WO2011130650A2 - Antibodies for the treatment of clostridium difficile-associated infection and disease - Google Patents
Antibodies for the treatment of clostridium difficile-associated infection and disease Download PDFInfo
- Publication number
- WO2011130650A2 WO2011130650A2 PCT/US2011/032713 US2011032713W WO2011130650A2 WO 2011130650 A2 WO2011130650 A2 WO 2011130650A2 US 2011032713 W US2011032713 W US 2011032713W WO 2011130650 A2 WO2011130650 A2 WO 2011130650A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- toxin
- antibody
- antigen
- difficile
- binding fragment
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
- C07K16/1282—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Clostridium (G)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/40—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum bacterial
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
- A61K49/10—Organic compounds
- A61K49/14—Peptides, e.g. proteins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
- A61K49/10—Organic compounds
- A61K49/14—Peptides, e.g. proteins
- A61K49/16—Antibodies; Immunoglobulins; Fragments thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/12—Antidiarrhoeals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
- C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/55—Fab or Fab'
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Definitions
- This invention relates generally to compositions and therapies that can be used to treat Clostridium difficile (C difficile) infection and C difficile-associated disease conditions and pathologies, such as C diff. -associated diarrhea (CD AD), which can result from infection by C difficile bacteria.
- the invention further relates to antibodies or antigen-binding fragments thereof that bind specifically to epitopes on toxin A and/or toxin B of C difficile, compositions comprising such antibodies, as well as methods of using the antibodies or the compositions.
- C difficile (or C diff.) is a Gram-positive, spore-forming, anaerobic bacterium that represents the leading cause of nosocomial (hospital- acquired) antibiotic-associated diarrhea and pseudomembranous colitis.
- C difficile infection is estimated to total more than 750,000 cases per year in the U.S., and it is responsible for more deaths than all other intestinal infections combined (1).
- C difficile constitutes a greater risk to patients than methicillin-resistant Staphylococcus aureus (MRS A) or any other bacteria (2).
- MRS A methicillin-resistant Staphylococcus aureus
- the annual costs for management of Clostridium difficile-associated disease (CDAD) are estimated to exceed 3.2 billion dollars in the U.S (3).
- Recent outbreaks of C difficile strains with increased virulence or antibiotic resistance have led to treatment failures, more frequent relapses and increased mortality rates (4).
- CDAD is typically induced by the disruption of the colonic flora through the use of antibiotics such as clindamycin, cephalosporins, and fluoroquinolones. (3, 8) This perturbation in the colonic microenvironment, along with exposure to C difficile spores, leads to colonization. Approximately one-third of all patients that become colonized develop CDAD(9), which can result in severe diarrhea, perforation of the colon, colectomy and death (10). CD AD results following the acquisition and proliferation of C. difficile in the gut, where C. difficile bacteria produce toxin A and toxin B, two important virulence factors of CD AD. Toxins A and B of C. difficile show considerable sequence and structural homology.
- Both have a C-terminal receptor-binding domain containing multiple repeating sequences, a central hydrophobic domain and an N-terminal glucosyltransferase domain.
- the receptor- binding domain mediates binding of the toxins to intestinal epithelial cells via host receptors that remain poorly defined in humans. Following internalization via an endosomal pathway, the central hydrophobic domain inserts into the membrane of the endosome. The acidic pH of the endosome triggers pore formation and translocation of the amino-terminal domains of the toxins into the cytosol. Glucosylation of the cytosolic target Rho GTPases leads to disruption of the cytoskeleton and cell death. Toxins A and B demonstrate different pathological profiles with potential synergy in causing disease.
- C. difficile infection has been increasing steadily, particularly in the elderly, who are often frail. Approximately one-third of patients with C. difficile infection have recurrences of their infection, usually within two months of the initial illness. Repeat infections tend to be more severe than the original disease; they are often more fatal. Older adults and people with weakened immune systems are particularly susceptible to recurring infections. If not treated promptly and appropriately, the complications of C. difficile infection include dehydration, kidney failure, bowel perforation, toxic megacolon, which can lead to rupture of the colon, and death.
- C. difficile infection is the most common infection acquired by hospitalized patients, it may also be acquired outside of hospitals in the community. It is estimated that 20,000 infections with C. difficile occur in the community each year in the United States. Internationally, the incidence is highly variable and depends on multiple factors, including the frequency with which endoscopy is used to establish the diagnosis, antimicrobial use patterns and epidemiologic patterns.
- the invention provides, at least in part, new antibody reagents and compositions comprising anti-C. difficile toxin A and/or toxin B antibodies.
- the reagents and compositions can be beneficial for treating the increasingly prevalent numbers of subjects afflicted with C. difficile associated infection and disease, providing improved quality of life, resolving CD AD and C. difficile infection and aiding in the survival of these infected individuals.
- an isolated antibody or an antigen-binding fragment thereof which specifically binds toxin A of C. difficile and which cross competes for binding to toxin A of C. difficile with a monoclonal antibody produced by a hybridoma cell line deposited under ATCC Accession No. PTA-9692, PTA-9694, or PTA-9888 is provided.
- the hybridoma cell line is deposited under ATCC Accession No. PTA-9692.
- the hybridoma cell line is deposited under ATCC Accession No. PTA-9694.
- the hybridoma cell line is deposited under ATCC Accession No. PTA-9888.
- the monoclonal antibody, or antigen-binding fragment thereof is in chimeric or humanized form.
- an isolated antibody or an antigen-binding fragment thereof which specifically binds to a C. difficile toxin A epitope defined by a monoclonal antibody produced by the hybridoma cell line deposited under ATCC Accession No. PTA-9692, PTA-9694, or PTA-9888 is provided.
- the hybridoma cell line is deposited under ATCC Accession No. PTA-9692.
- the hybridoma cell line is deposited under ATCC Accession No. PTA-9694.
- the hybridoma cell line is deposited under ATCC Accession No. PTA-9888.
- the monoclonal antibody, or antigen-binding fragment thereof is in chimeric or humanized form.
- an isolated antibody or an antigen-binding fragment thereof which specifically binds toxin B of C. difficile and which cross competes for binding to toxin B of C. difficile of a monoclonal antibody produced by the hybridoma cell line deposited under ATCC Accession No. PTA-9693 or PTA-9692 is provided.
- the hybridoma cell line is deposited under ATCC Accession No. PTA-9693.
- the hybridoma cell line is deposited under ATCC Accession No. PTA-9692.
- the monoclonal antibody, or antigen-binding fragment thereof is in chimeric or humanized form.
- an isolated antibody or an antigen-binding fragment thereof which specifically binds to a C. difficile toxin B epitope defined by a monoclonal antibody produced by the hybridoma cell line deposited under ATCC Accession No. PTA-9693 or PTA-9692 is provided.
- the hybridoma cell line is deposited under ATCC Accession No. PTA-9693.
- the hybridoma cell line is deposited under ATCC Accession No. PTA-9692.
- the monoclonal antibody, or antigen-binding fragment thereof is in chimeric or humanized form.
- the isolated antibody or antigen-binding fragment thereof neutralizes the in vivo toxicity of toxin B of C. difficile.
- the antibody or antigen-binding fragment thereof neutralizes the in vivo toxicity of toxin B of C. difficile in an amount of from 0.1-1000 ⁇ g.
- monoclonal antibody PA-39 (ATCC Accession No. 9692), or an antigen- binding fragment thereof is provided.
- monoclonal antibody PA-50 (ATCC Accession No. PTA-9694), or an antigen-binding fragment thereof is provided.
- monoclonal antibody PA-38 (ATCC Accession No. PTA-9888), or an antigen-binding fragment thereof is provided.
- monoclonal antibody PA-41 (ATCC Accession No. PTA-9693), or an antigen-binding fragment thereof is provided.
- the monoclonal antibody, or antigen-binding fragment thereof is in chimeric or humanized form.
- an expression vector comprising at least one nucleic acid molecule encoding the antibodies or antigen-binding fragments thereof as described above and herein is provided.
- an expression vector comprising a nucleic acid molecule encoding the heavy chain or portion thereof of the antibodies or antigen-binding fragments thereof as described above or herein is provided.
- an expression vector comprising a nucleic acid molecule encoding the light chain, or portion thereof, of the antibodies or antigen-binding fragments thereof as described above or herein is provided.
- an expression vector comprising at least one nucleic acid molecule encoding the heavy chain, or portion thereof, and light chain, or portion thereof, of the antibodies or antigen binding fragments thereof as described above or herein is provided.
- a host cell transformed or transfected by any of the expression vectors described above and herein is provided.
- a plasmid which encodes the any of the antibodies or antigen binding fragments thereof as described above and herein is provided.
- an isolated anti-C. difficile toxin A antibody or antigen-binding fragment as described above and herein, wherein the antibody or antigen-binding fragment neutralizes the in vivo toxicity of toxin A of C. difficile.
- the antibody or antigen-binding fragment neutralizes the in vivo toxicity of toxin A of C. difficile in an amount of from 0.1 ⁇ g to 1000 ⁇ g, or 1 ⁇ g/kg to 100,000 ⁇ g/kg.
- the isolated antibody or antigen-binding fragment neutralizes the in vivo toxicity of toxin A of C.
- the antibody is monoclonal antibody PA-39 (ATCC Accession No. 9692), or an antigen-binding fragment thereof.
- the antibody is monoclonal antibody PA-50 (ATCC Accession No. PTA-9694), or an antigen-binding fragment thereof.
- the antibody is monoclonal antibody PA-38 (ATCC Accession No. PTA-9888), or an antigen-binding fragment thereof.
- the monoclonal antibody, or antigen-binding fragment thereof is in chimeric or humanized form.
- an isolated anti-C. difficile toxin B antibody or antigen-binding fragment as described above and herein, wherein the antibody or antigen-binding fragment neutralizes the in vivo toxicity of toxin B of C. difficile.
- the isolated antibody or antigen-binding fragment thereof neutralizes the in vivo toxicity of toxin B of C. difficile in an amount selected from 0.5 ⁇ g-1000 ⁇ g, 0.5 ⁇ g, 5 ⁇ g, 40 ⁇ g, 50 ⁇ g, 100 ⁇ g, 200 ⁇ g, 1000 ⁇ g, or from 10 mg/kg-50 mg/kg.
- the antibody is monoclonal antibody PA-39 (ATCC Accession No.
- the antibody is monoclonal antibody PA-41 (ATCC Accession No. PTA-9693), or an antigen-binding fragment thereof.
- the monoclonal antibody, or antigen-binding fragment thereof is in chimeric or humanized form.
- Another aspect provides an isolated anti-C. difficile toxin A antibody or antigen-binding fragment as described above and herein, wherein the antibody or antigen-binding fragment, when administered to a C. difficile-infected subject in combination with an isolated antibody or antigen-binding fragment thereof that specifically binds and/or neutralizes toxin B of C. difficile, treats CDAD and/or increases the survivability of the subject.
- the anti-toxin A and anti-toxin B antibodies or fragments thereof are administered simultaneously.
- the anti-toxin A and anti-toxin B antibodies or fragments thereof are administered at different times.
- the anti-toxin A and anti-toxin B antibodies or fragments thereof are administered sequentially.
- the isolated antibody or antigen-binding fragment that specifically binds toxin A of C. difficile is an antibody produced by the hybridoma cell line deposited under ATCC Accession No. PTA- 9692, PTA-9694, or PTA-9888, an antigen-binding fragment thereof, a humanized form thereof, or a monoclonal antibody that cross -reacts therewith for binding toxin A.
- the isolated antibody or antigen-binding fragment that specifically binds toxin B of C. difficile is an antibody produced by the hybridoma cell line deposited under ATCC Accession No. PTA-9693 or PTA-9692, an antigen-binding fragment thereof, a humanized form thereof, or a monoclonal antibody that cross-reacts therewith for binding toxin B.
- Another aspect provides an isolated anti-C. difficile toxin B antibody or antigen-binding fragment as described above and herein, wherein the antibody or antigen-binding fragment, when administered to a C. difficile-infected subject in combination with an isolated antibody or antigen-binding fragment thereof that specifically binds and/or neutralizes toxin A of C. difficile, treats CD AD and/or increases the survivability of the subject.
- the anti-toxin A and anti-toxin B antibodies or fragments thereof are administered simultaneously.
- the anti-toxin A and anti-toxin B antibodies or fragments thereof are administered at different times.
- the anti-toxin A and anti-toxin B antibodies or fragments thereof are administered sequentially.
- the isolated antibody or antigen-binding fragment that specifically binds toxin A of C. difficile is an antibody produced by the hybridoma cell line deposited under ATCC Accession No. PTA- 9692, PTA-9694, or PTA-9888, an antigen-binding fragment thereof, a humanized form thereof, or a monoclonal antibody that cross -reacts therewith for binding toxin A.
- the isolated antibody or antigen-binding fragment that specifically binds toxin B of C. difficile is an antibody produced by the hybridoma cell line deposited under ATCC Accession No. PTA-9693 or PTA-9692, an antigen-binding fragment thereof, a humanized form thereof, or a monoclonal antibody that cross-reacts therewith for binding toxin B.
- Another aspect provides an isolated anti-C. difficile toxin A antibody or antigen-binding fragment as described above and herein, wherein the antibody or antigen-binding fragment, when administered to a C. difficile-infected subject in combination with an isolated antibody or antigen-binding fragment thereof that specifically binds toxin B of C. difficile, treats CD AD and/or improves the survivability of the subject.
- the anti-toxin A antibody or antigen-binding fragment thereof is administered in an amount of 1 ⁇ g-1000 ⁇ g, or from 1 ⁇ g-250 ⁇ g or from 5 ⁇ g-100 ⁇ g and the dose of the anti-toxin B antibody or antigen-binding fragment thereof is administered in an amount of from 0.1 ⁇ g-1000 ⁇ g. or from 1 ⁇ g-250 ⁇ g or from 5 ⁇ g-100 ⁇ g.
- the isolated antibody or antigen- binding fragment that specifically binds toxin A of C. difficile is an antibody produced by the hybridoma cell line deposited under ATCC Accession No.
- the isolated antibody or antigen-binding fragment that specifically binds toxin B of C. difficile is an antibody produced by the hybridoma cell line deposited under ATCC Accession No. PTA- 9693 or PTA-9692, an antigen-binding fragment thereof, a humanized form thereof, or a monoclonal antibody that cross-reacts therewith for binding toxin B.
- Another aspect provides an isolated anti-C. difficile toxin A antibody or antigen-binding fragment as described above and herein, wherein the antibody or antigen-binding fragment, when administered to a C. difficile-infected subject in combination with an isolated antibody or antigen-binding fragment thereof that specifically binds toxin B of C. difficile, treats CD AD and/or improves the survivability of the subject.
- the anti-toxin A antibody or antigen-binding fragment thereof is administered in an amount of 50 mg/kg
- the anti-toxin B antibody or antigen-binding fragment thereof is administered in an amount of 50 mg/kg.
- the isolated antibody or antigen-binding fragment that specifically binds toxin B of C. difficile is an antibody produced by the hybridoma cell line deposited under ATCC Accession No. PTA-9693 or PTA-9692, an antigen-binding fragment thereof, a humanized form thereof, or a monoclonal antibody that cross -reacts therewith for binding toxin B.
- Another aspect provides an isolated anti-C. difficile toxin A antibody or antigen-binding fragment as described above and herein, wherein the antibody or antigen-binding fragment, when administered to a C. difficile-infected subject in combination with an isolated antibody or antigen-binding fragment thereof that specifically binds toxin B of C. difficile, effects a cure or survivability rate of 50%, 60%, 70%, 80%, 90%, or 100%.
- the antibody or antigen-binding fragment is administered q2d x 4 at a dose of 40-50 mg/kg.
- the isolated antibody or antigen-binding fragment that specifically binds toxin A of C. difficile is an antibody produced by the hybridoma cell line deposited under ATCC Accession No.
- the isolated antibody or antigen-binding fragment that specifically binds toxin B of C. difficile is an antibody produced by the hybridoma cell line deposited under ATCC Accession No.
- PTA-9693 or PTA-9692 an antigen-binding fragment thereof, a humanized form thereof, or a monoclonal antibody that cross-competes for binding toxin B with one of more of the monoclonal antibodies deposited under ATCC Accession No. PTA-9692 or PTA-9693.
- Another aspect provides a bispecific antibody or an antigen-binding fragment thereof which comprises (i) a monoclonal antibody produced by the hybridoma cell line deposited under ATCC Accession No. PTA-9692, PTA-9694, or PTA-9888, an antigen-binding fragment thereof, a humanized version of the antibody or antigen-binding fragment thereof, a heavy chain variable domain of the antibody or antigen-binding fragment thereof, and/or a light chain variable domain of the antibody or antigen-binding fragment thereof; and (ii) a monoclonal antibody produced by the hybridoma cell line deposited under ATCC Accession No.
- PTA-9693 or PTA-9692 an antigen-binding fragment thereof, a humanized version of the antibody or antigen-binding fragment thereof, a heavy chain variable domain of the antibody or antigen-binding fragment thereof, and/or a light chain variable domain of the antibody or antigen-binding fragment thereof.
- Another aspect provides a bispecific antibody or antigen-binding fragment thereof, wherein the antibody comprises (i) a monoclonal antibody produced by the hybridoma cell line deposited under ATCC Accession No. PTA-9692, an antigen-binding fragment thereof, a humanized version of the antibody or antigen-binding fragment thereof, a heavy chain variable domain of the antibody or antigen-binding fragment thereof, and/or a light chain variable domain of the antibody or antigen-binding fragment thereof; and (ii) an isolated monoclonal antibody produced by the hybridoma cell line deposited under ATCC Accession No.
- PTA-9693 or PTA-9692 an antigen-binding fragment thereof, a humanized version of the antibody or antigen-binding fragment thereof, a heavy chain variable domain of the antibody or antigen-binding fragment thereof, and/or a light chain variable domain of the antibody or antigen-binding fragment thereof.
- an antibody or antigen-binding fragment thereof as described above and herein, wherein the antigen-binding fragment is selected from an Fab fragment, an F(ab') 2 fragment, and an Fv fragment is provided.
- an isolated antibody or antigen- binding fragment thereof as described above and herein, wherein the antibody or antigen- binding fragment thereof is or comprises a single chain antibody is provided.
- compositions comprising one or more of the antibodies or antigen-binding fragments thereof of the invention, as described above and herein, and a pharmaceutically acceptable carrier, excipient, vehicle, or diluent.
- the composition comprises at least one anti-toxin A antibody of the invention, for example, mAb PTA-9692, mAb PTA-9694, mAb PTA-9888, an antigen-binding fragment thereof, or a humanized form thereof, and at least one anti-toxin B antibody of the invention, for example, mAb PTA-9692, mAb PTA-9693, an antigen-binding fragment thereof, or a humanized form thereof.
- the composition comprises one anti-toxin A antibody of the invention, for example, mAb PTA-9888, an antigen-binding fragment thereof, or a humanized form thereof, and one anti-toxin B antibody of the invention, for example, mAb 9693, an antigen-binding fragment thereof, or a humanized form thereof.
- the composition comprises one anti-toxin A antibody of the invention, for example, mAb PTA-9694, an antigen-binding fragment thereof, or a humanized form thereof, and one antitoxin B antibody of the invention, for example, mAb 9693, an antigen-binding fragment thereof, or a humanized form thereof.
- each mAb is present in the composition in the same amount.
- each mAb is present in the composition in a 1:1 ratio, by weight, relative to each other. In an embodiment, each mAb is present in the composition in different amounts. In an embodiment, each mAb is present in the composition in ratios other than 1:1, by weight, relative to each other, wherein the ratios are as provided herein.
- the composition further comprises an additional therapeutic agent.
- the additional therapeutic agent is a/an antibiotic, antibacterial, bacteriocide, bacteriostat or a combination thereof. In an embodiment, the additional therapeutic agent is metronizadole, vancomycin, fidaxomicin, or a combination thereof.
- compositions comprising an expression vector of the invention, as described above and herein, and a pharmaceutically acceptable carrier, excipient, vehicle, or diluent is provided.
- a composition comprising a host cell harboring an expression vector of the invention, as described above and herein, and a pharmaceutically acceptable carrier, excipient, vehicle, or diluent is provided.
- a composition comprising a plasmid of the invention, as described above and herein, and a pharmaceutically acceptable carrier, excipient, vehicle, or diluent is provided.
