 10 15Phe Arg Val Gln Glu Asp Glu
Tyr Val Ala Ile Leu Asp Ala Leu Glu 20 25
30Glu Tyr His Asn Met Ser Glu Asn Thr Val Val Glu Lys Tyr
Leu Lys 35 40 45Leu Lys Asp Ile
Asn Ser Leu Thr Asp Thr Tyr Ile Asp Thr Tyr Lys 50 55
60Lys Ser Gly Arg Asn Lys Ala Leu Lys Lys Phe Lys Glu
Tyr Leu Val65 70 75
80Ile Glu Ile Leu Glu Leu Lys Asn Ser Asn Leu Thr Pro Val Glu Lys
85 90 95Asn Leu His Phe Ile Trp
Ile Gly Gly Gln Ile Asn Asp Thr Ala Ile 100
105 110Asn Tyr Ile Asn Gln Trp Lys Asp Val Asn Ser Asp
Tyr Asn Val Asn 115 120 125Val Phe
Tyr Asp Ser Asn Ala Phe Leu Ile Asn Thr Leu Lys Lys Thr 130
135 140Ile Ile Glu Ser Ala Ser Asn Asp Thr Leu Glu
Ser Phe Arg Glu Asn145 150 155
160Leu Asn Asp Pro Glu Phe Asn His Thr Ala Phe Phe Arg Lys Arg Met
165 170 175Gln Ile Ile Tyr
Asp Lys Gln Gln Asn Phe Ile Asn Tyr Tyr Lys Ala 180
185 190Gln Lys Glu Glu Asn Pro Asp Leu Ile Ile Asp
Asp Ile Val Lys Thr 195 200 205Tyr
Leu Ser Asn Glu Tyr Ser Lys Asp Ile Asp Glu Leu Asn Ala Tyr 210
215 220Ile Glu Glu Ser Leu Asn Lys Val Thr Glu
Asn Ser Gly Asn Asp Val225 230 235
240Arg Asn Phe Glu Glu Phe Lys Thr Gly Glu Val Phe Asn Leu Tyr
Glu 245 250 255Gln Glu Ser
Val Glu Arg Trp Asn Leu Ala Gly Ala Ser Asp Ile Leu 260
265 270Arg Val Ala Ile Leu Lys Asn Ile Gly Gly
Val Tyr Leu Asp Val Asp 275 280
285Met Leu Pro Gly Ile His Pro Asp Leu Phe Lys Asp Ile Asn Lys Pro 290
295 300Asp Ser Val Lys Thr Ala Val Asp
Trp Glu Glu Met Gln Leu Glu Ala305 310
315 320Ile Met Lys His Lys Glu Tyr Ile Pro Glu Tyr Thr
Ser Lys His Phe 325 330
335Asp Thr Leu Asp Glu Glu Val Gln Ser Ser Phe Glu Ser Val Leu Ala
340 345 350Ser Lys Ser Asp Lys Ser
Glu Ile Phe Leu Pro Leu Gly Asp Ile Glu 355 360
365Val Ser Pro Leu Glu Val Lys Ile Ala Phe Ala Lys Gly Ser
Ile Ile 370 375 380Asn Gln Ala Leu Ile
Ser Ala Lys Asp Ser Tyr Cys Ser Asp Leu Leu385 390
395 400Ile Lys Gln Ile Gln Asn Arg Tyr Lys Ile
Leu Asn Asp Thr Leu Gly 405 410
415Pro Ile Ile Ser Gln Gly Asn Asp Phe Asn Thr Thr Met Asn Asn Phe
420 425 430Gly Glu Ser Leu Gly
Ala Ile Ala Asn Glu Glu Asn Ile Ser Phe Ile 435
440 445Ala Lys Ile Gly Ser Tyr Leu Arg Val Gly Phe Tyr
Pro Glu Ala Asn 450 455 460Thr Thr Ile
Thr Leu Ser Gly Pro Thr Ile Tyr Ala Gly Ala Tyr Lys465
470 475 480Asp Leu Leu Thr Phe Lys Glu
Met Ser Ile Asp Thr Ser Ile Leu Ser 485
490 495Ser Glu Leu Arg Asn Phe Glu Phe Pro Lys Val Asn
Ile Ser Gln Ala 500 505 510Thr
Glu Gln Glu Lys Asn Ser Leu Trp Gln Phe Asn Glu Glu Arg Ala 515
520 525Lys Ile Gln Phe Glu Glu Tyr Lys Lys
Asn Tyr Phe Glu Gly Ala Leu 530 535
540Gly Glu Asp Asp Asn Leu Asp Phe Ser Gln Asn Thr Val Thr Asp Lys545
550 555 560Glu Tyr Leu Leu
Glu Lys Ile Ser Ser Ser Thr Lys Ser Ser Glu Gly 565
570 575Gly Tyr Val His Tyr Ile Val Gln Leu Gln
Gly Asp Lys Ile Ser Tyr 580 585
590Glu Ala Ala Cys Asn Leu Phe Ala Lys Asn Pro Tyr Asp Ser Ile Leu
595 600 605Phe Gln Arg Asn Ile Glu Asp
Ser Glu Val Ala Tyr Tyr Tyr Asn Pro 610 615
620Thr Asp Ser Glu Ile Gln Glu Ile Asp Lys Tyr Arg Ile Pro Asp
Arg625 630 635 640Ile Ser
Asp Arg Pro Lys Ile Lys Leu Thr Phe Ile Gly His Gly Lys
645 650 655Ala Glu Phe Asn Thr Asp Ile
Phe Ala Gly Leu Asp Val Asp Ser Leu 660 665
670Ser Ser Glu Ile Glu Thr Ala Ile Gly Leu Ala Lys Glu Asp
Ile Ser 675 680 685Pro Lys Ser Ile
Glu Ile Asn Leu Leu Gly Cys Asn Met Phe Ser Tyr 690
695 700Ser Val Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu
Leu Leu Arg Val705 710 715
720Lys Asp Lys Val Ser Glu Leu Met Pro Ser Met Ser Gln Asp Ser Ile
725 730 735Ile Val Ser Ala Asn
Gln Tyr Glu Val Arg Ile Asn Ser Glu Gly Arg 740
745 750Arg Glu Leu Leu Asp His Ser Gly Glu Trp Ile Asn
Lys Glu Glu Ser 755 760 765Ile Ile
Lys Asp Ile Ser Ser Lys Glu Tyr Ile Ser Phe Asn Pro Lys 770
775 780Glu Asn Lys Ile Ile Val Lys Ser Lys Asn Leu
Pro Glu Leu Ser Thr785 790 795
800Leu Leu Gln Glu Ile Arg Asn Asn Ser Asn Ser Ser Asp Ile Glu Leu
805 810 815Glu Glu Lys Val
Met Leu Ala Glu Cys Glu Ile Asn Val Ile Ser Asn 820
825 830Ile Glu Thr Gln Val Val Glu Glu Arg Ile Glu
Glu Ala Lys Ser Leu 835 840 845Thr
Ser Asp Ser Ile Asn Tyr Ile Lys Asn Glu Phe Lys Leu Ile Glu 850
855 860Ser Ile Ser Glu Ala Leu Cys Asp Leu Lys
Gln Gln Asn Glu Leu Glu865 870 875
880Asp Ser His Phe Ile Ser Phe Glu Asp Ile Ser Glu Thr Asp Glu
Gly 885 890 895Phe Ser Ile
Arg Phe Ile Asn Lys Glu Thr Gly Glu Ser Ile Phe Val 900
905 910Glu Thr Glu Lys Thr Ile Phe Ser Glu Tyr
Ala Asn His Ile Thr Glu 915 920
925Glu Ile Ser Lys Ile Lys Gly Thr Ile Phe Asp Thr Val Asn Gly Lys 930
935 940Leu Val Lys Lys Val Asn Leu Asp
Thr Thr His Glu Val Asn Thr Leu945 950
955 960Asn Ala Ala Phe Phe Ile Gln Ser Leu Ile Glu Tyr
Asn Ser Ser Lys 965 970
975Glu Ser Leu Ser Asn Leu Ser Val Ala Met Lys Val Gln Val Tyr Ala
980 985 990Gln Leu Phe Ser Thr Gly
Leu Asn Thr Ile Thr Asp Ala Ala Lys Val 995 1000
1005Val Glu Leu Val Ser Thr Ala Leu Asp Glu Thr Ile
Asp Leu Leu 1010 1015 1020Pro Thr Leu
Ser Glu Gly Leu Pro Ile Ile Ala Thr Ile Ile Asp 1025
1030 1035Gly Val Ser Leu Gly Ala Ala Ile Lys Glu Leu
Ser Glu Thr Ser 1040 1045 1050Asp Pro
Leu Leu Arg Gln Glu Ile Glu Ala Lys Ile Gly Ile Met 1055
1060 1065Ala Val Asn Leu Thr Thr Ala Thr Thr Ala
Ile Ile Thr Ser Ser 1070 1075 1080Leu
Gly Ile Ala Ser Gly Phe Ser Ile Leu Leu Val Pro Leu Ala 1085
1090 1095Gly Ile Ser Ala Gly Ile Pro Ser Leu
Val Asn Asn Glu Leu Val 1100 1105
1110Leu Arg Asp Lys Ala Thr Lys Val Val Asp Tyr Phe Lys His Val
1115 1120 1125Ser Leu Val Glu Thr Glu
Gly Val Phe Thr Leu Leu Asp Asp Lys 1130 1135
1140Val Met Met Gln Gln Asp Asp Leu Val Ile Ser Glu Ile Asp
Phe 1145 1150 1155Asn Asn Asn Ser Ile
Val Leu Gly Lys Cys Glu Ile Trp Arg Met 1160 1165
1170Glu Gly Gly Ser Gly His Thr Val Thr Asp Asp Ile Asp
His Phe 1175 1180 1185Phe Ser Ala Pro
Ser Ile Thr Tyr Arg Glu Pro His Leu Ser Ile 1190
1195 1200Tyr Asp Val Leu Glu Val Gln Lys Glu Glu Leu
Asp Leu Ser Lys 1205 1210 1215Asp Leu
Met Val Leu Pro Asn Ala Pro Asn Arg Val Phe Ala Trp 1220
1225 1230Glu Thr Gly Trp Thr Pro Gly Leu Arg Ser
Leu Glu Asn Asp Gly 1235 1240 1245Thr
Lys Leu Leu Asp Arg Ile Arg Asp Asn Tyr Glu Gly Glu Phe 1250
1255 1260Tyr Trp Arg Tyr Phe Ala Phe Ile Ala
Asp Ala Leu Ile Thr Thr 1265 1270
1275Leu Lys Pro Arg Tyr Glu Asp Thr Asn Ile Arg Ile Asn Leu Asp
1280 1285 1290Ser Asn Thr Arg Ser Phe
Ile Val Pro Ile Ile Thr Thr Glu Tyr 1295 1300
1305Ile Arg Glu Lys Leu Ser Tyr Ser Phe Tyr Gly Ser Gly Gly
Thr 1310 1315 1320Tyr Ala Leu Pro Leu
Ser Gln Tyr Asn Met Gly Ile Asn Ile Glu 1325 1330
1335Leu Ser Glu Ser Asp Val Trp Ile Ile Asp Val Asp Asn
Val Val 1340 1345 1350Arg Asp Val Thr
Ile Glu Ser Asp Lys Ile Lys Lys Gly Asp Leu 1355
1360 1365Ile Glu Gly Ile Leu Ser Thr Leu Ser Ile Glu
Glu Asn Lys Ile 1370 1375 1380Ile Leu
Asn Ser His Glu Ile Asn Phe Ser Gly Glu Val Asn Gly 1385