- Another aspect provides a binding protein comprising at least two polypeptide chains comprising binding sites for binding toxin A and toxin B of C. difficile, wherein at least one polypeptide chain comprises a first heavy chain variable domain, a second heavy chain variable domain, and a heavy chain constant domain or portion thereof; and at least one polypeptide chain comprises a first light chain variable domain, a second light chain variable domain, and a light chain constant domain or portion thereof, wherein the variable domains comprising the polypeptide chains form functional binding sites for toxin A and toxin B of C. difficile .
- the first heavy chain variable domain and the first light chain variable domain of the binding protein form a functional binding site for toxin A of C.
- the binding protein comprises an Fc region.
- the binding protein neutralizes the toxicity of toxin A and toxin B of C. difficile.
- the binding protein has an on rate constant (Kon) to toxin A or toxin B selected from at least
- the binding protein has an off rate constant (Koff) to toxin A or toxin B selected from at most 10 - " 3 s - " 1 ; at most 10 ' V 1 ; at most 10 ' V 1 ; or at most 10 ' V 1 , as measured by surface plasmon resonance.
- the binding protein has a dissociation constant (KD) to toxin A or toxin B selected from at most 10 "7 M; at most 10 "8 M; at most 10 "9 M; at most 10 "10 M; at most 10 "11 M; at most 10 "12 M; or at most 10 "13 M.
- composition comprising the binding protein as described above and herein and a pharmaceutically acceptable carrier, excipient, vehicle, or diluent.
- the composition further comprises an additional therapeutic agent.
- the additional therapeutic agent of the composition is a/an antibiotic, antibacterial, bacteriocide, bacteriostat, or combination thereof.
- the additional therapeutic agent of the composition is metronizadole, vancomycin, fidaxomicin, nitazoxanide, rifaximin ramoplanin, or a combination thereof.
- hybridoma cell line deposited under ATCC Accession No. PTA-9692, PTA-9693, PTA-9494, or PTA-9888 is provided.
- compositions include one or more the antibodies of the invention, preferably in humanized form.
- the compositions contain at least one anti-toxin A antibody provided herein in humanized form, or an antigen-binding fragment thereof, and at least one antitoxin B antibody of the invention in humanized form, or an antigen-binding fragment thereof.
- one or more additional therapeutic reagents, drugs, compounds, or ingredients may be included in the compositions.
- the compositions further include a pharmaceutically acceptable carrier, diluent, vehicle, or excipient.
- compositions are administered in an amount effective to treat the C. difficile infection or C. difficile-associated disease, for example, C. difficile-associated diarrhea (CDAD).
- CDAD C. difficile-associated diarrhea
- two compositions are administered to the subject in an amount effective to treat the C. difficile infection or C. difficile-associated disease.
- the two compositions are administered at the same time.
- the two compositions are administered at different times.
- Another aspect provides a method of inhibiting or neutralizing toxicity to a cell by C. difficile toxin A and toxin B, which comprises subjecting the cell to an effective C. difficile toxin A inhibiting or neutralizing dose of an anti-toxin A monoclonal antibody of the invention, or an antigen-binding fragment thereof, and an effective C.
- dijjicile toxin B inhibiting or neutralizing dose of an anti-toxin B monoclonal antibody of the invention, or an antigen- binding fragment thereof.
- the anti-toxin A antibody and the anti-toxin B antibody are in humanized form.
- the anti-toxin A antibody and the anti- toxin B antibody are in chimeric form.
- the antibodies or antigen-binding fragments thereof are administered at the same time.
- the antibodies or antigen-binding fragments thereof are administered at different times.
- the cell is present in a subject and the antibodies or antigen-binding fragments thereof are administered to the subject in amount effective to inhibit or neutralize the C. dijjicile toxin A and toxin B.
- Another aspect provides a method of inhibiting or neutralizing toxicity of a cell by a C. difficile toxin which comprises subjecting the cell to an effective C. difficile toxin inhibiting or neutralizing dose of at least one of the compositions of the invention as described herein.
- the cell is subjected to an effective C. difficile toxin inhibiting or neutralizing dose of two compositions, one of which comprises an anti-toxin A antibody or antigen-binding fragment thereof, and one of which comprises an anti-toxin B antibody or antigen-binding fragment thereof.
- the antibodies are humanized.
- the antibodies are chimeric.
- the two compositions are administered at the same time or at different times.
- the cell is present in a subject and the at least one composition is administered to the subject in amount effective to inhibit or neutralize the C. difficile toxin.
- Another aspect provides a method of neutralizing toxins produced by a hypervirulent strain of C. difficile, which comprises administering to a subject in need thereof (i) an antibody or antigen-binding fragment thereof of the invention, wherein the antibody binds and neutralizes toxin A of C. difficile and (ii) an antibody or antigen-binding fragment thereof of the invention, wherein the antibody binds and neutralizes toxin B of C. difficile, in an amount effective to neutralize the toxins produced by the hypervirluent strain.
- the antibodies of (i) and (ii) are humanized antibodies.
- the antibodies of (i) and (ii) are chimeric antibodies.
- the antibodies or antigen-binding fragments thereof are administered at the same time or at different times.
- the toxins of the hypervirulent strain are toxn A and toxin B.
- the hypervirulent strain of C. difficile are one or more of BI/NAP1/027, CCL676, HMC553, Pitt45, CD196, attachedal 5, or enclosureal 7.1.
- the anti-toxin A antibody or antigen-binding fragment thereof has a neutralizing activity against toxin A produced by hypervirulent strains of C. difficile as determined by an -12 -11
- the anti-toxin B antibody or antigen-binding fragment thereof has a neutralizing activity against toxin B produced by hypervirulent strains of C. difficile as determined by an EC 50 value of from 1. ⁇ ⁇ ⁇ to 6.5 "10 M.
- kits comprising an antibody or antigen-binding fragment thereof of the invention and as described herein, particularly in humanized form, and instructions for use is provided.
- the antibodies or antigen-binding fragments are contained in the same container in the kit.
- the antibodies or antigen-binding fragments are contained in separate containers in the kit.
- the kit comprises a linker for conjugating the antibodies or antigen-binding fragments thereof.
- the kit comprises an additional therapeutic agent, which may be a/an antibiotic, antibacterial, bacteriocide, or bacteriostat.
- the additional therapeutic agent is metronizadole, vancomycin, fidaxomicin, nitazoxanide , rifaximin ramoplanin, or a combination thereof.
- a monoclonal antibody, or an antigen-binding fragment thereof, particularly in humanized form, which neutralizes toxin A or toxin B of a hypervirulent strain of C. difficile is provided.
- the monoclonal antibody is designated by ATCC Accession number PTA-9692, PTA-9694, PTA-9888, or PTA-9693 and is produced by a hybridoma cell line deposited under ATCC Accession No. PTA-9692, PTA-9694, PTA-9888, or PTA-9693, respectively.
- the hypervirulent strain of C. difficile is one or more of BI/NAP1/027, CCL676, HMC553, Pitt45, CD196, axleal 5 and spirital 7.1.
- a method of treating a subject who is asymptomatic, but who is susceptible to, or at risk of, contracting C. difficile infection which comprises: administering to the subject (i) an anti-C. difficile toxin A antibody or antigen-binding fragment thereof provided and as described herein and (ii) an an anti-C. difficile toxin B antibody or antigen-binding fragment thereof provided and as described herein, in an amount effective to treat the subject is provided.
- the subject is hospitalized.
- a humanized monoclonal antibody generated against toxin A of C. dijjicile is provided. In an embodiment, such an anti-C.
- a antibody is composed of two heavy chain polypeptides, wherein each heavy chain contains a VH region comprising the amino acid sequence as set forth in SEQ ID NO:l and a human CH region, and two light chain polypeptides, wherein each light chain contains a VL region comprising the amino acid sequence as set forth in SEQ ID NO:3 and a human CL region.
- each heavy chain contains a VH region comprising the amino acid sequence as set forth in SEQ ID NO:l and a human CH region
- two light chain polypeptides wherein each light chain contains a VL region comprising the amino acid sequence as set forth in SEQ ID NO:3 and a human CL region.
- a antibody is composed of two heavy chain polypeptides, wherein each heavy chain contains a VH region comprising the amino acid sequence as set forth in SEQ ID NO:2 and a human CH region, and two light chain polypeptides, wherein each light chain contains a VL region comprising the amino acid sequence as set forth in SEQ ID NO: 3 and a human CL region.
- each heavy chain contains a VH region comprising the amino acid sequence as set forth in SEQ ID NO:2 and a human CH region
- two light chain polypeptides wherein each light chain contains a VL region comprising the amino acid sequence as set forth in SEQ ID NO: 3 and a human CL region.
- a antibody is composed of two heavy chain polypeptides, wherein each heavy chain contains a VH region comprising the amino acid sequence as set forth in SEQ ID NO:l and a human CH region, and two light chain polypeptides, wherein each light chain contains a VL region comprising the amino acid sequence as set forth in SEQ ID NO:4 and a human CL region.
- each heavy chain contains a VH region comprising the amino acid sequence as set forth in SEQ ID NO:l and a human CH region
- two light chain polypeptides wherein each light chain contains a VL region comprising the amino acid sequence as set forth in SEQ ID NO:4 and a human CL region.
- a antibody is composed of two heavy chain polypeptides, wherein each heavy chain contains a VH region comprising the amino acid sequence as set forth in SEQ ID NO:2 and a human CH region, and two light chain polypeptides, wherein each light chain contains a VL region comprising the amino acid sequence as set forth in SEQ ID NO:4 and a human CL region.
- each heavy chain contains a VH region comprising the amino acid sequence as set forth in SEQ ID NO:2 and a human CH region
- two light chain polypeptides wherein each light chain contains a VL region comprising the amino acid sequence as set forth in SEQ ID NO:4 and a human CL region.
- a antibody is composed of two heavy chain polypeptides, wherein each heavy chain contains a VH region comprising the amino acid sequence as set forth in SEQ ID NO: 5 and a human CH region, and two light chain polypeptides, wherein each light chain contains a VL region comprising the amino acid sequence as set forth in SEQ ID NO:7 and a human CL region.
- each heavy chain contains a VH region comprising the amino acid sequence as set forth in SEQ ID NO: 5 and a human CH region
- two light chain polypeptides wherein each light chain contains a VL region comprising the amino acid sequence as set forth in SEQ ID NO:7 and a human CL region.
- a antibody is composed of two heavy chain polypeptides, wherein each heavy chain contains a VH region comprising the amino acid sequence as set forth in SEQ ID NO: 6 and a human CH region, and two light chain polypeptides, wherein each light chain contains a VL region comprising the amino acid sequence as set forth in SEQ ID NO:7 and a human CL region.
- a humanized monoclonal antibody generated against toxin B of C. dijjicile is provided.
- an anti-C. dijjicile toxin B antibody is composed of two heavy chain polypeptides, wherein each heavy chain contains a VH region comprising the amino acid sequence as set forth in SEQ ID NO: 8 and a human CH region, and two light chain polypeptides, wherein each light chain contains a VL region comprising the amino acid sequence as set forth in SEQ ID NO: 10 and a human CL region.
- an anti-C is composed of two heavy chain polypeptides, wherein each heavy chain contains a VH region comprising the amino acid sequence as set forth in SEQ ID NO: 8 and a human CH region, and two light chain polypeptides, wherein each light chain contains a VL region comprising the amino acid sequence as set forth in SEQ ID NO: 10 and a human CL region.
- difficile toxin B antibody is composed of two heavy chain polypeptides, wherein each heavy chain containins a VH region comprising the amino acid sequence as set forth in SEQ ID NO: 9 and a human CH region, and two light chain polypeptides, wherein each light chain contains a VL region comprising the amino acid sequence as set forth in SEQ ID NO: 10 and a human CL region.
- a monoclonal antibody, or a fragment thereof, generated against toxin A of C. difficile wherein the antibody is composed of two heavy chain polypeptides, each heavy chain containing a VH region and a human CH region and two light chain polypeptides, each light chain containing a VL region and a human CL region is provided.
- the nucleic acid sequence (or cDNA) encoding the amino acid sequence of the antibody heavy chain polypeptide of SEQ ID NO: 14 is set forth in SEQ ID NO: 15 (Fig. 38B);
- the nucleic acid sequence (or cDNA) encoding the amino acid sequence of the antibody light chain polypeptide of SEQ ID NO: 16 is set forth in SEQ ID NO: 17 (Fig. 38A).
- a monoclonal antibody, or a fragment thereof, generated against toxin A of C. difficile wherein the antibody is composed of two heavy chain polypeptides, each heavy chain containing a VH region and a human CH region and two light chain polypeptides, each light chain containing a VL region and a human CL region is provided.
- the nucleic acid sequence (or cDNA) encoding the amino acid sequence of the antibody heavy chain polypeptide of SEQ ID NO: 18 is set forth in SEQ ID NO: 19 (Fig. 39B);
- the nucleic acid sequence (or cDNA) encoding the amino acid sequence of the antibody light chain polypeptide of SEQ ID NO:20 is set forth in SEQ ID NO:21 (Fig. 39A).
- a monoclonal antibody, or a fragment thereof, generated against toxin B of C. difficile wherein the antibody is composed of two heavy chain polypeptides, each heavy chain containing a VH region and a human CH region and two light chain polypeptides, each light chain containing a VL region and a human CL region is provided.
- the nucleic acid sequence (or cDNA) encoding the amino acid sequence of the antibody heavy chain polypeptide of SEQ ID NO:22 is set forth in SEQ ID NO:23 (Fig. 40B);
- the nucleic acid sequence (or cDNA) encoding the amino acid sequence of the antibody light chain polypeptide of SEQ ID NO:24 is set forth in SEQ ID NO:25 (Fig.
- the CH region of the monoclonal antibody is selected from IgGl, IgG2a, IgG2b, IgG3, IgG4, IgA, IgE, or IgM.
- the CH region is IgGl.
- the CL region is selected from the ⁇ or ⁇ isotype. In an embodiment, the CL region is of the ⁇ isotype.
- the CDRs i.e., CDR1, CDR2, and/or CDR3, of the humanized antibodies, or antigen-binding fragments thereof, as described herein, are embraced to bind and/or neutralize toxin A and/or toxin B of C. dijjicile in products and methods according to the invention.
- an anti-C. dijjicile toxin A antibody, or a fragment thereof, wherein the V region of the L chain comprises a sequence selected from one or more of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:7 is provided.
- an anti-C. dijjicile toxin A antibody, or a fragment thereof, wherein the V region of the H chain comprises a sequence selected from one or more of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:6.
- an isolated antibody or an antigen-binding fragment thereof which (i) specifically binds toxin A of C. dijjicile and which cross competes for binding to toxin A of C. dijjicile with a monoclonal antibody produced by a hybridoma cell line deposited under ATCC Accession No. PTA-9692, or which (ii) specifically binds to a C. dijjicile toxin A epitope defined by a monoclonal antibody produced by the hybridoma cell line deposited under ATCC Accession No. PTA-9692, wherein the epitope defined by the monoclonal antibody produced by the hybridoma cell line deposited under ATCC Accession No.
- PTA- 9692 comprises a region outside of the receptor binding domain, e.g., the translocation domain, of C. dijjicile toxin A is provided.
- the antibody is in humanized form. In an embodiment, the antibody is in chimeric form.
- an isolated antibody or an antigen-binding fragment thereof which (i) specifically binds toxin A of C. dijjicile and which cross competes for binding to toxin A of C. dijjicile with a monoclonal antibody produced by a hybridoma cell line deposited under ATCC Accession No. PTA-9694, or which (ii) specifically binds to a C. dijjicile toxin A epitope defined by a monoclonal antibody produced by the hybridoma cell line deposited under ATCC Accession No. PTA-9694, wherein the epitope defined by the monoclonal antibody produced by the hybridoma cell line deposited under ATCC Accession No.
- PTA- 9694 comprises at least two sites in the receptor binding domain, e.g., C-terminal receptor binding epitopes, of C. difficile toxin A is provided.
- the antibody is in humanized form. In an embodiment, the antibody is in chimeric form.
- an isolated antibody or an antigen-binding fragment thereof which (i) specifically binds toxin A of C. difficile and which cross competes for binding to toxin A of C. difficile with a monoclonal antibody produced by a hybridoma cell line deposited under ATCC Accession No. PTA-9888, or which (ii) specifically binds to a C. difficile toxin A epitope defined by a monoclonal antibody produced by the hybridoma cell line deposited under ATCC Accession No. PTA-9888, wherein the epitope defined by the monoclonal antibody produced by the hybridoma cell line deposited under ATCC Accession No. PTA- 9888 comprises C-terminal receptor binding epitopes of C. difficile toxin A is provided.
- the antibody is in humanized form.
- the antibody is in chimeric form.
- an isolated antibody or an antigen-binding fragment thereof which (i) specifically binds toxin B of C. difficile and which cross competes for binding to toxin B of C. difficile with a monoclonal antibody produced by a hybridoma cell line deposited under ATCC Accession No. PTA-9693, or which (ii) specifically binds to a C. difficile toxin B epitope defined by a monoclonal antibody produced by the hybridoma cell line deposited under ATCC Accession No. PTA-9693, wherein the epitope defined by the monoclonal antibody produced by the hybridoma cell line deposited under ATCC Accession No. PTA- 9693 comprises the N-terminal enzyme domain of C.
- the epitope defined by the monoclonal antibody produced by the hybridoma cell line deposited under ATCC Accession No. PTA-9693 comprises a 63 kDa fragment generated by caspase 1 -treatment of toxin B comprising the N-terminal enzyme domain of C. difficile toxin B.
- the epitope defined by the monoclonal antibody produced by the hybridoma cell line deposited under ATCC Accession No. PTA-9692 comprises the translocation domain of C. difficile toxin B.
- the epitope defined by the monoclonal antibody produced by the hybridoma cell line deposited under ATCC Accession No. PTA-9692 comprises a 167 kDa fragment generated by caspase 1 -treatment of toxin B and a 63 kDa protein which comprises untreated toxin B.
- the antibody is in humanized form. In an embodiment, the antibody is in chimeric form.
- a method of producing a monoclonal antibody which binds and neutralizes toxin A or toxin B of C. difficile involving immunizing one or more recipient animals with inactive toxoid A at periodic intervals; boosting the animals with increasing amounts of active toxin A or active toxin B at periodic intervals; obtaining hybridoma cells from immune cells of the immunized and boosted animal fused with a suitable immortalized cell line, wherein the hybridoma cells produce and secrete anti-toxin A antibodies which bind and neutralize toxin A of C. difficile or anti-toxin B antibodies which bind and neutralize toxin B of C. difficile is provided.
- the anti-C the anti-C.
- the immunizing and boosting steps include an adjuvant.
- the adjuvant is Quil A.
- the immunizing and boosting steps are performed at periodic intervals of every three weeks.
- the recipient animals are immunized with two or three doses of toxoid A, followed by three to five boosts of escalating doses of either active toxin A or active toxin B.
- an isolated antibody, or antigen-binding fragment thereof which inhibits, blocks, or prevents C. difficile toxin A toxicity by inhibiting, blocking, or preventing toxin A internalization and cytocellular toxicity.
- the antibody is a monoclonal antibody.
- the antibody is a humanized or chimeric antibody.
- the antibody is PA-39 (ATCC Accession No. PTA-9692) or humanized PA-39.
- the antibody is PA-50 (ATCC Accession No. PTA-964) or humanized PA-50.
- the antibody competes with PA-39, humanized PA-39, PA-50, or humanized PA-50 for binding to toxin A.
- the antibody binds a single site in a region of toxin A outside of the receptor binding domain of toxin A. In an embodiment, the antibody competes with PA-39 or a humanized form thereof by binding a single site in a region of toxin A outside of the receptor binding domain of toxin A. In an embodiment, the antibody binds to at least two sites in the receptor binding domain of toxin A. In an embodiment, the antibody competes with PA-50 or a humanized form thereof by binding to at least two sites in the receptor binding domain of toxin A. In an embodiment, the antibody inhibits toxin A toxicity via a mixed-competitive mechanism of action. In an embodiment, the antibody inhibits toxin A toxicity via a competitive mechanism of action. All of the above embodiments are meant to encompass the antigen -binding fragment of the antibody.
- an isolated antibody, or antigen-binding fragment thereof which inhibits, blocks, or prevents C. difficile toxin B toxicity by binding to an epitopic site in the N-terminal enzymatic region of toxin B.
- the antibody is a monoclonal antibody.
- the antibody is a humanized or chimeric antibody.
- the antibody is PA-41 (ATCC Accession No. PTA-9693) or a humanized form of PA-41.
- the antibody competes with PA-41 or humanized PA-41 for binding to the N-terminal enzymatic region of toxin B of C. difficile.
- the antibody competes with PA-41 or humanized PA-41 for binding to a single site in the N- terminal enzymatic region of toxin B of C. difficile. In an embodiment, the antibody inhibits toxin B toxicity via a mixed-competitive mechanism of action.
- Another aspect provides a vaccine or immunogen comprising portions, fragments, or peptides of toxin A and/or toxin B of C. difficile containing the epitopic regions recognized and/or bound by one or more of monoclonal antibody PA-39 (ATCC Accession No. PTA-9692), a humanized form of PA-39, monoclonal antibody PA-50 (ATCC Accession No. PTA-9694), a humanized form of PA-51, monoclonal antibody PA-41 (ATCC Accession No.
- the vaccine or immunogen comprises portions, fragments, or peptides of toxin A and toxin B of C. difficile containing the epitopic regions recognized and/or bound by one or more of monoclonal antibody PA-39 (ATCC Accession No.
- the epitope-containing portions, fragments, or peptides of toxin A and/or toxin B of the vaccine or immunogen are derived from the toxin A or toxin B protein by proteolytic cleavage.
- the toxin A fragments, portions, or peptides of the vaccine or immunogen are produced by proteolytic cleavage by enterokinase.
- the toxin B fragments, portions, or peptides of the vaccine or immunogen are produced by proteolytic cleavage by caspase (caspase 1).
- the epitope-containing portions or fragments of the vaccine or immunogen are chemically or recombinantly synthesized peptides of the toxin A or toxin B protein.
- the fragments, portions, or peptides of the vaccine or immunogen containing one or more epitopic regions of toxin A and/or toxin B that are recognized and bound by the antibody are derived from one or more of the amino terminus of toxin A; the amino terminus of toxin B; the carboxy terminus of toxin A; the carboxy terminus of toxin B; the receptor binding domain of toxin A; a region outside of the receptor binding domain of toxin A; the receptor binding domain of toxin B; the N-terminal enzymatic region of toxin B; the glucosyltransferase domain of toxin A; the glucosyltransferase domain of toxin B; the proteolytic domain of toxin A; the proteolytic domain of toxin B; the hydrophobic, pore-forming domain of toxin A; or the hydrophobic, pore-forming domain of toxin B.
- the epitope-containing fragments or portions of toxin A or toxin B are ⁇ 300 kDa, -158-160 kDa, -100-105 kDa, e.g., 103 kDa, -90-95 kDa, e.g., 91 kDa, and/or -63-68 kDa, e.g., 63 kDa or 68 kDa in size.
- the epitope-containing fragments or portions of toxin A are -158-160 kDa; -90- 95 kDa, e.g., 91 kDa, and/or -63-68 kDa, e.g., 68 kDa in size.
- the epitope-containing fragments or portions of toxin B are -100-105 kDa, e.g., 103 kDa and/or -63-68 kDa, e.g., 63 kDa in size.
- the toxin A or toxin B, or fragment, portion or peptide thereof is that of any of the strains provided herein.
- Another aspect provides a method of neutralizing, inhibiting, blocking, reducing, ameliorating, curing, or treating C. difficile infection or a C. difficile-associated disease in a subject in need thereof, comprising administering to the subject an effective amount of the above-described vaccine or immunogen.
- a humoral response to toxin A and/or toxin B of C. difficile following administration of the vaccine or immunogen is elicited in the subject, thereby producing anti-toxin A and/or anti-toxin B antibodies that can specifically neutralize, inhibit, block, reduce, ameliorate, cure, or treat C.
- the antibodies that are elicited via the subject's humoral response include antibodies having specificities and mechanisms of action similar or identical to the mAbs of the invention, or antibodies which compete with the mAbs of the invention in neutralizing toxin A and/or toxin B of C. difficile, or which compete with the mAbs of the invention in the mechanism of action involved in neutralizing toxin A and/or toxin B of C. difficile.
- a method of neutralizing, inhibiting, or blocking toxin A and/or toxin B activity in or against a cell susceptible to C. difficile infection comprising contacting the cell with an antibody, or antigen-binding fragment thereof, in accordance with the present invention, wherein the antibody, or antigen-binding fragment thereof, neutralizes, inhibits, or blocks the toxin A and/or toxin B activity in or against the cell by a competitive or a mixed competitive mechanism of action is provided.
- the antibody is one or more of a monoclonal antibody, a humanized antibody, or a chimeric antibody.
- the cell e.g., an intestinal epithelial cell
- the antibody, or antigen-binding fragment thereof is administered in an effective amount to the subject.
- the toxin is toxin A.
- the toxin is toxin B.
- the toxin is toxin A and the mechanism of action is a competitive inhibition mechanism of action.
- the antibody, or antigen binding fragment thereof is PA-50 (ATCC Accession No. PTA-9694), a humanized form thereof, or an antibody, or fragment thereof, which competes with PA-50 for neutralizing toxin A activity.
- the toxin is toxin A and the mechanism of action is a mixed-competitive inhibition mechanism of action.
- the antibody, or antigen binding fragment thereof is PA-39 (ATCC Accession No. PTA-9692), a humanized form thereof, or an antibody, or fragment thereof, which competes with PA-39 for neutralizing toxin A activity.
- the toxin is toxin B and the mechanism of action is a mixed competitive inhibition mechanism of action.
- the antibody, or antigen binding fragment thereof is PA-41 (ATCC Accession No. PTA-9693), a humanized form thereof, or an antibody, or fragment thereof, which competes with PA-41 for neutralizing toxin B activity.
- Figs. 1A-1C demonstrate the specificity of anti-C. difficile toxin mAbs of the invention for toxin A and/or toxin B via ELISA.
- ELISA plates were coated with toxin A (filled circles) or toxin B (open squares) overnight at 4°C. After the plates were washed and blocked, murine mAb PA-38 (A), PA-39 (B), or PA-41 (C) was titrated and added to the plates. Monoclonal antibody binding was detected with HRP conjugated goat anti-mouse IgG-Fc. OD was measured on a SpectraMax M5 Plate Reader (Molecular Devices).
- Figs. 2A-2D provide results from Biacore binding characterization assays using murine mAbs PA-38, PA-39, PA-41 and PA-50. Binding specificity was determined using a Biacore 3000 instrument (GE Healthcare). The mAbs (PA-38 (2A), PA-39 (2B), PA-50 (2C), PA-41 (2D), or nonspecific mAb as control) were covalently immobilized onto the CM5 sensor chip (GE Healthcare) surface at approximately 10,000 resonance units (RU) according to the manufacturer's instructions for amine coupling. Binding experiments were performed at 25 °C in PBS.
- Purified toxin A or toxin B (List Biological Laboratories) at 30 nM was passed over control (nonspecific mAb) and test flow cells at a flow rate of 5 ⁇ / ⁇ with an association phase (600s for PA-38, PA-39 and PA-41; and 300s for PA-50) and a dissociation phase (300s for PA-38, PA-39 and PA-41; and 600s for PA-50).
- Graphs are presented in RU over time.
- Figs. 3A-3E and 3F-3H show the results of antibody-toxin binding kinetics as determined by Biacore.
- murine mAbs were captured using a CM5 sensor chip prepared with Biacore' s mouse antibody capture kit. Toxin was then passed through the flow cells at varying concentrations (0.4 -100 nM, two-fold escalation) at a flow rate of 30 ⁇ / ⁇ . All mAb concentrations were tested in duplicate and the chip surface was regenerated after each run using the conditions specified in the kit. The changes in RU were recorded and analyzed using the Bia Evaluation Software 1:1 (Langmuir) binding model which calculated the KD of the mAb for the toxin.
- Figs. 3A binding of PA-38 to toxin A
- Fig. 3B binding of PA-50 to toxin A
- Fig. 3C binding of PA-39 to toxin A
- Fig. 3D binding of PA-39 to toxin B
- Fig. 3E binding of PA-41 to toxin B.
- murine mAbs i.e., mPA- 50, mPA-41, or mPA-39, were covalently linked to a CM5 sensor chip by the amine-coupling method.
- Toxin A (line designated “(red)") or toxin B (line designated “(blue)" at 30 nM was passed over test flow cells (mPA-50, mPA-41, or mPA-39) at a flow rate of 5 ⁇ / ⁇ .
- the results show that mPA-50 selectively binds toxin A (Fig. 3F), and mPA-41 selectively binds toxin B (Fig. 3G).
- mPA-39 binds preferentially to toxin A, but also demonstrates cross- reactivity to toxin B (Fig. 3H).
- Fig. 4 demonstrates the in vitro neutralization activity of toxin A activity using purified murine mAb PA-39 on CHO-K1 cells.
- toxin A was incubated with varying concentrations of PA-39 for 1 hour at 37°C (Example 3A).
- the mAb- toxin mixtures were then added to CHO-Kl cells plated in 96-well plates at 2,000 cells/well and incubated for 72 hours. Cell survival was compared in treated and untreated cultures and the concentration of mAbs required for 50% neutralization of cytotoxicity (EC 50 ) was calculated.
- Cell viability was determined via CellTiter-Blue; raw data were normalized to untreated control wells. The values were plotted using Prism and curves were calculated using a sigmoidal dose response (variable slope) model. The curve was then used to determine mAb EC 50 .
- the data points represent the average of three wells on the same plate.
- Fig. 5 demonstrates the in vitro neutralization activity of toxin B activity using purified murine mAb PA-41 on CHO-Kl cells.
- toxin B was incubated with varying concentrations of PA-41 for 1 hour at 37°C (Example 3B).
- the mAb- toxin mixtures were then added to CHO-Kl cells plated in 96-well plates at 2,000 cells/well and incubated for 72 hours. Cell survival was compared in treated and untreated cultures and the concentration of mAbs required for 50% neutralization of cytotoxicity (EC 50 ) was calculated.
- Cell viability was determined via CellTiter-Blue; raw data were normalized to untreated control wells. The values were plotted using Prism and curves were calculated using a sigmoidal dose response (variable slope) model. The curve was then used to determine mAB EC 50 .
- the data points represent the average of three wells on the same plate.
- Fig. 6 demonstrates the in vitro neutralization activity of toxin A activity using purified murine mAbs PA-38 and PA-50 on T-84 cells.
- T-84 cells were seeded (15,000 cells/well) in 96 well plates and treated with a combination of titrated mAb (PA-38 ( ⁇ ) or PA-50 (A )) and toxin A (60 ng/ml). After incubation (72 hours), cell survival was compared in treated and untreated cultures and the concentration of mAbs required for 50% neutralization of cytotoxicity (EC 50 ) was calculated.
- Cell viability was determined via CellTiter-Blue; raw data were normalized to untreated control wells. The values were plotted using Prism and curves were calculated using a sigmoidal dose response (variable slope) model. The curve was then used to determine mAb EC 50 .
- the data points represent the average of three wells on the same plate.
- Fig. 7 demonstrates the results of testing murine mAbs PA-38 ( ⁇ ) or PA-50 (A ) for their ability to block or prevent toxin A induced hemagglutination of rabbit red blood cells (RBCs).
- Toxin A (2 ⁇ g/ml) was combined with various dilutions of PA-38 or PA-50 and the mixture was added to plates containing 50 rabbit erythrocytes. Plates were incubated at 4°C for 4 hours. Hemagglutination was quantified as color intensity using ImageQuant 400 (GE Healthcare) dot array analysis. The data were rendered as % control, with 100% representing no hemagglutination. The data points represent the average of three wells assayed on the same plate.
- Fig. 8 demonstrates the activity of anti-C. difficile toxin mAbs of the invention in preventing disruption of a Caco-2 cell monolayer by toxin A.
- Caco-2 cells were seeded (25,000 cells/well) in the upper chamber of a 96-well Multiscreen Caco-2 Assay plate (Millipore). After an incubation of 10-14 days, the formation of a tight monolayer was confirmed by measuring transepithelial electrical resistance (TEER) using an epithelial voltohmmeter (World Precision Instruments).
- TEER transepithelial electrical resistance
- toxin A 25 ng/mL
- serially-diluted murine mAbs PA-38 ( ⁇ ) or PA-50 ( A )
- PA-38 ⁇
- PA-50 A
- Monolayer integrity was compared in untreated and toxin treated wells. Inhibition data were fit to a non-linear regression, sigmoidal dose-response curve using GraphPad Prism software in order to determine the concentration of mAb required for 50% toxin inhibition (EC 50 ).
- Figs. 9A-9C demonstrate the ability of the anti-toxin A mAbs PA-38 (9A) and PA-50 (9B) to neutralize toxin A activity in vivo.
- Female Swiss Webster mice (6-8-weeks old, 5 mice/group) were injected (i.p.) with murine mAb PA-38 or murine mAb PA-50 in the amounts indicated, or with PBS (200 ⁇ ) on Day 0.
- the neutralization activity of a comparator anti-toxin A monoclonal antibody, referred to herein as CDA-1 was evaluated in the antibody amounts indicated (9C).
- the anti-toxin A comparator mAb CDA-1 was produced by synthesizing (DNA2.0) nucleic acids encoding heavy and light chain variable regions of 3D8 (WO2006/121422 and US2005/0287150), which were cloned into full-length human IgGl expression vectors (pCON-gammal and pCON-kappa).
- the CDA-1 comparator mAb was expressed and produced in CHO-KSV1 cells and purified as described in the Examples section herein. The mice were then injected with 100 ng of toxin A (200 ⁇ ) on Day 1 and monitored daily for the first 72 hours and weekly thereafter.
- Fig. 10 demonstrates the ability of mAb PA-41 to neutralize toxin B activity in vivo.
- Female Swiss Webster mice (6-8-weeks old, 5 mice/group) were injected (i.p.) with either murine mAb PA-41 in the amounts indicated or with PBS (200 ⁇ ) on Day 0.
- the mice were then injected with 100 ng of toxin B (200 ⁇ ) on Day 1 and monitored daily for the first 72 hours and weekly thereafter.
- the results of this experiment show that the PA-41 mAb completely inhibits C. difficile toxin B-associated toxicity after a single dose of 5 ⁇ g of mAb/animal.
- CDB-1 comparator mAb a comparator anti-toxin B monoclonal antibody
- the anti-toxin B comparator mAb CDB-1 was produced by synthesizing (DNA2.0) nucleic acids encoding heavy and light chain variable regions of 124 (WO2006/121422 and US2005/0287150), which were cloned into full-length human IgGl expression vectors (pCON-gammal and pCON-kappa).
- the CDB-1 comparator mAb was expressed and produced in CHO-KSV1 cells and purified as described in the Examples section herein. The results of these experiments showed no toxin B neutralization activity by the comparator CDB-1 mAb, even in an amount of 250 ⁇ g.
- Fig. 11 demonstrates the ability of a combination of murine mAbs PA-38 and PA-41 (PA- 38+PA-41) of the invention to neutralize toxin A and toxin B activity in vivo.
- Female Swiss Webster mice (6-8-weeks old, 5 mice/group) were injected (i.p.) with the PA-38+PA-41 mAb combination or with PBS (200 ⁇ ) on Day 0.
- the mice were then injected with 100 ng of a combination of toxin A and toxin B (200 ⁇ ) on Day 1 and were monitored daily for the first 72 hours and weekly thereafter.
- the plots for toxin, PA-38 alone (open circles) and PA-41 alone (filled diamonds) overlap in the graph.
- Figs. 12A and 12B demonstrate pharmacokinetic (PK) results in hamsters for murine mAbs PA-38 and PA-41.
- Hamsters were dosed i.p. with 2 mg/kg (O) or 10 mg/kg ( ⁇ ) of mAb PA- 38 (12A) or PA41 (12B). Animals were bled at set intervals and serum was analyzed using an ELISA with toxin coated plates and goat anti-mouse IgG, HRP conjugated for detection. The resulting curves illustrate the dose dependent response in the 2 mg/kg and 10/mg/kg cohorts for each antibody. WinNonLin analysis was performed on each curve. Both monoclonal antibodies have a terminal half life of greater than 6 days.
- Example 13 illustrates the survival results of the hamster study described in Example 5B.
- hamsters were treated with clindamycin, inoculated with C. difficile ( ⁇ infected control, Group 3), and then treated with vancomycin ( ⁇ 20 mg/kg, Group 4), a combination of murine mAbs PA-38 + PA-41 ( ⁇ 50, 50 mg/kg, Group 6), or a combination of mAbs PA- 39 + PA-41(0 50, 40 mg/kg, Group 7). All animals in the uninfected control (Group 1) and goat polyclonal Abs treated control (group 5) survived. The animals treated with a combination of anti-toxin A and anti-toxin B mAbs of the invention survived and were protected against C. difficile toxicity for the duration of the study.
- Fig. 14 shows the mean body weight (in grams) of hamsters from the study described in Example 5B.
- the animal treatment groups are as follows: uninfected control ( ⁇ , Group 1); vancomycin-treated control ( ⁇ , Group 4); PA-38 + PA-41 murine mAb combination-treated group (x, Group 6), or PA-39 + PA-41 murine mAb combination-treated group ( ⁇ , Group 7). Animals in the infected control group (Group 3) did not survive for 5 days; therefore, a postinfection body weight measurement could not be made for Group 3.
- Figs. 15A-15D depict postmortem necropsy results from the hamster study described in Example 5B.
- Representative animals from each of the relevant groups of the study were evaluated: (A) Group 1, uninfected control; (B) Group 3, infected control; (C) Group 6, PA- 38 + PA-41 murine mAb combination-treated group; and (D) Group 7, PA-39 + PA-41 murine mAb combination-treated group.
- the arrows indicate the cecum of each hamster. The cecum was noticeably red and inflamed in the infected control Group 3 (B).
- the ceca of the hamsters in Group 6 (C) and Group 7 (D) were similar to the ceca in the healthy, uninfected control animals of Group 1 (A).
- Figs. 16A and 16B-1 and B-2 illustrates the survival results of the hamster study described in Example 5C.
- hamsters were treated with clindamycin, inoculated with C. difficile (m infected control, Group 1), and then different groups of animals were treated with vancomycin ( ⁇ 20 mg/kg, Group 2), the combination of murine mAbs PA-39 + PA-41 ( A 50 + 50 mg/kg, Group 3), murine mAb PA-41 alone (o 50 mg/kg, Group 4), murine mAb PA-38 alone (V 50 mg/kg, Group 5), murine mAb PA-39 alone ( ⁇ 50 mg/kg, Group 6), or murine mAb PA-50 alone ( 50 mg/kg, Group 7).
- Fig. 16B-1 illustrates the animal survival results of the hamster study described in Example 5E. Kaplan-Meier survival curves are shown for animals treated with clindamycin and then inoculated with C.
- Fig. 16B-2 illustrates the weight change results of the hamster study described in Example 5E. Mean ( ⁇ SD) body weights of animals over time in the different treatment groups described for Fig. 16B-1 compared
- Figs. 17A-17C show caspase 1 treatment of C. difficile toxin B.
- A Full length C. difficile toxin B and its domains.
- B 3-8% Tris-Acetate SDS-PAGE (reducing) analysis of toxin B (TcdB) and caspase 1-treated toxin B. Four toxin fragments were observed: 193, 167, 103 and 63 kDa.
- C The possible fragments generated following caspase 1 treatment of toxin B.
- Figs. 18A-18C show SDS-PAGE (A) and Western blot (B, C) detection of C. difficile toxin B fragments using anti-toxin B mAbs.
- FIG. 17B SDS-PAGE analysis of toxin B fragments generated by caspase 1 treatment (same as Fig. 17B);
- B Western blot detection of toxin B fragments using mAb PA-41.
- C Western blot detection of toxin B fragments using murine mAb PA-39.
- Figs. 19A-19E show the characterization of anti-C. difficile toxin murine mAbs by Biacore binding. Competitve binding of the anti-toxin mAbs was assessed.
- A mAb PA-41 binds a single epitope on toxin B.
- B mAb PA-39 binds a single epitope on toxin A.