1390 1395Ser Asn Gly Phe Val Ser Leu Thr Phe Ser
Ile Leu Glu Gly Ile 1400 1405 1410Asn
Ala Ile Ile Glu Val Asp Leu Leu Ser Lys Ser Tyr Lys Leu 1415
1420 1425Leu Ile Ser Gly Glu Leu Lys Ile Leu
Met Leu Asn Ser Asn His 1430 1435
1440Ile Gln Gln Lys Ile Asp Tyr Ile Gly Phe Asn Ser Glu Leu Gln
1445 1450 1455Lys Asn Ile Pro Tyr Ser
Phe Val Asp Ser Glu Gly Lys Glu Asn 1460 1465
1470Gly Phe Ile Asn Gly Ser Thr Lys Glu Gly Leu Phe Val Ser
Glu 1475 1480 1485Leu Pro Asp Val Val
Leu Ile Ser Lys Val Tyr Met Asp Asp Ser 1490 1495
1500Lys Pro Ser Phe Gly Tyr Tyr Ser Asn Asn Leu Lys Asp
Val Lys 1505 1510 1515Val Ile Thr Lys
Asp Asn Val Asn Ile Leu Thr Gly Tyr Tyr Leu 1520
1525 1530Lys Asp Asp Ile Lys Ile Ser Leu Ser Leu Thr
Leu Gln Asp Glu 1535 1540 1545Lys Thr
Ile Lys Leu Asn Ser Val His Leu Asp Glu Ser Gly Val 1550
1555 1560Ala Glu Ile Leu Lys Phe Met Asn Arg Lys
Gly Ser Thr Asn Thr 1565 1570 1575Ser
Asp Ser Leu Met Ser Phe Leu Glu Ser Met Asn Ile Lys Ser 1580
1585 1590Ile Phe Val Asn Phe Leu Gln Ser Asn
Ile Lys Phe Ile Leu Asp 1595 1600
1605Ala Asn Phe Ile Ile Ser Gly Thr Thr Ser Ile Gly Gln Phe Glu
1610 1615 1620Phe Ile Cys Asp Glu Asn
Asn Asn Ile Gln Pro Tyr Phe Ile Lys 1625 1630
1635Phe Asn Thr Leu Glu Thr Asn Tyr Thr Leu Tyr Val Gly Asn
Arg 1640 1645 1650Gln Asn Met Ile Val
Glu Pro Asn Tyr Asp Leu Asp Asp Ser Gly 1655 1660
1665Asp Ile Ser Ser Thr Val Ile Asn Phe Ser Gln Lys Tyr
Leu Tyr 1670 1675 1680Gly Ile Asp Ser
Cys Val Asn Lys Val Val Ile Ser Pro Asn Ile 1685
1690 1695Tyr Thr Asp Glu Ile Asn Ile Thr Pro Val Tyr
Glu Thr Asn Asn 1700 1705 1710Thr Tyr
Pro Glu Val Ile Val Leu Asp Ala Asn Tyr Ile Asn Glu 1715
1720 1725Lys Ile Asn Val Asn Ile Asn Asp Leu Ser
Ile Arg Tyr Val Trp 1730 1735 1740Ser
Asn Asp Gly Asn Asp Phe Ile Leu Met Ser Thr Ser Glu Glu 1745
1750 1755Asn Lys Val Ser Gln Val Lys Ile Arg
Phe Val Asn Val Phe Lys 1760 1765
1770Asp Lys Thr Leu Ala Asn Lys Leu Ser Phe Asn Phe Ser Asp Lys
1775 1780 1785Gln Asp Val Pro Val Ser
Glu Ile Ile Leu Ser Phe Thr Pro Ser 1790 1795
1800Tyr Tyr Glu Asp Gly Leu Ile Gly Tyr Asp Leu Gly Leu Val
Ser 1805 1810 1815Leu Tyr Asn Glu Lys
Phe Tyr Ile Asn Asn Phe Gly Met Met Val 1820 1825
1830Ser Gly Leu Ile Tyr Ile Asn Asp Ser Leu Tyr Tyr Phe
Lys Pro 1835 1840 1845Pro Val Asn Asn
Leu Ile Thr Gly Phe Val Thr Val Gly Asp Asp 1850
1855 1860Lys Tyr Tyr Phe Asn Pro Ile Asn Gly Gly Ala
Ala Ser Ile Gly 1865 1870 1875Glu Thr
Ile Ile Asp Asp Lys Asn Tyr Tyr Phe Asn Gln Ser Gly 1880
1885 1890Val Leu Gln Thr Gly Val Phe Ser Thr Glu
Asp Gly Phe Lys Tyr 1895 1900 1905Phe
Ala Pro Ala Asn Thr Leu Asp Glu Asn Leu Glu Gly Glu Ala 1910
1915 1920Ile Asp Phe Thr Gly Lys Leu Ile Ile
Asp Glu Asn Ile Tyr Tyr 1925 1930
1935Phe Glu Asp Asn Tyr Arg Gly Ala Val Glu Trp Lys Glu Leu Asp
1940 1945 1950Gly Glu Met His Tyr Phe
Ser Pro Glu Thr Gly Lys Ala Phe Lys 1955 1960
1965Gly Leu Asn Gln Ile Gly Asp Asp Lys Tyr Tyr Phe Asn Ser
Asp 1970 1975 1980Gly Val Met Gln Lys
Gly Phe Val Ser Ile Asn Asp Asn Lys His 1985 1990
1995Tyr Phe Asp Asp Ser Gly Val Met Lys Val Gly Tyr Thr
Glu Ile 2000 2005 2010Asp Gly Lys His
Phe Tyr Phe Ala Glu Asn Gly Glu Met Gln Ile 2015
2020 2025Gly Val Phe Asn Thr Glu Asp Gly Phe Lys Tyr
Phe Ala His His 2030 2035 2040Asn Glu
Asp Leu Gly Asn Glu Glu Gly Glu Glu Ile Ser Tyr Ser 2045