- C mAbs PA-39 and PA-41 bind to different epitopes on toxin B. The binding of mAb PA-41 to toxin B is epitopically different from the binding of the comparator CDB-1 anti-toxin B antibody to toxin B.
- EK enterokinase
- A Full length C. difficile toxin A and its domains.
- B 3-8% Tris-Acetate SDS-PAGE (reducing) analysis of toxin A (TcdA) and EK-treated toxin A.
- C The possible fragments generated following EK treatment of toxin A at 25°C for 48 hours.
- Figs. 21A-21C show Coomassie Blue staining (SDS-PAGE), (A), and Western blot (B, C) detection of C. difficile toxin A fragments using anti-toxin A murine mAbs.
- A SDS- PAGE analysis of toxin A fragments generated by EK treatment (same as Fig. 20B).
- B Western blot detection of toxin A fragments using mAb PA-50.
- C Western blot detection of toxin A fragments using mAb PA-39.
- the kDa band is -158 kDa and may be considered to be -158-160 kDa.
- Figs. 22A-1 and A-2-22F show the characterization of murine anti-toxin A mAb binding to toxin A of C. difficile using a Biacore binding assay.
- Fig. 22A- 1 PA-50 mAb was observed to bind multiple sites on toxin A.
- Fig. 22A-2 PA-50 mAb was immobilized onto the sensor chip and then sequentially contacted with purified toxin A, additional PA-50 and comparator mAb CDA-1 (WO/2006/121422; US2005/0287150).
- toxin A captured by comparator mAb CDA-1 on the Biacore chip further binds additional CDA-1 and PA-50 mAb, showing the differences in toxin A epitopes bound by the antibodies.
- Fig. 22C the PA-39 mAb binding epitope on toxin A is different from the comparator mAb CDA-1 binding epitope on toxin A.
- Fig. 22D competitive binding of murine mAbs PA-50 and PA-39 to toxin A was performed using Biacore.
- Figs. 22E and 22F The Biacore results were confirmed by Western Blot analysis utilizing toxin A that was untreated or treated with the enzyme enterokinase (EK).
- E mPA-39 and comparator mAb CDA-1 show different binding patterns to EK-treated toxin A (Lane: TcdA/EK), thus indicating different binding domains and epitopes on toxin A.
- F mPA-50 and comparator mAb CDA-1 bind to the same domain of toxin A, but to different epitopes.
- Figs. 23A and 23B demonstrate the neutralizing activity of PA-41 in vitro against a diverse panel of twenty C. difficile toxigenic clinical isolates, including 6 BI/NAP1/027 isolates, 3 reference strains (VPI 10463, ATCC 43596, and 630), 2 toxin A-negative/toxin B-positive (toxA-/toxB+) isolates, 3 outpatient isolates, and 6 other common clinical isolates.
- Fig. 23A shows the neutralizing activity of murine mAb PA-41 on CHO-Kl cells against supematants generated from the different C. difficile clinical isolate cultures as presented in Example 8, Table 6.
- mAb PA-41 Purified mAb PA-41 was serially diluted and then mixed with a supernatant with predefined dilution factor that can cause >90% cell death. The mixtures were incubated for lhour at 37°C and then added to CHO-Kl cells. The cells were incuated for 72 hours, and cell viability was measured using Cell-Titer Blue.
- Fig. 23B illustrates the neutralizing activity of humanized mAb hPA-41 as well as that of comparator mAb CDB-1 against the 2 C. difficile reference strains (VPI 10463 and ATCC 43596) and 6 C. difficile BI/027/027 strains (CCL678, HMC553, Pitt 45, CD196, Montreal 5.1 and Montreal 7.1).
- Figs. 24A and 24B demonstrate the neutralizing activity of mAb PA-50 in vitro against the C. difficile toxigenic clinical isolates described for Figs. 23 A and B.
- Fig. 24A shows the neutralizing activity of murine mAb PA-50 on T-84 cells against supematants generated from different C. difficile clinical isolate cultures as presented in Example 8, Table 6.
- Purified mAb PA-50 was serially diluted and then mixed with a supernatant with predefined dilution factor that can cause >90% cell death. The mixtures were incubated for lhour at 37°C and then added to T-84 cells. The cells were incuated for 72 hours, and cell viability was measured using Cell-Titer Blue.
- 24B shows the results of similar experiments performed using humanized mAb hPA-50 and the comparator mAb CDA-1 on T-84 cells.
- the neutralization activity of hPA-50 (filled squares) and CDA-1 comparator (filled triangles) on T-84 cells against supematants generated from six BI/NAP1/027 strains (CCL678, HMC553, Pitt 45, CD196, Montreal 5.1 and Montreal 7.1) is shown.
- BI/NAP1/027 strains CCL678, HMC553, Pitt 45, CD196, Montreal 5.1 and Montreal 7.1
- Figs. 25A-25D demonstrate neutralization of toxins produced by diverse strains of C. difficile.
- Figs. 25A and 25B show neutralizing activity of murine mAb PA-39 on T-84 cells against supematants generated from different C. difficile clinical isolate cultures as presented in Example 8, Table 6.
- mAb PA-39 (hybridoma supernatant) was serially diluted and the dilution factor of each supernatant is shown.
- Fig. 25B shows the results of similar experiments performed using murine mAb PA-39 and the comparator CDA-1 mAb on T-84 cells.
- humanized anti-toxin B mAb PA-41 and comparator anti-toxin A mAb CDB-1 were tested for neutralization of cytoxicity of C. difficile culture supernatants against T-84 cells. (Example 8, Table 7).
- Fig. 26 shows that the chimeric mAb PA-41 (cPA-41 mAb) effectively neutralizes the toxicity of C. difficile toxin B on CHO-K1 compared to the counterpart murine mAb PA-41.
- Two chimeric PA-41 mAbs were generated; one which had a glycosylation site removed in the VL region, designated as cPA-41(NG), and one which had no glycosylation site removal, designated as cPA-41(G) in the figure.
- cPA-41(NG) and cPA-41(G) showed similar neutralization levels for toxin B (2 pg/mL, TechLab) on CHO-K1 cells, and both chimeric mAbs neutralized toxin B at a level similar to that of the parent murine mAb (mPA-41).
- Fig. 27 shows that chimeric PA-39 (cPA-39) mAb effectively neutralizes the toxicity of C. difficile toxin A (1 ⁇ g/mL, Listlab) on CHO-K1 cells compared with the parent murine PA-39 (mPA-39) mAb.
- Fig. 28 shows that chimeric PA-50 (cPA-50) mAb effectively neutralizes the toxicity of C. difficile toxin A (60 ng/mL, TechLab) on T-84 cells compared with the parent murine PA-50 (mPA-50) mAb.
- Fig. 29 shows the in vitro neutralization activity of murine PA-41 (mPA-41) and humanized PA-41 (hPA-41) mAbs against C. difficile toxin B. Percent cell survival compared with controls was measured using CellTiter Blue. hPA-41 mAb effectively neutralizes the toxicity of toxin B (2 pg/mL, Techlab) on CHO-K1 cells with an EC 50 of 6 pM and was virtually equipotent to the parental murine monoclonal antibody (mPA-41).
- Fig. 30 shows the in vitro neutralization activity of murine PA-39 (mPA-39) and humanized PA-39 (hPA-39) mAbs against C. difficile toxin A.
- hPA-39 mAb effectively neutralizes the toxicity of toxin A (20 ng/mL, TechLab) on CHO-K1 cells with an EC 50 of 50 pM and was virtually equipotent to the parental murine monoclonal antibody (mPA-39).
- Figs. 31A-31H show the results of in vitro neutralization activity and mechanism of action (MOA) studies using the described mAbs.
- the cell-based assays using CHO-K1 cells and T- 84 cells were carried out as described in Examples 1, 3 and 7.
- Fig. 31A shows that humanized PA-50 (hPA-50) mAb effectively neutralized the toxicity of toxin A (60 ng/mL, TechLab) on T-84 cells compared with the parental murine PA-50 mAb (mPA-50).
- Figs. 31B-D show the neutralization activities of anti-toxin A mAbs PA-39 and PA-50 compared with that of comparator anti-toxin A mAb CDA-1.
- Figs. 31E and F show the neutralization activity of anti-toxin B mAb PA-41 compared with that of comparator antitoxin B mAb CDB-1.
- the EC 50 and maximum percent inhibition values are as presented in Table B of Example 7C.
- Figs. 31G and H show an ELISA method and results for assessing the ability of the anti-toxin A mAbs to block toxin A internalization into cells and for evaluating the activities of these mAbs in preventing toxin A internalization relative to polyclonal goat anti-toxin A antibody control and no antibody control.
- Figs. 32A and 32B depict the amino acid sequences of humanized PA-39 (hPA-39) VH regions, SEQ ID NO:l and SEQ ID NO:2. Amino acid residues are shown in single letter code. Numbers above the sequences indicate the locations according to Kabat et al. The locations of the CDRs are underlined.
- Figs. 33A and 33B depict the amino acid sequences of humanized PA-39 (hPA-39) VL regions, SEQ ID NO:3 and SEQ ID NO:4. Amino acid residues are shown in single letter code. Numbers above the sequences indicate the locations according to Kabat et al. The locations of the CDRs are underlined.
- Figs. 34A and 34B depict the amino acid sequences of humanized PA-50 (hPA-50) VH regions, SEQ ID NO:5 and SEQ ID NO:6. Amino acid residues are shown in single letter code. Numbers above the sequences indicate the locations according to Kabat et al. The locations of the CDRs are underlined.
- Fig. 35 depicts the amino acid sequence of humanized PA-50 VL region, SEQ ID NO:7. Amino acid residues are shown in single letter code. Numbers above the sequences indicate the locations according to Kabat et al. The locations of the CDRs are underlined.
- Figs. 36A and 36B depict the amino acid sequences of humanized PA-41 (hPA-41) VH regions, SEQ ID NO:8 and SEQ ID NO:9. Amino acid residues are shown in single letter code. Numbers above the sequences indicate the locations according to Kabat et al. The locations of the CDRs are underlined.
- Fig. 37 depicts the amino acid sequence of humanized PA-41 VL region, SEQ ID NO: 10. Amino acid residues are shown in single letter code. Numbers above the sequences indicate the locations according to Kabat et al. The locations of the CDRs are underlined.
- Figs. 38A and 38B show the nucleic acid sequence and encoded amino acid sequence of a humanized anti-C. difficile toxin A monoclonal antibody.
- Fig. 38A depicts the amino acid sequence of the light chain of the humanized anti-toxin A monoclonal antibody as set forth in SEQ ID NO: 16, which is encoded by the nucleic acid sequence as set forth in SEQ ID NO: 17.
- Fig. 38B depicts the amino acid sequence of the heavy chain of the humanized monoclonal antibody as set forth in SEQ ID NO: 14, which is encoded by the nucleic acid sequence as set forth in SEQ ID NO: 15.
- Figs. 39A and 39B show the nucleic acid sequence and encoded amino acid sequence of a humanized anti-C. difficile toxin A monoclonal antibody.
- Fig. 39A depicts the amino acid sequence of the light chain of the humanized anti-toxin A monoclonal antibody as set forth in SEQ ID NO:20, which is encoded by the nucleic acid sequence as set forth in SEQ ID NO:21.
- Fig. 39B depicts the amino acid sequence of the heavy chain of the humanized antitoxin A monoclonal antibody as set forth in SEQ ID NO: 18, which is encoded by the nucleic acid sequence as set forth in SEQ ID NO: 19.
- Figs. 40A and 40B show the nucleic acid sequence and encoded amino acid sequence of a humanized anti-C. difficile toxin B monoclonal antibody.
- Fig. 40A depicts the amino acid sequence of the light chain of the humanized anti-toxin B monoclonal antibody as set forth in SEQ ID NO:24, which is encoded by the nucleic acid sequence as set forth in SEQ ID NO:25.
- Fig. 40B depicts the amino acid sequence of the heavy chain of the humanized anti- toxin B monoclonal antibody as set forth in SEQ ID NO:22, which is encoded by the nucleic acid sequence as set forth in SEQ ID NO:23.
- Figs. 41A-41C demonstrate the in vitro neutralization activity against C.
- A murine mAb PA-39 and PA-39 Fab neutralization activity on CHO- Kl cells; toxin A (Techlab, 60 ng/ml);
- B murine mAb PA-41 and PA-41 Fab neutralization activity on CHO-K1 cells; toxin B (Techlab, 2 pg/ml);
- C murine mAb PA- 50 and PA-50 Fab neutralization activity on T-84 cells; toxin A (Techlab, 60 ng/ml).
- Figs. 42A and 42B show the antibody concentration profiles resulting from the pharmacokinetic (PK) study described in Example 13 herein.
- Fig. 42A depicts the PK results (serum antibody concentration in ⁇ g/mL) over 29 days from animals receiving a single dose of purified, humanized, anti-toxin A mAb PA-50 at a concentration of 1 mg/kg ( A ) or 5 mg/kg ( ⁇ ) on day 0.
- Fig. 42B depicts the PK results (serum antibody concentration in ⁇ g/mL) over 29 days from animals receiving a single dose of purified, humanized, anti-toxin B mAb PA-41 at a concentration of 1 mg/kg ( A ) or 5 mg/kg ( ⁇ ) on day 0.
- the present invention encompasses antibodies and antigen-binding fragments thereof that provide non-antibiotic therapies and treatments which block the pathogenic effects of C. difficile infection and, preferably, provide time for the colon to heal and/or the normal microflora of the gastrointestinal tract, e.g., colon, bowel, intestine, etc., to become reestablished.
- the monoclonal antibodies as described herein, antigen-binding fragments thereof, such as humanized or chimeric forms thereof provide non- antibiotic therapeutics and medicaments both to treat active disease and to prevent recurrent disease so as to allow patients suffering from C. difficile infection and CDAD to resolve their disease without relapse to further disease or to more severe disease or symptoms.
- antibodies of the invention have therapeutic activity against active disease caused by, or associated with, infection of subjects by C. difficile. In an embodiment, antibodies of the invention resolve active disease caused by, or associated with, infection of subjects by C. difficile. In an embodiment, antibodies of the invention have therapeutic effects in decreasing the duration and/or severity of active disease caused by, or associated with, infection by C. difficile in a subject. In an embodiment, antibodies of the invention or portions or fragments thereof can be provided in combination with antibiotic therapeutics.
- Monoclonal antibodies (mAbs) against C. difficile toxins A and B have been generated as described herein.
- the anti-toxin mAbs exhibit potent activity both in in vitro assays and in preclinical animal models of C. difficile infection in vivo. More specifically, mAbs of the invention potently and durably protect hamsters from mortality in a relevant and stringent hamster model of C. difficile infection.
- the antibodies provide non-antibiotic approaches for the treatment of CD AD and may allow the discontinuation of antibiotics and block the pathogenic effects of C. difficile toxins, thereby providing time for the colon to heal and the normal bowel microflora to become reestablished.
- the mAbs as described herein can provide therapeutic benefit by their ability to neutralize the toxins of C. difficile and may be employed in passive or active strategies to treat patients with multiple recurrences of C. difficile.
- the mAbs may be utilized for preventing recurrence of infection, for treating severe, active forms of disease, and for treating patients with multiple relapses of C. difficile-associated diseases.
- the mAbs as described herein can provide an effective means to neutralize C. difficile toxins A and B so as to prevent, block, or inhibit recurrence of infection, and/or severe and active forms of the disease and multiple relapses.
- toxin A and toxin B refer to the cytotoxic enterotoxins produced by the C. difficile microorganism. Toxins A and B are the major virulence determinants of C. difficile, and toxin-negative strains are nonpathogenic. Toxins A and B are transcribed from a pathogenicity locus that includes the toxin genes, tcdA (toxin A) and tcdB (toxin B), and three regulatory genes, one of which (tcdC) encodes a putative negative regulator of toxin transcription. TcdC protein appears to inhibit toxin transcription during the early, exponential-growth phase of the bacterial life cycle.
- toxin B an autocatalytic cleavage site between leucine 5 43 and glycine 5 44 has been described. Cleavage results from activation of an aspartyl protease domain by host cytosolic inositol phosphate, and releases the active glucosyltransferase domain.
- antibodies and antigen-binding fragments thereof that bind specifically to toxin A and/or toxin B of C. difficile, compositions containing one or more of such antibodies or antigen-binding fragments thereof, vectors containing nucleic acid sequences that encode the antibodies or antigen-binding fragments thereof, hybridoma cell lines that produce the antibodies, and methods of using the antibodies or antigen -binding fragments thereof for treatment or prevention of C. difficile infection or C. difficile-associated disease.
- antibodies or “immunoglobulins” is referred to herein in describing the present invention and its various aspects and embodiments, this term is also generally meant to embrace antigen-binding fragments of such antibodies or immunoglobulins, so as to avoid excessive repetition of the associated phrase "antigen- binding fragments" whenever the term “antibodies” or “immunoglobulins” is mentioned.
- the present invention emcompasses not only antibodies directed against toxin A and toxin B of C. difficile, i.e., toxin A and toxin B antigens, but also fragments of such antibodies which bind the C. difficile toxin A and toxin B antigens, as described further herein.
- such antigen-binding fragments are capable of neutralizing the toxicity of toxin A and/or toxin B in a manner similar to that of the intact antibody.
- the antibodies embraced by the invention include isolated antibodies which specifically bind toxin A of C. difficile and which competitively inhibit, or cross compete for, the specific binding to toxin A of C. difficile of an isolated monoclonal antibody produced by the hybridoma cell line deposited under ATCC Accession No. PTA-9692, PTA-9694, or PTA- 9888.
- the antibodies also include isolated antibodies which specifically bind to toxin A of C. difficile and which specifically bind to an epitope on toxin A of C. difficile defined by the binding of an isolated monoclonal antibody produced by the hybridoma cell line deposited under ATCC Accession No. PTA-9692, PTA-9694, or PTA-9888.
- the epitope resides in the C-terminal receptor binding domain of tcdA.
- the antibodies competitively inhibit, or cross compete for, the binding of PA- 50 to tcdA.
- the eptiope resides in the translocation domain of tcdA.
- the antibodies competitively inhibit, or cross compete for, the binding of PA-39 to tcdA.
- Such isolated antibodies may comprise monoclonal antibodies, polyclonal antibodies, chimeric antibodies, human antibodies, humanized antibodies, and antigen binding fragments or portions thereof.
- Biacore assays support a single binding site on toxin A for PA-39.
- Western blot detection of enzymatically-digested toxin A demonstrates that PA-39 binds a region on toxin A that is separate from the regions bound by PA-50 and the CDA-1 comparator mAb.
- the in vitro activity of PA-39 in the toxin potency assay shows shifts in both EC 50 and the maximum percent inhibition as more toxin A is added to culture, indicating a mixed-competitive mechanism of inhibition for PA-39.
- ELISA detection of toxin A after protection by 100-fold excess of PA-39 confirmed that inhibition of toxin by PA-39 occurs by preventing toxin internalization and adverse cytocellular toxin effects, e.g., cytoxicity.
- Biacore assays support at least two binding sites on toxin A for PA-50.
- Western blot detection of enzymatically-digested toxin A demonstrates that PA-50 binds a region on toxin A similar to that bound by comparator mAb CDA-1.
- the in vitro activity of PA-50 in the toxin potency assay shows a shift in EC50 as more toxin A is added to culture, indicating a competitive mechanism of inhibition for PA-50.
- ELISA detection of toxin A after protection by 100-fold excess of PA-50 confirmed that inhibition of toxin by PA-50 occurs by preventing toxin internalization and subsequent cytotoxicity.
- the antibodies of the invention also include isolated antibodies which specifically bind toxin B of C. difficile and which competitively inhibit, or cross compete for, the specific binding to toxin B of C. difficile of an isolated monoclonal antibody produced by the hybridoma cell line deposited under ATCC Accession No. PTA-9693 or PTA-9692.
- the antibodies also include isolated antibodies which specifically bind to toxin B of C. difficile and which specifically bind to an epitope on toxin B of C. difficile defined by the binding of an isolated monoclonal antibody produced by the hybridoma cell line deposited under ATCC Accession No. PTA- 9693 or PTA-9692.
- the epitope resides in the N-terminal enzyme domain of tcdB.
- the antibodies competitively inhibit, or cross compete for, the binding of PA-41 to tcdB.
- the epitope resides in the translocation domain, e.g., amino acids 850-1330 of tcdB.
- the antibodies competitively inhibit, or cross compete for, the binding of PA-39 to tcdB.
- Biacore assays support a single binding site on toxin B for PA-41.
- Western blot detection of enzymatically-digested toxin B shows that PA-41 binds a different region on toxin B that is different from that bound by comparator mAb CDB-1.
- the in vitro activity of PA-41 in the toxin potency assay shows shifts in both EC 50 and the maximum percent inhibition as more toxin B is added to culture, indicating a mixed-competitive mechanism of inhibition PA-41.
- Antibodies provided herein include monoclonal antibodies produced by hybridomas that were deposited and given the following Patent Deposit Designations: PTA-9692 (for PA-39), PTA-9693 (for PA-41), PTA-9694 (for PA-50), and PTA-9888 (for PA-38). These hybridomas were deposited pursuant to, and in satisfaction of, the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure with the American Type Culture Collection (“ATCC”), P.O.
- ATCC American Type Culture Collection
- both the deposited hybridomas and the monoclonal antibodies produced by the hybridomas may be referred to by the same ATCC Deposit Designations or to the numbers found within the ATCC Deposit Designations.
- PTA-9888 or 9888 may be used to refer to the deposited hybridoma or to the monoclonal antibody produced by the hybridoma.
- the names of the monoclonal antibodies described herein may be used interchangeably with the names of the hybridomas that produce them. It will be clear to one of skill in the art when the name is intended to refer to the antibody or to the hybridoma that produces the antibody.
- the antigen-binding fragments provided herein include the antigen-binding fragments of the aforementioned deposited antibodies.
- the antibodies of the invention exhibit a number of beneficial characteristics.
- the anti-toxin A antibodies neutralize or inhibit the toxicity of toxin A both in vitro and in vivo.
- humanized PA-39 and humanized PA-41 demonstrated neutralization potencies (i.e., EC 50 values) of 46 pM against toxin A on cells and 5 pM against toxin B, respectively, on these cells.
- neutralization potencies i.e., EC 50 values
- humanized anti-C. difficile toxin A and anti-C. difficile toxin B monoclonal antibodies of the invention show increased anti-toxin neutralization characteristics compared with those of other anti-toxin antibodies that have been reported.
- an anti-toxin A antibody of the invention neutralizes or inhibits the in vivo toxicity of C. difficile toxin A at an effective dose.
- the antitoxin B antibodies neutralize or inhibit the in vivo toxicity of toxin B.
- an effective dose of one or more anti-toxin A antibodies of the invention is provided to a C. difficile-infected subject.
- an effective dose of one or more anti-toxin A antibodies of the invention is provided in combination with an effective dose of one or more anti-toxin B antibodies of the invention to a C. difficile-infected subject.
- an anti-toxin A antibody of the invention in a 1 : 1 combination with an anti-toxin B antibody of the invention is provided as an effective dose to a C. difficile-infected subject.
- an effective dose of an anti-toxin A antibody and an anti-toxin B antibody of the invention may be, for example, a 1 ⁇ 2:1, 1: 1, 2:1, 3:1, 4:1, etc., combination of the antibodies provided to a C. difficile-infected subject.
- the antibodies are humanized.
- the antibodies are included in a composition.
- an effective dose of the anti-toxin A and/or anti-toxin B antibodies may range from 0.1 ⁇ g to 1000 milligrams (mg).
- the anti-toxin A antibodies and anti-toxin B antibodies or antigen- binding fragments thereof may be administered to a subject in an amount of, for example, 0.1 mg/kg-150 mg/kg; in an amount of 0.5 mg/kg-75 mg/kg; in an amount of 1 mg/kg-100 mg/kg; in an amount of 1 mg/kg-50 mg/kg; in an amount of 2 mg/kg-40 mg/kg; in an amount of 2 mg/kg-50 mg/kg; in an amount of 5 mg/kg -50 mg/kg; in an amount of 5 mg/kg-25 mg/kg; in an amount of 10 mg/kg-40 mg/kg; in an amount of 10 mg/kg-50 mg/kg; in an amount of 10 mg/kg-25 mg/kg; or in an amount of 15 mg/kg-50 mg/kg.
- the aforementioned amounts may comprise the varying ratios of anti-toxin A antibody and anti-toxin B antibody provided in combination.
- neutralize refers to the reduction, inhibition, blocking, amelioration, or elimination of adverse effect(s) of the toxin(s) which the antibody(ies) specifically bind.
- Neutralization of adverse effect(s) of the toxin(s) includes 1) delaying, reducing, inhibiting, or preventing onset or progression of C. difficile infection or C. difficile-associated diarrhea or disease, 2) increasing survival of a subject as compared to the median survival of subjects who have not been treated with the antibody(ies) and who have C. dijjicile infection or C. difficile-associated disease, 3) eliminating one or more symptoms or adverse effects or reducing the severity of one or more symptoms or adverse effects associated with C. difficile infection or C.
- C. difficile-associated diarrhea or disease 4) allowing for the repopulation of the normal microflora of the gastrointestinal tract of subjects who are or have been infected with C. difficile, 5) preventing a recurrence of C. difficile infection or C. difficile-associated disease in subjects who have been afflicted with C. difficile infection or C. difficile-associated disease, 6) effecting a cure rate of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% in subjects who have C. difficile infection or C. difficile- associated disease, and/or 7) preventing death due to CDAD or other adverse events associated with C. difficile infection.
- Anti-C. difficile toxin A and toxin B antibodies of the invention may be used to treat a number of species of subjects, including man (humans) and other non-human (mammalian) animals.
- Subjects treatable in accordance with the invention include human beings, non- human primates, dogs, cats, mice, rats, hamsters, guinea pigs, cows, goats, sheep, pigs, horses, and the like.
- Human subjects may also be referred to as patients or individuals herein.
- subjects include a human patient having a C. difficile infection or a C. difficile- associated disease.
- Such human patients include those who are elderly or immune- compromised.
- anti-C. difficile toxin A and toxin B antibodies can resolve C. difficile disease and increase the survival of a subject.
- one or more anti-toxin A antibodies and/or one or more anti-toxin B antibodies when administered to a subject, improve(s) the survival of the subject compared with the median survival of subjects who have not been treated with the antibody(ies) and who have C. difficile infection or C. difficile-associated disease.
- the dose or amount of the one or more anti-toxin A or anti-toxin B antibodies may range for example from 0.2 ⁇ g-250 ⁇ g, or from 2 ⁇ g -50 , ⁇ g, or from 5 ⁇ g - 50 , ⁇ g, e.g., based on in vivo mouse studies.
- the dose or amount of one or more anti-toxin A or anti-toxin B antibodies may range for example from 2 mg/kg-40 mg/kg, 2 mg/kg-50 mg/kg, 5 mg/kg-40 mg/kg, 5 mg/kg-50 mg/kg, 10 mg/kg-40 mg/kg, or 10 mg/kg-50 mg/kg, e.g., based on in vivo hamster studies.
- antibodies of the invention can effect a cure or survival rate of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99%.
- the antibodies can effect a cure or survival rate of 100%.
- one or more anti-toxin A antibodies when administered to a subject, together with one or more anti-toxin B antibodies, effect a cure or survival rate of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%.
- cure rate refers to the percentage of subjects that a clinician would determine to no longer have the infection or disease out of a population of subjects with the infection or disease administered one or more antibodies, or one or more compositions thereof, of the invention.
- Sudvival rate refers to the percentage of subjects that survive for a desired period of time out of a population of subjects administered one or more antibodies, or one or more compositions thereof, of the invention. Examples of such desired periods of time are provided elsewhere herein.
- Anti-toxin A and anti-toxin B antibodies provided by the invention can allow for the restoration of normal gut flora in a subject infected with C. difficile. In this way, such antibodies can resolve disease in patients undergoing treatment. Anti-toxin A and anti-toxin B antibodies of the invention can also demonstrate beneficial in vivo pharmacokinetics. Antitoxin A and anti-toxin B antibodies of the invention can also provide prolonged or long lasting therapy for a subject who has been infected with C. difficile. As used herein, "long lasting" refers to therapy that results in an absence of C. difficile infection or C. dijficile- associated disease one month or more after cessation of treatment. Preferably, the therapy results in an absence of C. difficile infection or C.
- therapy with mAbs of the invention results in treating or depressing active C. difficile infection and in reducing or diminishing the robustness of infection.
- therapy provided by the invention results in an absence of C. difficile infection or C. difficile-associated disease in a subject for 1, 2, 3, 4, 5, or 6 months.
- therapy provided by the invention results in an absence of C. difficile infection or C. difficile-associated disease in a subject for longer than 6 months.
- Anti-toxin A and anti-toxin B antibodies of the invention can prevent recurrence of C. difficile infection and/or C. difficile-associated disease.
- Hypervirulent strains comprise a group of closely related isolates characterized as North American Pulsed Field Type 1 (NAP1), restriction enzyme analysis type "BI”, and PCR Ribotype 027 known collectively as BI/NAPl/027 (5).
- NAP1 North American Pulsed Field Type 1
- BI restriction enzyme analysis type
- PCR Ribotype 027 known collectively as BI/NAPl/027 (5).
- the hypervirulence of the BI/NAPl/027 strains has been attributed at least in part to increased production of toxins A and B, two virulent factors of CDAD (6).
- BI/NAPl/027 isolates produce 16-23-fold higher levels of toxins A and B than other strains (6).
- the apparent fitness of these strains creates the threat of worldwide spread compromising the potential for antibiotic treatment for other diseases, as well as increasing CDAD recurrence rates and severity.
- antibiotics under development for the treatment of CDAD such as nitazoxanide, rifaximin, ramoplanin and fidaxomicin, clinical isolates of C. difficile that are resistant to rifaximin have been reported.
- the mAbs of the invention are employed in the treatment of infection caused by various strains of C. difficile.
- the C. difficile strains are highly infectious and their toxins are neutralized by the mAbs of the invention.
- the toxins of hypervirulent strains of C. difficile, including BI/NAPl/027 are neutralized by the mAbs of the invention.
- the mAbs of the invention provide therapeutic effects in neutralizing toxins from a broad range of toxigenic clinical isolates, including strains from outpatient isolates.
- the mAbs neutralize the toxins of hypervirulent isolates, such as BI/NAPl/027, and at least 90% or more of other clinically relevant isolates of C. difficile.
- mAbs of the invention have been shown to neutralize significantly the toxicity/activity of nineteen different clinical isolates of C. difficile, including BI/NAPl/027 and other hypervirulent C. difficile strains, e.g., CCL676, HMC553, Pitt45, CD196, accordinglyal 5 and spirital 7.1.
- antibodies are provided which neutralize toxin A and toxin B of hypervirulent strains of C.
- mAbs of the invention are provided for use in neutralizing hypervirulent strains of C. difficile, including hospital and non-hospital derived isolates, as treatment for C. difficile infection and diseases related thereto.
- mAbs of the invention exhibit an EC 50 neutralization value in the range of 93 pM-30 nM, or an EC 50 of 46 pM, for neutralizing toxin A and an EC 50 value in the range of 4 pM-9.5 pM, or an EC 50 of 5 pM, for neutralizing toxin B depending on the in vitro cell-based assay employed.
- an assay comprising CHO-K1 cells in which 8 ⁇ g/ml of toxin A was used, anti-toxin A mAbs of the invention exhibited an EC 50 value of 93 pM.
- anti-toxin B mAb of the invention In an assay comprising CHO-K1 cells in which 8 pg/ml of toxin B was used, anti-toxin B mAb of the invention exhibited an EC 50 value of 9.2 pM. In an assay comprising T-84 cells in which 240 ng/ml of toxin A was used, anti-toxin A mAbs of the invention exhibited EC 50 values of 146 pM and 175 pM. In a Caco- 2 cell-based assay, anti-toxin A mAbs of the invention neutralized toxin A toxicity at EC 50 levels of 196 pM and 485 pM.
- anti-toxin A mAbs of the invention had an EC 50 neutralization value of 1.8 nM and 30 nM for preventing RBC hemagglutination.
- the antibodies of the invention can have any one of, a combination of, or all of, the aforementioned features.
- mAbs of the invention demonstrate superior toxin- neutralizing potency both in vitro and in vivo in the best available preclinical models of CDAD.
- mAbs of the invention demonstrate uniquely broad and potent neutralization of toxins from numerous B I/NAP 1/027 strains.
- such mAbs have demonstrated complete and durable protection from mortality in a highly stringent hamster model of CDAD.
- antibodies to toxin A include those that competitively inhibit, or cross compete for, the specific binding to toxin A of C. difficile of an isolated monoclonal antibody produced by the hybridoma cell line deposited under ATCC Accession Nos. PTA-9692, PTA-9694, or PTA-9888.
- Preferred antibodies to toxin B include those that competitively inhibit, or cross compete for, the specific binding to toxin B of C. dijjicile of an isolated monoclonal antibody produced by the hybridoma cell line deposited under ATCC Accession No. PTA-9693 or PTA-9692.
- antibodies include those that competitively inhibit, or cross compete for, the specific binding to toxin A of C.
- cross-competition assays can be used to determine if an antibody competitively inhibits binding to toxin A and/or toxin B by another antibody.
- Such methods can be cell-based methods employing flow cytometry or solid phase binding analysis.
- Other assays that evaluate the ability of antibodies to cross-compete for binding toxin A and/or toxin B in solid phase or in solution phase also can be used.
- antibodies or antigen-binding fragments thereof encompassed by the invention include those that competitively inhibit the specific binding by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. Inhibition can be assessed at various molar ratios or mass ratios; for example, competitive binding experiments can be conducted with a 2-fold, 3-fold, 4-fold, 5-fold, 7-fold, 10-fold or more molar excess of the first antibody over the second antibody.
- antibodies encompassed by the invention include those that specifically bind to an epitope on toxin A of C. difficile defined by the binding of an isolated monoclonal antibody produced by the hybridoma cell line deposited under ATCC Accession No. PTA-9692, PTA- 9694, or PTA-9888. Still other antibodies or antigen-binding fragments encompassed by the invention include those that specifically bind to an epitope on toxin B of C. difficile defined by the binding of an isolated monoclonal antibody produced by the hybridoma cell line deposited under ATCC Accession No. PTA-9693 or PTA-9692. Still other antibodies or antigen-binding fragments encompassed by the invention include those that specifically bind to an epitope on toxin A of C.
- fragments (peptides) of the toxin preferably synthetic peptides
- fragments (peptides) that bind an antibody can be used to determine whether a candidate antibody or antigen-binding fragment thereof binds the same epitope.
- fragments (peptides) of the toxin preferably synthetic peptides
- overlapping peptides of a defined length e.g., 8 or more amino acids
- the peptides preferably are offset by 1 amino acid, such that a series of peptides covering every 8 amino acid fragment of the toxin sequence are prepared. Fewer peptides can be prepared by using larger offsets, e.g., 2 or 3 amino acids.
- peptides e.g., 9-, 10- or 11-mers
- Binding of peptides to antibodies can be determined using standard methodologies including surface plasmon resonance (e.g., Biacore) and ELISA assays.
- surface plasmon resonance e.g., Biacore
- ELISA assays For examination of conformational epitopes, which the antibodies provided herein may, in some embodiments, bind, larger peptide fragments can be used.
- Other methods that use mass spectrometry to define conformational epitopes have been described and can be used (see, e.g., Baerga-Ortiz et al., Protein Science 11 : 1300-1308, 2002 and references cited therein).
- Epitopes can be confirmed by introducing one or more point mutations or deletions into a known epitope, and then testing binding with one or more antibodies to determine which mutations reduce binding of the antibodies.
- the antibodies or antigen-binding fragments provided by the invention may specifically bind toxin A and/or toxin B with sub-nanomolar affinity.
- the antibodies or antigen-binding fragments may have binding affinities of about 1 x 10 "9 M or less, about 1 x 10 "10 M or less, or about 1 x 10 ⁇ U M or less. In a particular embodiment, the binding affinity is less than about 5 x 10 "10 M.
- the antibodies or antigen-binding fragments may have an on rate constant (Kon) to toxin A or toxin B of at least K ⁇ NT 1 ; at least ⁇ 1 ; at least lOV ' Y 1 ; at least lO ⁇ ' Y 1 ; at least lO ⁇ ' Y 1 ; or at least lO ⁇ M ' Y 1 , as measured by surface plasmon resonance.
- the antibodies or antigen-binding fragments may have an off rate constant (Koff) to toxin A or toxin B of at most 10 ' V 1 ; at most 10 ' V 1 ; at most 10 ' V 1 ; or at most 10 ' V 1 , as measured by surface plasmon resonance.
- the antibodies or antigen-binding fragments may have a dissociation constant (K D ) to toxin A or toxin B of at most 10 "7 M; at most 10 "8 M; at most 10 "9 M; at most 10 "10 M; at most 10 "11 M; at most 10 "12 M; or at most 10 "13 M.
- K D dissociation constant
- the terms "antibody” or “immunoglobulin” include glycoproteins comprising at least two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by disulfide bonds.
- Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or V H ) and a heavy chain constant region (C H ).
- the heavy chain constant region is comprised of three domains, CHI, CH2 and CH3.
- Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or V L ) and a light chain constant region (C L ).
- the light chain constant region is comprised of one domain, C L .
- V H and V L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs).
- CDRs complementarity determining regions
- FRs framework regions
- Each V H and V L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4.
- the variable regions of the heavy and light chain polypeptides contain or form a binding domain that interacts with/binds an antigen.
- the constant regions of the antibodies may mediate the binding of the immunoglobulin 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 invention further provides other forms of antibodies, such as single chain antibodies, recombinantly produced antibodies, bispecific, hetero specific, or multimeric antibodies, diabodies, etc., as further described herein.
- antigen-binding fragment of an antibody as used herein, refers to one or more portions of an antibody that retain the ability to specifically bind to an antigen (e.g., toxin A, toxin B, toxin A and toxin B, etc.) or to epitopic regions of an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
- the monoclonal antibody fragments function in a manner similar to the intact counterpart monoclonal antibodies.
- the monoclonal antibody fragments cross-react with the intact counterpart monoclonal antibodies.
- the monoclonal antibody fragments can be used interchangeably with the intact counterpart monoclonal antibodies.
- binding fragments encompassed within the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the V L , V H , C L , and C H I domains; (ii) a F(ab') 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 V H and CHI domains; (iv) a Fv fragment consisting of the V L and V H domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546) which consists of a V H domain; and (vi) an isolated complementarity determining region (CDR).
- a Fab fragment a monovalent fragment consisting of the V L , V H , C L , and C H I domains
- F(ab') 2 fragment a bivalent fragment
- the two domains of the Fv fragment, V L and V H are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V L and V H 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).
- single chain Fv single chain Fv
- Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody.
- antibody fragments are obtained using conventional procedures, such as proteolytic fragmentation procedures, as described in J. Goding, Monoclonal Antibodies: Principles and Practice, pp 98-118 (N.Y. Academic Press 1983), which is hereby incorporated by reference, as well as by other techniques known to those having skill in the art.
- the fragments are screened for activity or utility in the same manner as are intact antibodies.
- Fab fragments of mAbs of the invention were generated and tested for their neutralization activity in cell-based assays, as described in Example 10 herein.
- antibody fragments, such as Fab fragments, of the mAbs of the invention may also be utilized to bind and neutralize toxin A and/or toxin B of C. difficile.
- an "isolated antibody”, as used herein, is intended to refer to an antibody which is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to toxin A is substantially free of antibodies that specifically bind antigens other than toxin A).
- An isolated antibody that specifically binds to an epitope, isoform or variant of toxin A or toxin B generally has cross -reactivity to other related antigens, e.g., from other C. difficile strains.
- an isolated antibody that specifically binds to an epitope, isoform, or variant of toxin A may also specifically bind toxin B, and an isolated antibody that specifically binds to an epitope, isoform, or variant of toxin B may also specifically bind toxin A.
- the isolated antibody or antigen-binding fragment thereof that specifically binds to an epitope, isoform, or variant of toxin A does not also specifically bind toxin B.
- the isolated antibody or antigen-binding fragment thereof that specifically binds to an epitope, isoform, or variant of toxin B does not also specifically bind toxin A.