2050 2055Gly Ile Leu Asn Phe Asn Asn Lys Ile Tyr
Tyr Phe Asp Asp Ser 2060 2065 2070Phe
Thr Ala Val Val Gly Trp Lys Asp Leu Glu Asp Gly Ser Lys 2075
2080 2085Tyr Tyr Phe Asp Glu Asp Thr Ala Glu
Ala Tyr Ile Gly Leu Ser 2090 2095
2100Leu Ile Asn Asp Gly Gln Tyr Tyr Phe Asn Asp Asp Gly Ile Met
2105 2110 2115Gln Val Gly Phe Val Thr
Ile Asn Asp Lys Val Phe Tyr Phe Ser 2120 2125
2130Asp Ser Gly Ile Ile Glu Ser Gly Val Gln Asn Ile Asp Asp
Asn 2135 2140 2145Tyr Phe Tyr Ile Asp
Asp Asn Gly Ile Val Gln Ile Gly Val Phe 2150 2155
2160Asp Thr Ser Asp Gly Tyr Lys Tyr Phe Ala Pro Ala Asn
Thr Val 2165 2170 2175Asn Asp Asn Ile
Tyr Gly Gln Ala Val Glu Tyr Ser Gly Leu Val 2180
2185 2190Arg Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu
Thr Tyr Thr Ile 2195 2200 2205Glu Thr
Gly Trp Ile Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr 2210
2215 2220Tyr Phe Val Pro Glu Thr Lys Lys Ala Cys
Lys Gly Ile Asn Leu 2225 2230 2235Ile
Asp Asp Ile Lys Tyr Tyr Phe Asp Glu Lys Gly Ile Met Arg 2240
2245 2250Thr Gly Leu Ile Ser Phe Glu Asn Asn
Asn Tyr Tyr Phe Asn Glu 2255 2260
2265Asn Gly Glu Ile Gln Phe Gly Tyr Ile Asn Ile Glu Asp Lys Met
2270 2275 2280Phe Tyr Phe Gly Glu Asp
Gly Val Met Gln Ile Gly Val Phe Asn 2285 2290
2295Thr Pro Asp Gly Phe Lys Tyr Phe Ala His Gln Asn Thr Leu
Asp 2300 2305 2310Glu Asn Phe Glu Gly
Glu Ser Ile Asn Tyr Thr Gly Trp Leu Gly 2315 2320
2325Leu Asp Glu Lys Arg Tyr Tyr Phe Thr Asp Glu Tyr Ile
Ala Ala 2330 2335 2340Thr Gly Ser Val
Ile Ile Asp Gly Glu Glu Tyr Tyr Phe Asp Pro 2345
2350 2355Asp Thr Ala Gln Leu Val Ile Ser Glu 2360
236543129PRTHomo sapiens 43Met Asp Met Met Val Pro Ala Gln
Leu Leu Gly Leu Leu Leu Leu Trp1 5 10
15Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser 20 25 30Val Ser Ala
Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 35
40 45Gln Gly Ile Ser Ser Trp Leu Ala Trp Tyr Gln
His Lys Pro Gly Lys 50 55 60Ala Pro
Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val65
70 75 80Pro Ser Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr 85 90
95Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln 100 105 110Ala Asn
Ser Phe Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 115
120 125Lys44129PRTHomo sapiens 44Met Asp Met Arg
Val Leu Ala Gln Leu Leu Gly Leu Leu Leu Leu Cys1 5
10 15Phe Pro Gly Ala Arg Cys Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser 20 25
30Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
35 40 45Gln Gly Ile Ser Ser Trp Leu Ala
Trp Tyr Gln Gln Lys Pro Glu Lys 50 55
60Ala Pro Lys Ser Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val65
70 75 80Pro Ser Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 85
90 95Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln 100 105
110Tyr Asn Ser Tyr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
115 120 125Lys45129PRTHomo sapiens 45Met
Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp1
5 10 15Leu Pro Gly Ala Arg Cys Val
Ile Trp Met Thr Gln Ser Pro Ser Leu 20 25
30Leu Ser Ala Ser Thr Gly Asp Arg Val Thr Ile Ser Cys Arg
Met Ser 35 40 45Gln Gly Ile Ser
Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 50 55
60Ala Pro Glu Leu Leu Ile Tyr Ala Ala Ser Thr Leu Gln
Ser Gly Val65 70 75
80Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
85 90 95Ile Ser Ser Leu Gln Pro
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 100