- an isolated antibody may be substantially free of other cellular material and/or chemicals.
- Antibodies which are substantially free of other antibodies having different antigenic specificities, or other materials and/or chemicals and/or proteins may be isolated and/or purified antibodies.
- Antibodies may be purified by methods commonly performed by those having skill in the art, e.g., affinity chromatography, Protein A chromatography, and the like.
- affinity chromatography Protein A chromatography, and the like.
- “specific binding” refers to antibody binding to a predetermined or cognate antigen.
- the antibody binds with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen.
- a non-specific antigen e.g., BSA, casein
- an antibody of the invention may bind a linear epitope of the target antigen, e.g., toxin A and/or toxin B. In an embodiment, an antibody of the invention may bind a conformational epitope of the target antigen, e.g., toxin A and/or toxin B.
- the isolated antibodies of the invention encompass various antibody (immunoglobulin) heavy and light chain isotypes, such as the heavy chain classes or isotypes IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgAsec, IgD, IgE, and subtypes thereof, e.g., IgG2a, IgG2b; and the light chain isotypes ⁇ and ⁇ , and subtypes thereof.
- the isolated antibodies are of the IgG2a or IgGl ⁇ isotype.
- isotype refers to the antibody class (e.g., IgM or IgGl or ⁇ ) that is encoded by heavy and light chain constant region genes.
- the antibodies or antigen-binding fragments thereof can be full length or can include only an antigen-binding fragment, such as the antibody constant and/or variable domain of IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgAsec, IgD, or IgE, or can consist of a Fab fragment, a F(ab') 2 fragment, and a Fv fragment.
- the antibodies of the present invention can be polyclonal, monoclonal, or a mixture of polyclonal and monoclonal antibodies.
- the antibodies can be produced by a variety of techniques well known in the art. Procedures for raising polyclonal antibodies are well known.
- polyclonal antibodies are raised by administering toxin A and/or toxin B protein subcutaneously to New Zealand white rabbits which have first been bled to obtain pre-immune serum.
- the toxin A and/or toxin B can be injected at a total volume of 100 ⁇ per site at six different sites, typically with one or more adjuvants.
- the rabbits are then bled two weeks after the first injection and periodically boosted with the same antigen three times every six weeks.
- a sample of serum is collected 10 days after each boost.
- Polyclonal antibodies are recovered from the serum, preferably by affinity chromatography using toxin A and/or toxin B to capture the antibody. This and other procedures for raising polyclonal antibodies are described in Harlow, E. and Lane, D., Eds., Antibodies: A Laboratory Manual (1988), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, the contents of which are hereby incorporated by reference.
- Monoclonal antibody production may be effected by techniques which are also well known in the art.
- the term "monoclonal antibody”, as used herein, refers to a preparation of antibody molecules of single molecular composition.
- a monoclonal antibody displays a single binding specificity and affinity for a particular epitope of a given antigen or immunogen.
- the process of monoclonal antibody production involves obtaining immune somatic cells with the potential for producing antibody, in particular B lymphocytes, which have been previously immunized with the antigen of interest either in vivo or in vitro or both, and that are suitable for fusion with a B-cell myeloma line.
- Monoclonal antibodies can be produced using immune cells and myeloma cells from different species, such as murine and human cells and cell lines, or for example, in mouse strains which have been genetically engineered to harbor a human immune system, as further described below.
- mAbs of the present invention were generated by designing and employing a new immunization strategy.
- the mAbs described herein and deposited were produced by immunizing animals with toxoid, followed by boosting the animals with the active (non toxoid) form of toxin A and/or toxin B (see Example 1 herein).
- Boosting with the active form of toxin A or toxin B served to identify those immunized animals that had developed uniquely protective antibodies by virtue of the novel immunization scheme.
- the active toxin A and/or toxin B boosting regimen was more highly immunogenic in recipient animals.
- Nonlimiting examples of adjuvants that are suitable for use include incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, Ribi (i.e., monophosphoryl lipid A, trehalose dimycolate, Mycobacterium cell wall skeleton, and Tween® 80, with 2% squalene), saponins, Quil A, or alum.
- a cytotoxic T lymphocyte (CTL) response can be primed by conjugating toxins (or fragments, inactive derivatives or analogs thereof) to lipids, such as, for example, tripalmitoyl-S-glycerylcysteinyl-seryl-serine.
- additional immunization methods can be utilized for generating monoclonal antibodies directed against toxin A and/or toxin B.
- animals e.g., mice
- in vivo immunization of animals can be carried out with the desired type and amount of protein or polypeptide, e.g., toxoid or toxin.
- protein or polypeptide e.g., toxoid or toxin.
- immunizations are repeated as necessary at intervals of up to several weeks to obtain a sufficient titer of antibodies.
- animals can be used as a source of antibody-producing lymphocytes. Following the last antigen boost, the animals are sacrificed and spleen cells removed.
- Mouse lymphocytes give a higher percentage of stable fusions with the mouse myeloma lines described herein.
- mice are suitable.
- other mouse strains rabbit, hamster, sheep, goat and frog may also be used as hosts for preparing antibody-producing cells. See; Goding (in Monoclonal Antibodies: Principles and Practice, 2d ed., pp. 60-61, Orlando, Fla., Academic Press, 1986).
- mouse strains that have human immunoglobulin genes inserted in the genome (and which cannot produce mouse immunoglobulins) can be used. Examples include the HumAb mouse strains produced by Medarex (now Bristol Myers Squibb)/GenPharm International, and the XenoMouse strains produced by Abgenix. Such mice produce fully human immunoglobulin molecules in response to immunization.
- Somatic cells may be obtained from, for example, the lymph nodes, spleens, and peripheral blood of antigen-primed animals, and the lymphatic cells of choice depend to a large extent on their empirical usefulness in the particular fusion system.
- the antibody-secreting lymphocytes are then fused with (mouse) B cell myeloma cells or transformed cells, which are capable of replicating indefinitely in cell culture, thereby producing an immortal, immunoglobulin-secreting cell line.
- the resulting fused cells, or hybridomas are cultured, and the resulting colonies screened for the production of the desired monoclonal antibodies.
- Colonies producing such antibodies are cloned, subcloned and grown either in vivo (as ascites) or in vitro to produce large quantities of antibody. Descriptions of hybridoma methodology and technology may be found in Kohler and Milstein, Nature 256:495 (1975) or Harlow, E. and Lane, D., Eds., Antibodies: A Laboratory Manual (1988), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, which are hereby incorporated by reference.
- human somatic cells capable of producing antibody are suitable for fusion with myeloma cell lines. While B lymphocytes from biopsied spleens, tonsils or lymph nodes of an individual may be used, the more easily accessible peripheral blood B lymphocytes (PBLs) are preferred.
- human B cells may be directly immortalized by the Epstein-Barr virus (Cole et al., 1995, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Although somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibodies can be employed, such as viral or oncogenic transformation of B lymphocytes.
- Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non- antibody-producing, have high fusion efficiency, and enzyme deficiencies that render them incapable of growing in certain selective media that support the growth of the desired hybridomas.
- myeloma cell lines that may be used for the production of fused cell lines include P3-X63/Ag8, X63-Ag8.653, NSl/l.Ag 4.1, Sp2/0-Agl4, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7, S194/5XX0 Bui, derived from mice; R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210 derived from rats; and U-266, GM1500-GRG2, LICR-LON-HMy2, UC729-6, derived from humans (Goding, in Monoclonal Antibodies: Principles and Practice, 2d ed., pp.
- Fusion with mammalian myeloma cells or other fusion partners capable of replicating indefinitely in cell culture is effected by standard and well-known techniques, for example, by using polyethylene glycol (“PEG”) or other fusing agents (See Milstein and Kohler, Eur. J. Immunol. 6:511 (1976), which is hereby incorporated by reference).
- PEG polyethylene glycol
- the antibodies can be recombinant antibodies.
- the term "recombinant antibody”, as used herein, is intended to include antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic for another species' immunoglobulin genes, antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial antibody library, or antibodies prepared, expressed, created, or isolated by any other means that involves splicing of immunoglobulin gene sequences to other DNA sequences.
- the antibodies can be chimeric or humanized antibodies.
- the term "chimeric antibody” refers to an antibody that combines a murine immunoglobulin (Ig) variable or hypervariable regions with a human Ig constant region or constant and variable framework regions.
- the chimeric antibody comprises the variable region of any of the deposited antibodies provided herein and a human contant region.
- the human constant region is an human IgG constant region, such as an human IgGl constant region.
- the chimeric antibodies can be produced by the method provided below in the Examples or by any method known to those of skill in the art.
- humanized antibody refers to an antibody that retains substantially only the antigen -binding CDRs from the parent antibody, e.g., murine monoclonal antibody, in association with human framework regions (see, e.g., Waldmann, 1991, Science 252:1657).
- Such chimeric or humanized antibodies which retain the binding specificity of the murine antibody, but have human Ig constant/framework regions, are expected to have reduced immunogenicity when administered in vivo. Therefore, the chimeric and humanized antibodies preferably retain the toxin-neutralizing activities of the monoclonal antibodies provided and are suitable for repeat dosing (e.g., in humans).
- One of ordinary skill in the art can use known methods (e.g., in vitro cell-based assays) for comparing the activity of the humanized antibodies to the deposited monoclonal antibodies provided herein and for determining whether or not the humanized antibodies treat and/or prevent relapse of an established C. difficile infection.
- One of ordinary skill in the art can also use the methods described herein including the hamster model of C. difficile infection described below.
- the sequences of the humanized mAbs can be designed by the following illustrative, non- limiting method.
- human V H and V L sequences having high homology to the murine V H and V L , respectively, are selected from among known human immunoglobulin (germline) sequences.
- CDR sequences from the murine mAb, together with framework amino acid residues important for maintaining the structure of the CDRs are grafted into the selected human framework sequences.
- human framework amino acid residues that are found to be atypical in the corresponding V region subgroup are substituted with the typical residues to reduce potential immunogenicity of the resulting humanized mAb.
- V H and V L regions are cloned into the expression vectors, e.g., pCON Gammal and pCON kappa (Lonza Biologies, Berkshire, UK), respectively. These vectors encode the constant region(s) of the human immunoglobulin heavy and light chain genes.
- 293T cells can be transiently transfected with these expression vectors using the Effectene system (Qiagen, Valencia, CA). Cell supernatants containing secreted chimeric mAb can be collected following transfection, e.g., after three days, and purified using Protein A chromatography.
- Other expression vectors and host cells may be used to recombinantly produce the described antibodies, as understood by those having skill in the art.
- a humanized anti-C. difficile toxin A mAb (hmAb) of the invention encompasses an immunoglobulin protein, or a fragment thereof, which is composed of (i) two heavy (H) chain polypeptides, wherein each H chain contains a VH region comprising the amino acid sequence as set forth in SEQ ID NO:l and a human CH region, e.g., an IgGl C region, and (ii) two light (L) chain polypeptides, wherein each L chain contains a VL region comprising the amino acid sequence as set forth in SEQ ID NO: 3 and a human CL region, e.g., a K chain C region.
- a humanized anti-C is a fragment thereof, which is composed of (i) two heavy (H) chain polypeptides, wherein each H chain contains a VH region comprising the amino acid sequence as set forth in SEQ ID NO:l and a human CH region, e.g., an IgGl C region, and (ii
- a mAb of the invention encompasses an immunoglobulin protein, or a fragment thereof, which is composed of (i) two heavy (H) chain polypeptides, wherein each H chain contains a VH region comprising the amino acid sequence as set forth in SEQ ID NO:2 and a human CH region, e.g., an IgGl C region, and (ii) two light (L) chain polypeptides, wherein each L chain contains a VL region comprising the amino acid sequence as set forth in SEQ ID NO: 3 and a human CL region, e.g., a ⁇ chain C region.
- a humanized anti-C In an embodiment, a humanized anti-C.
- a mAb of the invention encompasses an immunoglobulin protein which is composed of (i) two heavy (H) chain polypeptides, wherein each H chain contains a VH region comprising the amino acid sequence as set forth in SEQ ID NO:l and a human CH region, e.g., an IgGl C region, and (ii) two light (L) chain polypeptides, wherein each L chain contains a VL region comprising the amino acid sequence as set forth in SEQ ID NO: 4 and a human CL region, e.g., a ⁇ chain C region.
- a humanized anti-C is composed of (i) two heavy (H) chain polypeptides, wherein each H chain contains a VH region comprising the amino acid sequence as set forth in SEQ ID NO:l and a human CH region, e.g., an IgGl C region, and (ii) two light (L) chain polypeptides, wherein each L chain contains a VL region comprising the amino acid sequence as
- a mAb of the invention encompasses an immunoglobulin protein which is composed of (i) two heavy (H) chain polypeptides, wherein each H chain contains a VH region comprising the amino acid sequence as set forth in SEQ ID NO:2 and a human CH region, e.g., an IgGl C region, and (ii) two light (L) chain polypeptides, wherein each L chain contains a VL region comprising the amino acid sequence as set forth in SEQ ID NO:4 and a human CL region, e.g., a K chain C region.
- H heavy chain polypeptides
- L light chain polypeptides
- each L chain contains a VL region comprising the amino acid sequence as set forth in SEQ ID NO:4 and a human CL region, e.g., a K chain C region.
- Such humanized anti-C. difficile toxin A mAbs embrace a hPA-39 mAb of the invention.
- a humanized anti-C. difficile toxin A mAb of the invention encompasses an immunoglobulin protein which is composed of (i) two heavy (H) chain polypeptides, wherein each H chain contains a VH region comprising the amino acid sequence as set forth in SEQ ID NO:5 and a human CH region, e.g., a IgGl C region, and (ii) two light (L) chain polypeptides, wherein each L chain contains a VL region comprising the amino acid sequence as set forth in SEQ ID NO:7 and a human CL region, e.g., a ⁇ chain C region.
- a humanized anti-C is composed of (i) two heavy (H) chain polypeptides, wherein each H chain contains a VH region comprising the amino acid sequence as set forth in SEQ ID NO:5 and a human CH region, e.g., a IgGl C region, and (ii) two light (L) chain polypeptides, wherein each L
- a mAb of the invention encompasses an immunoglobulin protein which is composed of (i) two heavy (H) chain polypeptides, wherein each H chain contains a VH region comprising the amino acid sequence as set forth in SEQ ID NO:6 and a human CH region, e.g., an IgGl C region, and (ii) two light (L) chain polypeptides, wherein each L chain contains a VL region comprising the amino acid sequence as set forth in SEQ ID NO:7 and a human CL region, e.g., a ⁇ chain C region.
- Such humanized anti-C. difficile toxin A mAbs embrace a hPA-50 mAb of the invention.
- a humanized anti-C. difficile toxin B mAb of the invention encompasses an immunoglobulin protein which is composed of (i) two heavy (H) chain polypeptides, wherein each H chain contains a VH region comprising the amino acid sequence as set forth in SEQ ID NO:8 and a human CH region, e.g., an IgGl C region, and (ii) two light (L) chain polypeptides, wherein each L chain contains a VL region comprising the amino acid sequence as set forth in SEQ ID NO: 10 and a human CL region, e.g., a ⁇ chain C region.
- a humanized anti-C is composed of (i) two heavy (H) chain polypeptides, wherein each H chain contains a VH region comprising the amino acid sequence as set forth in SEQ ID NO:8 and a human CH region, e.g., an IgGl C region, and (ii) two light (L) chain polypeptides, wherein each L chain contains
- difficile toxin B mAb of the invention encompasses an immunoglobulin protein which is composed of (i) two heavy (H) chain polypeptides, wherein each H chain contains a VH region comprising the amino acid sequence as set forth in SEQ ID NO:9 and a human CH region, e.g., an IgGl C region, and (ii) two light (L) chain polypeptides, wherein each L chain contains a VL region comprising the amino acid sequence as set forth in SEQ ID NO: 10 and a human CL region, e.g., a ⁇ chain C region.
- Such humanized anti-C. difficile toxin B mAbs embrace a hPA-41 mAb of the invention.
- the L chain and H chain C regions of the above-described humanized antibodies of the invention may comprise the human ⁇ L chain C region (C L ) and human IgGl H chain C region (C H ) having sequences contained in Genbank Accession No. NW_001838785 and in Genbank Accession No. MW_001838121, respectively.
- the humanized antibodies comprise a human H chain C region selected from the IgG2a, IgG2b, IgG3, or IgG4 isotypes.
- the invention embraces a monoclonal antibody, or a fragment thereof, generated against toxin A of C. difficile, wherein the antibody is composed of two heavy chain polypeptides, each heavy chain containing a VH region and a human CH region and two light chain polypeptides, each light chain containing a VL region and a human CL region.
- the nucleic acid sequence (or cDNA) encoding the consecutive amino acid sequence of the heavy chain polypeptide of SEQ ID NO: 14 is set forth in SEQ ID NO: 15, (Fig. 38B);
- the nucleic acid sequence (or cDNA) encoding the consecutive amino acid sequence of the light chain polypeptide of SEQ ID NO: 16 is set forth in SEQ ID NO: 17 (Fig. 38A).
- the invention embraces a monoclonal antibody, or a fragment thereof, generated against toxin A of C. difficile, wherein the antibody is composed of two heavy chain polypeptides, each heavy chain containing a VH region and a human CH region and two light chain polypeptides, each light chain containing a VL region and a human CL region.
- the nucleic acid sequence (or cDNA) encoding the consecutive amino acid sequence of the heavy chain polypeptide of SEQ ID NO: 18 is set forth in SEQ ID NO:19, (Fig. 39B);
- the nucleic acid sequence (or cDNA) encoding the consecutive amino acid sequence of the light chain polypeptide of SEQ ID NO:20 is set forth in SEQ ID NO:21 (Fig. 39A).
- the invention embraces a monoclonal antibody, or a fragment thereof, generated against toxin B of C. difficile, wherein the antibody is composed of two heavy chain polypeptides, each heavy chain containing a VH region and a human CH region and two light chain polypeptides, each light chain containing a VL region and a human CL region.
- the nucleic acid sequence (or cDNA) encoding the consecutive amino acid sequence of the heavy chain polypeptide of SEQ ID NO:22 is set forth in SEQ ID NO:23 (Fig. 40B);
- the nucleic acid sequence (or cDNA) encoding the consecutive amino acid sequence of the light chain polypeptide of SEQ ID NO:24 is set forth in SEQ ID NO:25 (Fig. 40A).
- portions or fragments of the above-described anti-C. difficile toxin A and anti- toxin B humanized antibodies include the complementarity determining regions (CDRs) of the V regions of both the H and L chain polypeptides, as may be conventionally determined by those having skill in the art; F(ab) fragments, F(ab') fragments, F(ab')2 fragments, Fc fragments, Fd fragments, and the like.
- portions or fragments of the humanized antibodies containing V regions, or functional portions thereof will optimally bind to the respective toxin and neutralize the activity of the toxin.
- such functional portions or fragments of the humanized antibodies optimally neutralize toxin activity at a level similar to, if not better than, that of the complete humanized antibody.
- humanized mAbs directed against toxins A or B of C. difficile are provided. Such humanized mAbs were isolated and characterized as described in Example 9, Sections D and E hereinbelow.
- the light chain constant region (CL) of each of the humanized antibodies is of the kappa ( ⁇ ) class.
- the heavy chain constant region (CH) of each of the humanized antibodies is of the IgGl isotype.
- the CH region of the humanized antibodies is of the IgG2a, IgG2b, IgG3, IgG4, IgA, IgE, IgA, or IgM isotype.
- the humanized mAbs containing unique variable (V) regions were found to bind and neutralize the activity of either toxin A or toxin B of C. difficile.
- the VL and VH regions of the humanized mAbs may form a part of a complete immunoglobulin (Ig) or antibody molecule, or they may be used as portions or fragments of the antibody, in particular, portions or fragments having binding and/or neutralizing activity.
- Nonlimiting examples of antibody fragments include Fab, F(ab) 2 and F(ab'), or F(ab') 2 fragments.
- Embodiments of the invention are directed to anti-C. difficile toxin A humanized mAbs, or fragments thereof, having activity against toxin A of C.
- the V region of the L chain is selected from one or more of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:7.
- the invention is directed to the CDRs, namely, CDR1, CDR2, and/or CDR3, in the VH and VL regions of the described antibodies.