105 110Tyr Asn Ser Tyr Pro Trp Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile 115 120
125Lys46129PRTHomo sapiens 46Met Asp Met Met Val Pro Ala Gln Leu Leu Gly
Leu Leu Leu Leu Trp1 5 10
15Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
20 25 30Val Ser Ala Ser Val Gly Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40
45Gln Gly Ile Ser Ser Trp Leu Ala Trp Tyr Gln His Lys Pro Gly
Lys 50 55 60Ala Pro Lys Leu Leu Ile
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val65 70
75 80Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr 85 90
95Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
100 105 110Tyr Asn Ser Tyr Pro Trp
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 115 120
125Lys47129PRTHomo sapiens 47Met Asp Met Arg Val Pro Ala Gln
Leu Leu Gly Leu Leu Leu Leu Cys1 5 10
15Phe Pro Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser 20 25 30Val Ser Ala
Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 35
40 45Gln Gly Ile Ser Ser Trp Leu Ala Trp Tyr Gln
Gln Lys Pro Glu Lys 50 55 60Ala Pro
Lys Ser Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val65
70 75 80Pro Ser Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr 85 90
95Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln 100 105 110Tyr Asn
Ser Tyr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 115
120 125Lys48129PRTHomo sapiens 48Met Asp Met Arg
Val Leu Ala Gln Leu Leu Gly Leu Leu Leu Leu Cys1 5
10 15Phe Pro Gly Ala Arg Cys Asp Ile Gln Met
Thr Gln Ser Leu Ser Ser 20 25
30Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
35 40 45Gln Gly Ile Ser Ser Trp Leu Ala
Trp Tyr Gln Gln Lys Pro Glu Lys 50 55
60Ala Pro Lys Ser Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val65
70 75 80Pro Ser Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 85
90 95Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln 100 105
110Ala Asn Ser Phe Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
115 120 125Lys49141PRTHomo sapiens 49Met
Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly1
5 10 15Val Gln Cys Gln Val Gln Leu
Val Glu Ser Gly Gly Gly Val Val Gln 20 25
30Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Ser Phe 35 40 45Ser Asn Tyr Gly
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55
60Glu Trp Val Ala Leu Ile Trp Tyr Asp Gly Ser Asn Glu
Asp Tyr Thr65 70 75
80Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
85 90 95Thr Leu Tyr Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100
105 110Tyr Tyr Cys Ala Arg Trp Gly Met Val Arg Gly Val
Ile Asp Val Phe 115 120 125Asp Ile
Trp Gly Gln Gly Thr Val Val Thr Val Ser Ser 130 135
14050128PRTHomo sapiens 50Met Glu Ala Pro Ala Gln Leu Leu
Phe Leu Leu Leu Leu Trp Leu Pro1 5 10
15Asp Thr Thr Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr
Leu Ser 20 25 30Leu Ser Pro
Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser 35
40 45Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro 50 55 60Arg Leu
Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala65
70 75 80Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90
95Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln
Gln Arg Ser 100 105 110Asn Trp
Ser Gln Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 115
120 12551133PRTHomo sapiens 51Met Glu Phe Gly
Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly1 5