- Embodiments of the invention are directed to anti-C. difficile toxin B humanized mAbs, or fragments thereof, having activity against toxin B of C. difficile, wherein the V region of the L chain is set forth in SEQ ID NO: 10.
- Embodiments of the invention are directed to anti-C. difficile toxin A humanized mAbs, or fragments thereof, having activity against toxin A of C.
- Embodiments of the invention are directed to anti-C. difficile toxin B humanized mAbs, or fragments thereof, having activity against toxin B of C. difficile, wherein the V region of the H chain is selected from one or more of SEQ ID NO: 8 or SEQ ID NO:9.
- the invention is directed to the CDRs, namely, CDRl, CDR2, and/or CDR3, in the VH and VL regions of the described antibodies.
- the invention encompasses nucleic acids which encode antigen- binding portions, CDRs, or variable (V) regions of the anti-C. difficile toxin A and/or antitoxin B antibodies of the invention.
- the portions, CDRs, or V regions are derived from PA-38, PA-39, PA-41, or PA-50, or humanized versions thereof, as described herein.
- the invention encompasses the amino acid sequences of the antigen-binding portions, CDRs, or V regions that are encoded by the respective nucleic acids.
- the monoclonal antibodies of the present invention can be modified to be in the form of a bispecific antibody, a bifunctional antibody, a multispecific antibody, or a heterofunctional antibody.
- bispecific and hetero specific antibodies and procedures for making such antibodies may be found in a number of illustrative publications, for example, UA20090060910, WO2009/058383, WO2009/030734, WO2007/093630, USP 6,071,517, WO2008/024188, UA20070071675, USP 7,442,778, USP 7,235,641, USP 5,932,448 and USP 5,292,668.
- bispecific antibody is intended to include any agent, e.g., a protein, peptide, or protein or peptide complex, which has two different binding specificities and which binds to or interacts with (a) toxin A of C. difficile and (b) toxin B of C. difficile.
- the bispecific antibody comprises PA-39 or PA-50 or an antigen-binding fragment thereof and PA-41, or an antigen-binding fragment thereof.
- the bispecific antibody comprises a chimeric or humanized form of PA-39 or PA-50, or an antigen-binding fragment thereof and PA-41, or an antigen-binding fragment thereof.
- bispecific antibody comprising PA-39 and PA-41, or chimeric or humanized forms thereof, or an antigen-binding fragment thereof, would bind both toxin A and toxin B of C. difficile.
- a bispecific antibody comprising PA-50 and PA-41, or chimeric or humanized forms thereof, or an antigen-binding fragment thereof, would bind both toxin A and toxin B of C. difficile.
- the term "multispecific antibody” is intended to include any agent, e.g., a protein, peptide, or protein or peptide complex, which has more than two different binding specificities and which binds to or interacts with (a) toxin A of C. difficile, (b) toxin B of C.
- the invention includes, but is not limited to, bispecific, trispecific, tetraspecific, and other multispecific antibodies.
- the antibodies or antigen-binding fragments of the bispecific or multispecific antibodies are humanized.
- Diabodies provide therapeutic antibodies having dual specificity and being capable of targeting multiple different epitopes with a single molecule.
- Diabodies are bivalent, bispecific antibodies in which the V H and V L domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen-binding sites (see e.g., HoUiger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poijak, R.J., et al. (1994) Structure 2:1121-1123).
- the two antigen-binding regions of the bispecific antibody are either chemically linked or is expressed by a cell genetically engineered to produce the bispecific antibody. (See generally, Fanger et al., 1995 Drug News & Perspec. 8(3):133-137).
- an effective amount of a bispecific antibody can be administered to a subject with C. difficile infection and/or a C. difficile-associated disease, and the bispecific antibody neutralizes the toxicity of toxin A and toxin B in the subject.
- the antibodies may be human antibodies.
- the term "human antibody”, as used herein, is intended to include antibodies having variable and constant Ig regions derived from human germline immunoglobulin sequences.
- the human antibodies 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).
- the term "human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (referred to herein as "humanized antibodies”).
- Human antibodies directed against toxin A and/or toxin B can be generated using transgenic mice genetically modified and bred to express components of the human immune system rather than the mouse system.
- Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. patents 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference. These animals have been genetically modified such that there is a functional deletion in the production of endogenous (e.g., murine) antibodies. The animals are further modified to contain all or a portion of the human germ-line immunoglobulin gene locus such that immunization of these animals results in the production of fully human antibodies to the antigen of interest.
- mice e.g., XenoMouse (Abgenix), HumAb mice (Medarex/GenPharm)
- monoclonal antibodies are prepared according to standard hybridoma technology. These monoclonal antibodies have human immunoglobulin amino acid sequences and, therefore, will not provoke human anti-mouse antibody (HAMA) responses when administered to humans.
- HAMA human anti-mouse antibody
- nucleic acids and polynucleotides that encode the described antibodies or antigen-binding fragments thereof. It will also be appreciated that provided herein are nucleic acids and polynucleotides that comprise a sequence encoding the antibodies or antigen-binding fragments thereof. Vectors and plasmids engineered to contain and/or express antibody-encoding nucleic acids and polynucleotides are, therefore, provided by the invention.
- a "coding region" refers to a region of a nucleotide sequence that encodes a polypeptide sequence; the coding region can include a region coding for a portion of a protein that is later cleaved off, such as a signal peptide.
- the nucleotide and amino acid sequences may include sequences that encode or that are signal peptides. The invention embraces each of these sequences with, or without, the portion of the sequence that encodes or is a signal peptide.
- RNA is generated from hybridoma calls, and cDNA is reverse transcribed using an oligo-dT primer.
- RNase H can be used to remove RNA to make single- stranded cDNA.
- Spin column purification can be used to remove free nucleotides.
- a 3' poly-dG tail can be added with terminal transferase.
- PCR amplification can be performed using an oligo-dC primer plus a degenerate primer to the constant region. Approximately, 40 cycles can be performed for robust heavy chain amplification. Direct sequencing of the PCR products can then be performed.
- the antibody or antigen-binding fragment thereof is encoded by a nucleic acid molecule that is highly homologous to the foregoing nucleic acid molecules.
- the homologous nucleic acid molecule can comprise a nucleotide sequence that is at least about 90% identical to the nucleotide sequence provided herein.
- the homologous nucleic acid molecule can comprise a nucleotide sequence is at least about 95% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the nucleotide sequence provided herein.
- the homology can be calculated using various, publicly available software tools well known to one of ordinary skill in the art. Exemplary tools include the BLAST system available from the website of the National Center for Biotechnology Information (NCBI) at the National Institutes of Health.
- nucleic acid hybridization One method of identifying highly homologous nucleotide sequences is via nucleic acid hybridization.
- antibodies having the toxin A and/or toxin B binding properties and other functional properties described herein which are encoded by nucleic acid molecules that hybridize under high stringency conditions to the nucleic acid molecules encoding the antibodies of the invention.
- Identification of related sequences can also be achieved using polymerase chain reaction (PCR) and other amplification techniques suitable for cloning related nucleic acid sequences.
- PCR primers are typically selected to amplify portions of a nucleic acid sequence of interest, such as a CDR.
- high stringency conditions refers to parameters with which the art is familiar. Nucleic acid hybridization parameters may be found in references that compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York.
- hybridization buffer 3.5X SSC, 0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% Bovine Serum Albumin, 2.5mM NaH 2 P0 4 (pH7), 0.5% SDS, 2mM EDTA.
- SSC is 0.15M sodium chloride/0.015M sodium citrate, pH7; SDS is sodium dodecyl sulphate; and EDTA is ethylenediaminetetracetic acid.
- a membrane upon which the nucleic acid is transferred is washed, for example, in 2X SSC at room temperature and then at 0.1 - 0.5X SSC/0.1X SDS at temperatures up to 68°C.
- vectors e.g., expression vectors
- plasmids comprising the nucleic acid molecules described, in addition to other nucleic acid sequences, e.g., ORI, promoter, enhancer, termination sequences, required for protein, polypeptide, or peptide expression.
- the vectors can be used to transform or transfect host cells for producing the antibodies or antigen-binding fragments thereof with the binding specificity and/or characteristics of the antibodies or antigen-binding fragments described herein.
- the vectors can comprise an isolated nucleic acid molecule encoding the heavy chain or portion thereof of the antibodies and antigen-binding fragments provided.
- the vectors can comprise the nucleic acid sequences encoding the light chain or portion thereof.
- the vectors of the invention may comprise a sequence for a heavy chain or a portion thereof and a sequence of a light chain or portion thereof.
- plasmids are given which produce the antibodies or antigen binding fragments described herein.
- Modified versions of the antibodies of the invention are also provided. Modifications to an antibody or antigen-binding fragment thereof are typically made to the nucleic acid which encodes the antibody or antigen-binding fragment thereof, and can include deletions, point mutations, truncations, amino acid substitutions and additions of amino acids or non-amino acid moieties. Alternatively, modifications can be made directly to the polypeptide, such as by cleavage, addition of a linker molecule, addition of a detectable moiety, such as biotin, addition of a fatty acid, and the like. Modifications also embrace fusion proteins comprising all or part of the antibody or antigen-binding fragment amino acid sequence. Modifications further embrance coupling or joining of the antibody to another agent, such as a cytotoxic agent, drug, or therapeutic.
- Modified polypeptides include polypeptides which are modified specifically to alter a feature of the polypeptide unrelated to its physiological activity. For example, cysteine residues can be substituted or deleted to prevent unwanted disulfide linkages. Similarly, certain amino acids can be changed to enhance expression of a polypeptide by eliminating proteolysis by proteases in an expression system (e.g., dibasic amino acid residues in yeast expression systems in which KEX2 protease activity is present). Additionally, one or more amino acids can be changed, particularly in the Ig constant region, to prevent proteolytic degration of the antibody by enzymes following certain routes of administration, e.g., oral administration, as described for example, in WO2006/071877, published 6 July 2006.
- routes of administration e.g., oral administration, as described for example, in WO2006/071877, published 6 July 2006.
- Modifications conveniently are prepared by altering a nucleic acid molecule that encodes the polypeptide. Mutations of a nucleic acid which encode a polypeptide preferably preserve the amino acid reading frame of the coding sequence, and preferably do not create regions in the nucleic acid which are likely to hybridize to form secondary structures, such a hairpins or loops, which can be deleterious to expression of the modified polypeptide.
- Modifications can be made to any of the antibodies or antigen-binding fragments thereof by selecting an amino acid substitution, or by random mutagenesis of a selected site in a nucleic acid which encodes the polypeptide. Modified polypeptides then can be expressed and tested for one or more activities to determine which mutation provides a modified polypeptide with the desired properties. Further mutations can be made to modified polypeptides (or to non- modified polypeptides) which are silent as to the amino acid sequence of the polypeptide, but which provide preferred codons for translation in a particular host. The preferred codons for translation of a nucleic acid in, e.g., E. coli, are well known to those of ordinary skill in the art.
- modified polypeptides can be tested by cloning the gene encoding the modified polypeptide into an expression vector, introducing the vector into an appropriate host cell, expressing the modified polypeptide, and testing for a functional capability of the polypeptides as disclosed herein.
- the foregoing procedures are well known to one of ordinary skill in the art.
- a "conservative amino acid substitution” refers to an amino acid substitution which does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made.
- Modified polypeptides can be prepared according to methods for altering a polypeptide sequence as known to one of ordinary skill in the art, such as can be found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M.
- Conservative substitutions of amino acids include substitutions made among amino acids within the following exemplary groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.
- amino acid substitutions in polypeptides typically are made by alteration of a nucleic acid encoding a polypeptide. Such substitutions can be made by a variety of methods known to one of ordinary skill in the art. For example, amino acid substitutions may be made by PCR-directed mutation, site-directed mutagenesis, or by chemical synthesis of a gene encoding a polypeptide. Where amino acid substitutions are made to a small fragment of a polypeptide, the substitutions can be made by directly synthesizing the peptide.
- the activity of functionally equivalent fragments of polypeptides can be tested by cloning the gene encoding the altered polypeptide into a bacterial or mammalian expression vector, introducing the vector into an appropriate host cell, expressing the altered polypeptide, and testing for a functional capability of the polypeptides as disclosed herein.
- An anti-toxin antibody, or antigen-binding portion thereof, of the invention can be derivatized or linked to another functional molecule, for example, another peptide or protein.
- an antibody or antibody portion can be functionally linked, e.g., by chemical coupling, genetic fusion, noncovalent association, etc., to one or more other molecular entities, such as another antibody, a detectable agent, a cytotoxic agent, a therapeutic agent, a pharmaceutical agent, and/or a protein or peptide that can mediate association with another molecule, for example, a streptavidin core region or a polyhistidine tag.
- a derivatized protein or antibody may be produced by cros slinking or coupling two or more proteins or antibodies of the same or different types.
- Suitable crosslinkers or coupling agents 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), and commercially available (Pierce Chemical Company, Rockford, IL).
- An anti-toxin antibody or antigen -binding fragment thereof of the invention may be conjugated to another molecular entity, such as a label.
- Detectable agents or labels with which a protein can be derivatized or labeled include fluorescent compounds, enzymes, prosthetic groups (e.g., streptavidin/biotin and avidin/biotin), chemiluminescent materials, bioluminescent materials, chemical entities and radioactive materials.
- detectable fluorescent compounds include fluorescein, fluorescein isothiocyanate (FITC), rhodamine and phycoerythrin.
- a protein or antibody can also be derivatized with detectable enzymes, such as alkaline phosphatase (AP), horseradish peroxidase, beta-galactosidase, acetylcholinesterase, glucose oxidase, etc.
- Such enzymatically derivatized proteins or antibodies become detectable upon addition of a specific substrate of the enzyme so as to produce a detectable reaction product.
- Proteins derivatized with a prosthetic group, such as biotin can be detected by indirect measurement of avidin or streptavidin binding.
- Labeled proteins and antibodies may be used as diagnostic and/or experimental agents or reagents to isolate a known or predetermined antigen by standard techniques, such as affinity chromatography or immunoprecipitation, or to detect a known or predetermined antigen so as to determine protein levels in tissue as part of a clinical testing procedure, e.g., to monitor the efficacy of a given treatment regimen.
- the antigen to be detected may be a toxin in a cellular lysate or in a patient sample.
- the antibodies or antigen-binding fragments of the invention are used in combination, e.g., as a pharmaceutical composition comprising two or more different antibodies or antigen-binding fragments thereof (e.g., one or more directed against toxin A and one or more directed against toxin B, two or more directed against toxin A, or two or more directed against toxin B, etc.).
- Combinations of antibodies or antigen-binding fragments thereof can be combined in a single therapy (i.e., administered simultaneously) to achieve a desired therapeutic effect.
- the antibodies or antigen-binding fragments thereof can be administered separately (i.e., at different times). It follows, therefore, that the antibodies or antigen-binding fragments thereof can be stored together or separately.
- the antibodies or antigen-binding fragments thereof can be stored in an aqueous medium or as a lyophilized form, which can be reconstituted prior to use.
- compositions comprising one or more isolated antibodies or an antigen-binding fragment thereof are provided. Also provided are compositions comprising a combination of one or more of the aforementioned antibodies or antigen-binding fragments thereof. Also provided are compositions, each containing one or more of the aforementioned antibodies or antigen-binding fragments thereof, which compositions are intended for use in combination.
- Such compositions may include a physiologically or pharmaceutically acceptable carrier, excipient, vehicle, or diluent.
- the physiologically or pharmaceutically acceptable carrier, excipient, vehicle, or diluent can be mixed with the isolated antibody or antigen-binding fragment thereof.
- the compositions include a combination of multiple (e.g., two or more) isolated antibodies or antigen-binding fragments thereof.
- one or more of the antibodies or antigen-binding fragments thereof of the composition specifically bind toxin A of C. difficile and neutralize its toxic effects, while one or more of the antibodies or antigen-binding fragments thereof specifically bind toxin B of C. difficile and neutralize its toxic effects.
- both the one or more of the antibodies or antigen-binding fragments thereof that specifically bind toxin A of C. difficile and neutralize its toxic effects and the one or more that specifically bind toxin B of C. difficile and neutralizes its toxic effects are humanized.
- the composition comprises a combination of one anti-toxin A antibody or antigen-binding fragment thereof as described herein and one anti-toxin B antibody or antigen-binding fragment thereof as described herein.
- the anti-toxin A antibody and the anti-toxin B antibody may be present in equal amounts or ratios, e.g., 1:1.
- the anti-toxin A antibody and the anti- toxin B antibody may be present in different amounts or ratios, such as 1/2:1; 2:1; 3:1; 4:1, etc.
- the antibodies of the composition are humanized.
- the composition comprises a combination of mAb PTA-9888, an antigen- binding fragment thereof, or a humanized form thereof, and mAb 9693, an antigen-binding fragment thereof, or a humanized form thereof.
- the composition comprises a combination of mAb PTA-9694, an antigen-binding fragment thereof, or a humanized form thereof, and mAb 9693, an antigen-binding fragment thereof, or a humanized form thereof.
- the composition comprises a combination of any one of the following: Mab PTA-9692, an antigen-binding fragment thereof, or a humanized form thereof and mAb 9693, an antigen-binding fragment thereof, or a humanized form thereof; or mAb PTA-9888, an antigen-binding fragment thereof, or a humanized form thereof and mAb 9692, an antigen-binding fragment thereof, or a humanized form thereof; or mAb PTA-9694, an antigen-binding fragment thereof, or a humanized form thereof and mAb 9692, an antigen-binding fragment thereof, or a humanized form thereof.
- compositions also can be administered in combination therapy, i.e., combined with other therapeutic agents.
- the combination therapy can include a composition comprising one or more antibodies or antigen-binding fragments thereof as provided herein with at least one other conventional therapy.
- additional therapeutic agents include antibiotic therapeutics and non- antibiotic therapeutics. Additional therapeutic agents include C.
- difimicin also called PAR- 101 or OPT-80
- clindamycin cephalosporins (such as second and third generation cephalosporins), fluoroquinolones (such as gatifloxacin or moxifloxacin)
- macrolides e.g., erythromycin, clarithromycin, azithromycin
- penicillins aminoglycosides, trimethoprim- sulfamethoxazole, chloramphenicol, tetracycline, imipenem, and meropenem.
- Additional therapeutics also include antibiotics, antibacterial agents, bactericides, or bacteriostats.
- the additional therapeutic agent may be a small molecule or chemical compound of low molecular weight, which targets C. difficile and/or its toxins.
- the additional therapeutic agent is OPT-80.
- Non- antibiotic therapeutics include tolevamer, a high-molecular-weight anionic polymer that binds toxins A and B via nonspecific charge mechanisms.
- antibodies or antigen-binding fragments thereof of the invention may be used in combination with other antibodies or antigen-binding fragments thereof.
- additional therapeutic agents include normal pooled immunoglobulin, intravenous immunoglobulin, or polyclonal anti-toxin A and anti-toxin B immunoglobulins in sera.
- Other antibodies include human mAbs directed against toxin A or toxin B of C. difficile, as described and reported in the published literature (e.g., WO/2006/121422; US2005/0287150).
- CD AD typically is precipitated by the disruption of the colonic flora through the use of antibiotics such as clindamycin, cephalosporins, and fluoroquinolones. This perturbation in the colonic microenvironment, along with exposure to C. difficile spores, leads to colonization in afflicted individuals.
- CD AD Crohn's disease
- treat refers to any benefit to a subject with C. difficile infection or C. difficile-associated disease conferred through the administration of the antibodies or a composition or combination of compositions provided herein.
- a benefit can be the elimination of one or more symptoms or adverse effects, or a reduction in, or amelioration of, the severity of the one or more symptoms or adverse effects that result from the infection or disease; a delay, halt, or reversal in the progression of the infection or disease; a recolonization, resurgence, or repopulation of the normal and natural microflora of the gastrointestinal tract, colon, bowel, etc., or the cure of the infection or disease (i.e., a clinician would evaluate the subject and determine that the subject no longer has the infection or disease).
- Symptoms or adverse effects associated with C. difficile infection include dehydration, diarrhea, cramping, kidney failure, bowel perforation, toxic megacolon, which can lead to rupture of the colon, and death.
- the compositions provided can be used to reduce, diminish, ameliorate, or eliminate any or all of the symptoms or adverse effects provided herein.