10 15Val Gln Cys Gln Met Gln Leu Val Glu Ser
Gly Gly Gly Val Val Gln 20 25
30Pro Gly Arg Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Ser Phe
35 40 45Asn Ser Tyr Gly Met His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu 50 55
60Glu Trp Val Ser Val Ile Trp Ala Ser Gly Asn Lys Lys Tyr Tyr Ile65
70 75 80Glu Ser Val Glu Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85
90 95Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val 100 105
110Tyr Tyr Cys Ala Arg Ala Asn Phe Asp Tyr Trp Gly Gln Gly Thr Leu
115 120 125Val Thr Val Ser Ser
13052129PRTHomo sapiens 52Met Asp Met Arg Val Leu Ala Gln Leu Leu Gly Leu
Leu Leu Leu Cys1 5 10
15Phe Pro Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
20 25 30Leu Ser Ala Ser Val Gly Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40
45Gln Gly Ile Ser Ser Trp Leu Ala Trp Tyr Gln Gln Lys Pro Glu
Lys 50 55 60Ala Pro Lys Ser Leu Ile
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val65 70
75 80Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr 85 90
95Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
100 105 110Tyr Lys Ser Tyr Pro Val
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 115 120
125Lys53135PRTHomo sapiens 53Met Glu Phe Gly Leu Ser Trp Val
Phe Leu Val Ala Leu Leu Arg Gly1 5 10
15Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val
Val Gln 20 25 30Pro Gly Arg
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35
40 45Asn Lys Tyr Gly Met His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu 50 55 60Glu Trp
Val Ala Val Ile Trp Tyr Asp Gly Thr Asn Lys Tyr Tyr Ala65
70 75 80Asp Ser Met Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90
95Met Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val 100 105 110Tyr Tyr
Cys Ala Arg Asp Pro Pro Thr Ala Asn Tyr Trp Gly Gln Gly 115
120 125Thr Leu Val Thr Val Ser Ser 130
13554119PRTHomo sapiens 54Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Ser Gly Glu1 5 10
15Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser
Tyr 20 25 30Trp Ile Gly Trp
Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35
40 45Gly Ile Phe Tyr Pro Gly Asp Ser Ser Thr Arg Tyr
Ser Pro Ser Phe 50 55 60Gln Gly Gln
Val Thr Ile Ser Ala Asp Lys Ser Val Asn Thr Ala Tyr65 70
75 80Leu Gln Trp Ser Ser Leu Lys Ala
Ser Asp Thr Ala Met Tyr Tyr Cys 85 90
95Ala Arg Arg Arg Asn Trp Gly Asn Ala Phe Asp Ile Trp Gly
Gln Gly 100 105 110Thr Met Val
Thr Val Ser Ser 11555357DNAHomo sapiensCDS(1)..(357) 55gag gtg cag
ctg gtg cag tct gga gca gag gtg aaa aag tcc ggg gag 48Glu Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Ser Gly Glu1 5
10 15tct ctg aag atc tcc tgt aag ggt tct
gga tac agc ttt acc agc tac 96Ser Leu Lys Ile Ser Cys Lys Gly Ser
Gly Tyr Ser Phe Thr Ser Tyr 20 25
30tgg atc ggc tgg gtg cgc cag atg ccc ggg aag ggc ctg gag tgg atg
144Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
35 40 45ggg atc ttc tat cct ggt gac
tct agt acc aga tac agc ccg tcc ttc 192Gly Ile Phe Tyr Pro Gly Asp
Ser Ser Thr Arg Tyr Ser Pro Ser Phe 50 55
60caa ggc cag gtc acc atc tca gcc gac aag tcc gtc aac acc gcc tac
240Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Val Asn Thr Ala Tyr65
70 75 80ctg cag tgg agc
agc ctg aag gcc tcg gac acc gcc atg tat tac tgt 288Leu Gln Trp Ser
Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85
90 95gcg aga cgt cga aac tgg gga aat gct ttt
gat atc tgg ggc caa ggg 336Ala Arg Arg Arg Asn Trp Gly Asn Ala Phe
Asp Ile Trp Gly Gln Gly 100 105
110aca atg gtc acc gtc tct tca
357Thr Met Val Thr Val Ser Ser 11556138PRTHomo sapiens 56Met Gly
Ser Thr Ala Ile Leu Ala Leu Leu Leu Ala Val Leu Gln Gly1 5
10 15Val Cys Ala Glu Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys 20 25
30Ser Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser
Phe 35 40 45Thr Ser Tyr Trp Ile
Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu 50 55
60Glu Trp Met Gly Ile Phe Tyr Pro Gly Asp Ser Ser Thr Arg
Tyr Ser65 70 75 80Pro
Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Val Asn
85 90 95Thr Ala Tyr Leu Gln Trp Ser
Ser Leu Lys Ala Ser Asp Thr Ala Met 100 105
110Tyr Tyr Cys Ala Arg Arg Arg Asn Trp Gly Asn Ala Phe Asp
Ile Trp 115 120 125Gly Gln Gly Thr
Met Val Thr Val Ser Ser 130 13557414DNAHomo sapiens
57atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccgag
60gtgcagctgg tgcagtctgg agcagaggtg aaaaagtccg gggagtctct gaagatctcc
120tgtaagggtt ctggatacag ctttaccagc tactggatcg gctgggtgcg ccagatgccc
180gggaagggcc tggagtggat ggggatcttc tatcctggtg actctagtac cagatacagc
240ccgtccttcc aaggccaggt caccatctca gccgacaagt ccgtcaacac cgcctacctg
300cagtggagca gcctgaaggc ctcggacacc gccatgtatt actgtgcgag acgtcgaaac
360tggggaaatg cttttgatat ctggggccaa gggacaatgg tcaccgtctc ttca
41458108PRTHomo sapiens 58Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser
Leu Ser Pro Gly1 5 10
15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser
20 25 30Tyr Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40
45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe
Ser 50 55 60Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70
75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Tyr Gly Ser Ser Thr 85 90
95Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
10559324DNAHomo sapiensCDS(1)..(324) 59gaa att gtg ttg acg cag
tct cca ggc acc ctg tct ttg tct cca ggg 48Glu Ile Val Leu Thr Gln
Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5
10 15gaa aga gcc acc ctc tcc tgc agg gcc agt cag agt
gtt agc agc agc 96Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
Val Ser Ser Ser 20 25 30tac
tta gcc tgg tac cag cag aaa cct ggc cag gct ccc agg ctc ctc 144Tyr
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35
40 45atc tat ggt gca tcc agc agg gcc act
ggc atc cca gac agg ttc agt 192Ile Tyr Gly Ala Ser Ser Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser 50 55
60ggc agt ggg tct ggg aca gac ttc act ctc acc atc agc aga ctg gag
240Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65
70 75 80cct gaa gat ttt gca
gtg tat tac tgt cag cag tat ggt agc tca acg 288Pro Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Thr 85
90 95tgg acg ttc ggc caa ggg acc aag gtg gaa atc
aaa 324Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 10560128PRTHomo sapiens 60Met Glu Thr
Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro1 5
10 15Asp Thr Thr Gly Glu Ile Val Leu Thr
Gln Ser Pro Gly Thr Leu Ser 20 25
30Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
35 40 45Val Ser Ser Ser Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala 50 55
60Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro65
70 75 80Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 85
90 95Ser Arg Leu Glu Pro Glu Asp Phe Ala Val
Tyr Tyr Cys Gln Gln Tyr 100 105
110Gly Ser Ser Thr Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
115 120 12561384DNAHomo sapiens
61atggaaaccc cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccgga
60gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc
120ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa
180cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca
240gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag
300cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcaacgtg gacgttcggc
360caagggacca aggtggaaat caaa
384625PRTHomo sapiens 62Ser Tyr Trp Ile Gly1 56315DNAHomo
sapiensCDS(1)..(15) 63agc tac tgg atc ggc
15Ser Tyr Trp Ile Gly1 56417PRTHomo
sapiens 64Ile Phe Tyr Pro Gly Asp Ser Ser Thr Arg Tyr Ser Pro Ser Phe
Gln1 5 10
15Gly6551DNAHomo sapiensCDS(1)..(51) 65atc ttc tat cct ggt gac tct agt
acc aga tac agc ccg tcc ttc caa 48Ile Phe Tyr Pro Gly Asp Ser Ser
Thr Arg Tyr Ser Pro Ser Phe Gln1 5 10
15ggc
51Gly6610PRTHomo sapiens 66Arg Arg Asn Trp Gly Asn Ala Phe Asp
Ile1 5 106730DNAHomo sapiensCDS(1)..(30)
67cgt cga aac tgg gga aat gct ttt gat atc
30Arg Arg Asn Trp Gly Asn Ala Phe Asp Ile1 5
106812PRTHomo sapiens 68Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu
Ala1 5 106936DNAHomo sapiensCDS(1)..(36)
69agg gcc agt cag agt gtt agc agc agc tac tta gcc
36Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala1 5
10707PRTHomo sapiens 70Gly Ala Ser Ser Arg Ala Thr1
57121DNAHomo sapiensCDS(1)..(21) 71ggt gca tcc agc agg gcc act
21Gly Ala Ser Ser Arg Ala Thr1
5729PRTHomo sapiens 72Gln Gln Tyr Gly Ser Ser Thr Trp Thr1
57327DNAHomo sapiensCDS(1)..(27) 73cag cag tat ggt agc tca acg tgg
acg 27Gln Gln Tyr Gly Ser Ser Thr Trp
Thr1 57415PRTHomo sapiens 74Ala Phe Asp Ile Trp Gly Gln Gly
Thr Met Val Thr Val Ser Ser1 5 10
15755PRTHomo sapiens 75Ser Tyr Trp Ile Gly1
57617PRTHomo sapiens 76Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser
Pro Ser Phe Gln1 5 10
15Gly7710PRTHomo sapiens 77Ala Arg Arg Arg Asn Trp Gly Asn Ala Phe1
5 107812PRTHomo sapiens 78Arg Ala Ser Gln Ser
Val Ser Ser Ser Tyr Leu Ala1 5
107912PRTHomo sapiens 79Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys1
5 10807PRTHomo sapiens 80Gln Gln Tyr Gly
Ser Ser Pro1 58198PRTHomo sapiens 81Glu Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Glu1 5
10 15Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser
Phe Thr Ser Tyr 20 25 30Trp
Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35
40 45Gly Ile Ile Tyr Pro Gly Asp Ser Asp
Thr Arg Tyr Ser Pro Ser Phe 50 55
60Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr65
70 75 80Leu Gln Trp Ser Ser
Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85
90 95Ala Arg8296PRTHomo sapiens 82Glu Ile Val Leu
Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5
10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser
Gln Ser Val Ser Ser Ser 20 25
30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala
Thr Gly Ile Pro Asp Arg Phe Ser 50 55
60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65
70 75 80Pro Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85
90 95836PRTArtificial SequenceDescription of
Artificial Sequence Synthetic 6xHis tag 83His His His His His His1
5
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