- a "C. difficile infection” refers to an infection that results from the presence of C. difficile in the intestinal flora where it was not previously present or a change in the presence of C. difficile in the intestinal flora (e.g., an increase in the total amount of C. difficile relative to one or more other bacteria, etc.), which gives rise or may give rise to adverse effect(s) and/or an increase in the level of toxins A and/or B in the intestine or other organs and tissues comprising the gastrointestinal tract.
- CD AD results from the acquisition and proliferation of C. difficile in the gut.
- toxins A and B demonstrate different pathological profiles with potential synergy in causing disease.
- toxin A is an enterotoxin that induces diarrhea, while toxin B does not elicit a fluid response in this species.
- toxin B is more potently cytotoxic in vitro.
- Toxin A-negative, toxin B-positive (A- B+) strains of C. difficile have been increasingly reported.
- A-/B+ strains fail to produce toxin A due to deletion of the repetitive domain of the tcdA gene, yet are still capable of causing clinical disease.
- there are to date no reports of toxin A-positive, toxin B-negative (A+/B-) strains in humans.
- C. difficile infection commonly manifests as mild-to-moderate diarrhea, occasionally with abdominal cramping. Pseudomembranes, which are adherent yellowish- white plaques on the intestinal mucosa, are occasionally observed.
- patients with C. difficile infection can present with an acute abdomen and fulminant life-threatening colitis, which results from a disruption of the normal bacterial flora of the colon, colonization with C. difficile and release of toxins that cause mucosal inflammation and damage.
- Antibiotic therapy is the key factor that alters the colonic flora. While normal gut flora resists colonization and overgrowth with C. difficile, antibiotic use, which suppresses the normal flora, allows C. difficile bacteria to proliferate.
- the mAbs of the present invention are utilized for the treatment of subjects who are asymptomatic, but who are susceptible to, or at risk of, contracting C. difficile infection and becoming afflicted with its associated diseases. Such subjects may be hospitalized or may be outside of a hospital setting.
- C. difficile-related disease The chief risk factor for C. difficile-related disease is prior exposure to antibiotics.
- the most common antibiotics implicated in C. difficile colitis include cephalosporins (especially second and third generation), ampicillin/amoxicillin and clindamycin. Less commonly implicated antibiotics are the macrolides (i.e., erythromycin, clarithromycin, azithromycin) and other penicillins.
- Compounds or other agents which are occasionally reported to cause the disease include aminoglycosides, fluoroquinolones, trimethoprim-sulfamethoxazole, metronidazole, chloramphenicol, tetracycline, imipenem, and meropenem.
- C. difficile colitis Even brief exposure to any single antibiotic can cause C difficile colitis, particularly if normal intestinal flora are adversely affected or killed. A prolonged antibiotic course, or the use of two or more antibiotics, increases the risk of disease. Antibiotics traditionally used to treat C. difficile colitis have been shown to cause disease. Other risk factors associated with infection by C. difficile include advanced age (>65 years); weakened immune system; recent hospitalization (particularly sharing a hospital room with an infected patient, intensive care unit stays and prolonged hospital stays); living in a nursing home, hospice, or other longterm care facility; abdominal surgery; chronic colon disease, (e.g., inflammatory bowel disease (IBD) or colorectal cancer); taking prescription or over the counter antacids which may reduce stomach acid and allow C.
- IBD inflammatory bowel disease
- colorectal cancer colorectal cancer
- C. difficile to pass more easily into the intestine; and a previous C. difficile infection.
- More factors associated with C. difficile disease include antineoplastic agents, principally methotrexate, hemolytic -uremic syndrome, malignancies, intestinal ischemia, renal failure, necrotizing enterocolitis, Hirschsprung disease, IBD and nonsurgical gastrointestinal procedures, including nasogastric tubes.
- the subjects that can be administered the compositions provided herein include any of the subjects described that are at risk for C. difficile infection.
- C. difficile-associated diarrhea can be a serious condition with a mortality rate of up to 25% in elderly patients who are frail. Reports that focus on more seriously ill patients indicate mortality rates of 10-30%.
- C. difficile infection is more common in elderly people, and old age may promote susceptibility to colonization and disease. While infants and young children frequently harbor C. difficile and its toxins, clinical infection is uncommon. Cross -infection by C. difficile is common in neonatal units, but neonates do not seem to develop C. difficile associated diarrhea.
- a method for treating a subject who has C. difficile infection or disease exhibits any of the symptoms or the adverse effects provided herein, or has any of diseases provided herein is provided.
- the method reduces, diminishes, or ameliorates the severity of disease associated with C. difficile infection or C. difficile-associate disease in a subject.
- a method of treating a subject who is afflicted with C. difficile-associated diarrhea is provided.
- a method of neutralizing toxin A and/or toxin B of C. difficile in a subject As an example, a method of neutralizing combined systemic toxin A and toxin B of C. dijficile is provided.
- combined systemic toxin A and toxin B of C. difficile are neutralized by administering both a humanized anti-toxin A antibody or antigen- binding fragment thereof and a humanized anti-toxin B antibody or antigen-binding fragment thereof, or a composition comprising these antibodies.
- the combined systemic toxin A and toxin B of C are neutralized by administering both a humanized anti-toxin A antibody or antigen- binding fragment thereof and a humanized anti-toxin B antibody or antigen-binding fragment thereof, or a composition comprising these antibodies.
- the combined systemic toxin A and toxin B of C are neutralized by administering both a humanized anti-toxin A antibody or antigen- binding fragment thereof and a humanized anti-toxin
- difficile are neutralized through the administration of an antibody or antigen-binding fragment thereof that specifically binds both toxin A and toxin B, or a composition comprising the antibody (e.g., in humanized form).
- the humanized antibodies or compositions are administered in conjunction with another therapeutic agent which targets C. difficile.
- a method of restoring normal gastrointestinal flora in a subject infected with C. difficile, thereby effectively treating infection caused by C. difficile and/or and its toxins is also provided.
- a method of reducing the susceptibility of a subject to C. difficile infection or C. difficile-associated disease is also provided.
- a method of preventing C. difficile infection or C. difficile-associated disease in a subject is provided.
- the subject is administered one or more of the antibodies or the compositions provided herein (e.g., a composition comprising a monoclonal antibody or an antigen-binding fragment thereof directed against C. difficile toxin A and a composition comprising a monoclonal antibody or antigen-binding fragment directed against C. difficile toxin B).
- the compositions can be administered to the subject at the same time or at different times.
- the compositions can be administered to the subject as a mixture in a composition comprising a pharmaceutically acceptable carrier, vehicle, or excipient, and optionally another antibiotic, non- antibiotic, drug or therapeutic effective against C. difficile and/or its enterotoxins.
- a humanized anti-toxin A monoclonal antibody or antigen-binding fragment thereof and/or a humanized anti-toxin B monoclonal antibody or antigen-binding fragment thereof, or a pharmaceutically acceptable composition comprising the humanized antibodies or antigen- binding fragments thereof, separately or together, can be used in any of the described methods according to the invention.
- pharmaceutically acceptable carrier or “physiologically acceptable carrier” includes any and all salts, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
- the carrier is suitable for oral, intravenous, intraperitoneal, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).
- the active compound i.e., antibody may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.
- the pharmaceutical preparations of the invention When administered, the pharmaceutical preparations of the invention are applied in pharmaceutically-acceptable amounts and in pharmaceutically-acceptable compositions.
- pharmaceutically acceptable means a non-toxic, physiologically acceptable material that does not interfere with the effectiveness of the biological activity of the active ingredients.
- Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents, such as supplementary immune potentiating agents, including adjuvants, chemokines and cytokines.
- the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention.
- a salt retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S.M., et al. (1977) J. Pharm. Sci. 66: 1-19).
- Examples of such salts include acid addition salts and base addition salts.
- Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
- Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as ⁇ , ⁇ '-dibenzylethylenediamine, N-methylglucamine, chioroprocaine, choline, diethanolamine, ethylenediamine, ethylenediamine acetate (EDTA), with or without a counterion, such as sodium or calcium, procaine and the like.
- alkaline earth metals such as sodium, potassium, magnesium, calcium and the like
- nontoxic organic amines such as ⁇ , ⁇ '-dibenzylethylenediamine, N-methylglucamine, chioroprocaine, choline, diethanolamine, ethylenediamine, ethylenediamine acetate (EDTA), with or without a counterion, such as sodium or calcium, procaine and the like.
- compositions of the invention may be combined, if desired, with a pharmaceutically-acceptable carrier.
- pharmaceutically-acceptable carrier means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a human.
- carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
- the components of the pharmaceutical compositions also are capable of being admixed with the molecules of the compositions provided, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.
- the pharmaceutical compositions may contain suitable buffering agents, including: acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt.
- suitable buffering agents including: acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt.
- compositions also may contain, optionally, suitable preservatives, such as: benzalkonium chloride; chlorobutanol; and parabens.
- suitable preservatives such as: benzalkonium chloride; chlorobutanol; and parabens.
- compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing the active agent into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
- Anti-toxin A and anti-toxin B antibodies of the invention, or portions thereof, can be provided according to dosage regimens that may be adjusted to provide the optimum desired response, such as a therapeutic or prophylactic response, in an individual subject.
- a single bolus may be administered, several divided doses may be administered over time, or the dose may be reduced or increased proportionally, as may be indicated by a particular therapeutic situation.
- Parenteral compositions may be packaged or prepared in unit dosage form for ease of administration and uniformity of dosage.
- Unit dosage form refers to physically discrete units provided as unitary dosages for the subjects to be treated, wherein each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier, vehicle, excipient, or diluent.
- the specification for the unit dosage 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.
- compositions suitable for parenteral administration conveniently comprise a sterile aqueous or non-aqueous preparation, which is preferably isotonic with the blood of the recipient.
- This preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents.
- the sterile injectable preparation also may be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butane diol.
- acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
- sterile, fixed oils are conventionally employed as a solvent or suspending medium.
- any bland fixed oil may be employed including synthetic mono-or di- glycerides.
- fatty acids such as oleic acid may be used in the preparation of injectables.
- Carrier formulations suitable for oral, subcutaneous, intraperitoneal, intravenous, intramuscular, etc. administration can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.
- the active components can be prepared with carriers that will protect the components against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems.
- a controlled release formulation including implants and microencapsulated delivery systems.
- Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
- the antibody and compositions of the invention as therapeutics can be administered by any conventional route, including injection or by gradual infusion over time.
- the administration route may, as nonlimiting examples, be oral, intravenous, subcutaneous, intraperitoneal, intramuscular, intrathecal, intracavity, retroorbital, vaginal, rectal, inhalation, aspiration, dermal, via suppository, or transdermal.
- an "effective amount” is that amount of an antibody or antigen-binding fragment thereof or composition(s) as provided herein that alone, or together with further doses, or other therapeutic agent(s), produce the desired response, e.g., treats, ameliorates, eradicates, resolves, or prevents C. difficile infection, diarrhea, or a C. difficile-associated disease in a subject. This may involve only slowing the progression of the infection, diarrhea, or disease for a sustained period, e.g., longer than one week, two weeks, three weeks, one month, two months, three months, or more than three months. However, such effective amounts optimally treat or halt the progression of the infection, diarrhea, or disease permanently. This can be monitored by routine methods.
- the desired response to treatment of the disease or condition also can be delaying the onset or even preventing the onset of the infection or disease.
- Effective amounts will depend, of course, on the particular infection or disease being treated, the severity of the infection or disease, the individual patient parameters including age, physical condition, size, and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine testing or experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.
- compositions used in the foregoing methods preferably are sterile and contain an effective amount of one or more antibodies or antigen-binding fragments provided herein for producing the desired response in a unit of weight or volume suitable for administration to a patient.
- the response can, for example, be measured by determining the physiological effects of the composition, such as decrease of disease symptoms.
- Other assays will be known to one of ordinary skill in the art and can be employed for measuring the level of the response.
- the doses or amounts of the compositions administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. Other factors include the desired period of treatment. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits.
- doses or amounts can range from about 1 ⁇ g/kg to about 100,000 ⁇ g/kg.
- Nonlimiting examples of dose ranges constituting a therapeutically or prophylactically effective amount of an antibody, antibody portion, or composition of the invention include 0.1 mg/kg-100 mg/kg; 0.1 mg/kg-60 mg/kg; of 0.5 mg/kg-75 mg/kg; 0.5 mg/kg-25 mg/kg; 0.75 mg/kg-40 mg/kg; 1 mg/kg-50 mg/kg; or 1 mg/kg-5 mg/kg. It will be appreciated that for any particular individual, patient, or subject, specific doses and dosage regimens should be adjusted over time according to individual need and the professional judgment of the skilled practitioner who is administering or supervising the administration of the antibodies and/or compositions.
- the dose can be delivered continuously, such as by continuous pump, or at periodic intervals. Desired time intervals of multiple doses of a particular composition can be determined without undue experimentation by one skilled in the art.
- Other protocols for the administration of the compositions will be known to one of ordinary skill in the art, in which the dose amount, schedule of administration, sites of administration, mode of administration and the like vary from the foregoing.
- compositions administered to mammals other than humans, e.g., for testing purposes or veterinary therapeutic purposes, is carried out under substantially the same conditions as described above.
- kits comprising antibodies of the invention or compositions comprising antibodies of the invention, and instructions for use.
- the kits can further contain at least one additional reagent, such as an additional therapeutic agent, or one or more additional antibodies or antigen-binding fragments as provided herein (e.g., an antibody or antigen-binding fragment thereof to toxin A when the first antibody or antigen-binding fragment thereof in the kit is an antibody or antigen-binding fragment thereof to toxin B, and vice versa).
- kits can be packaged either in aqueous medium or in lyophilized form.
- conjugates e.g., a bispecific antibody conjugate
- the components of such conjugates can be supplied either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user or the kit in accordance with the provided instructions for use.
- a kit may comprise a carrier being compartmentalized to receive in close confinement therein one or more container means, or series of container means, such as test tubes, vials, flasks, bottles, syringes, or the like.
- a first container means or series of container means may contain one or more antibodies or antigen-binding fragments thereof.
- a second container means or series of container means may contain one or more antibodies or antigen-binding fragments thereof, wherein the antibodies or antigen-binding fragments thereof are different from those in the first container means, or some other additional therapeutic agent.
- the kits provided herein can further include a third container that contains a molecule to link the antibodies or antigen-binding fragments contained in the first and second containers.
- isolated means separated from its native environment and present in sufficient quantity to permit its identification or use. Isolated, when referring to a protein or polypeptide, means, for example: (i) selectively produced by expression cloning or (ii) purified as by chromatography or electrophoresis. Isolated proteins or polypeptides may be, but need not be, substantially pure. The term “substantially pure” means that the proteins or polypeptides are essentially free of other substances with which they may be found in nature, or from in vivo systems to an extent practical and appropriate for their intended use. Substantially pure polypeptides may be produced by techniques well known in the art.
- an isolated protein may be admixed with a pharmaceutically acceptable carrier in a pharmaceutical preparation, the protein may comprise only a small percentage by weight of the preparation.
- the protein is nonetheless isolated in that it has been separated from the substances with which it may be naturally associated in living systems, i.e. isolated from other naturally-occurring proteins.
- Methods for evaluating a candidate agent for efficacy in the treatment of C. difficile infection or C. difficile-associated disease are also provided. Such methods may comprise the steps of treating a subject with an agent that increases the risk of C. difficile infection or C. difficile- associated disease in the subject, inoculating the subject with C. difficile, treating the subject with the candidate agent, and evaluating the efficacy of treatment with the candidate agent.
- an "agent that increases the risk of C. difficile infection or C. difficile- associated disease” is any agent that is thought to promote the onset or progression of C. difficile infection or C. difficile-associated disease.
- Such an agent can be an antibiotic or non- antibiotic agent.
- the agent can be any of the antibiotics described herein.
- such an antibiotic may be clindamycin metronizadole, vancomycin, fidaxomicin, nitazoxanide , rifaximin ramoplanin, or a combination thereof.
- the candidate agent can be administered to the subject prior to or after inoculation with C. difficile.
- the candidate agent can be any agent that is thought to have the potential for treating or preventing C. difficile infection or C. difficile-associated disease.
- the candidate agents which can be an antibiotic or a non- antibiotic, include antibodies or antigen-binding fragments thereof that specifically bind toxin A and/or toxin B of C. difficile. These methods include any of the in vitro and in vivo methods described in the Examples hereinbelow.
- anti-toxin A and B mAbs described herein can provide non-antibiotic therapies designed to block the pathogenic effects of C. difficile toxins, allow the discontinuation of antibiotics and thereby provide time for the colon to heal and the normal bowel microflora to become re-established.
- Monoclonal antiboidies of the invention have demonstrated complete and durable (>37 days) protection in a stringent hamster model of CDAD. Based on their exceptional characteristics and properties, anti-C. difficile toxin A and toxin B mAbs of the invention provide new treatment options and medicaments for patients poorly served by existing therapies, including those individuals afflicted with the severest cases of disease.
- the present invention further encompasses a vaccine or immunogen comprising portions, fragments, or peptides of toxin A and/or toxin B of C. difficile containing the epitopic regions recognized and/or bound by one or more of monoclonal antibody PA-39 (ATCC Accession No. PTA-9692), a humanized form of PA-39, monoclonal antibody PA-50 (ATCC Accession No. PTA-9694), a humanized form of PA-50, monoclonal antibody PA-41 (ATCC Accession No.
- the vaccine or immunogen comprises portions, fragments, or peptides of toxin A and toxin B of C. difficile containing the epitopic regions recognized and/or bound by one or more of monoclonal antibody PA-39 (ATCC Accession No.
- the epitope-containing portions, fragments, or peptides of toxin A and/or toxin B of the vaccine or immunogen are derived from the toxin A or toxin B protein by proteolytic cleavage.
- the toxin A fragments, portions, or peptides of the vaccine or immunogen are produced by proteolytic cleavage by enterokinase.
- the toxin B fragments, portions, or peptides of the vaccine or immunogen are produced by proteolytic cleavage by caspase (caspase 1).
- the epitope-containing portions or fragments of the vaccine or immunogen are chemically or recombinantly synthesized peptides of the toxin A or toxin B protein.
- the fragments, portions, or peptides of the vaccine or immunogen containing one or more epitopic regions of toxin A and/or toxin B that are recognized and bound by the antibody are derived from one or more of the amino terminus of toxin A; the amino terminus of toxin B; the carboxy terminus of toxin A; the carboxy terminus of toxin B; the receptor binding domain of toxin A; a region outside of the receptor binding domain of toxin A; the N-terminal enzymatic region of toxin B; the glucosyltransferase domain of toxin A; the glucosyltransferase domain of toxin B; the proteolytic domain of toxin A; the proteolytic domain of toxin B; the hydrophobic, pore- forming domain of toxin A; or the hydrophobic, pore-forming domain of toxin B.
- the fragments containing one or more epitopic regions recognized and bound by the antibodies are derived from the amino terminus of toxin A or toxin B. In some embodiments, the fragments containing one or more epitopic regions recognized and bound by the antibodies are derived from the carboxy terminus of toxin A or toxin B. In some embodiments, the fragments containing one or more epitopic regions recognized and bound by the antibodies are derived from the glucosyltransferase domain of toxin A or toxin B. In some embodiments, the fragments containing one or more epitopic regions recognized and bound by the antibodies are derived from the proteolytic domain of toxin A or toxin B.
- the fragments containing one or more epitopic regions recognized and bound by the antibodies are derived from the hydrophobic, pore-forming domain of toxin A or toxin B. In some embodiments, the fragments containing one or more epitopic regions recognized and bound by the antibodies are derived from the receptor binding domain of toxin A. In some embodiments, the fragments containing one or more epitopic regions recognized and bound by the antibodies are derived from the receptor binding domain of toxin B. In some embodiments, the fragments containing one or more epitopic regions recognized and bound by the antibodies are derived from a region outside of the receptor binding domain of toxin A.
- the fragments containing one or more epitopic regions recognized and bound by the antibodies are derived from the N-terminal enzymatic region of toxin B.
- the epitope-containing fragments or portions of toxin A and/or toxin B are ⁇ 300 kDa in size.
- the epitope- containing fragments or portions of toxin A and/or toxin B are -158-160 kDa, -100-105 kDa, e.g., 103 kDa, -90-95 kDa, e.g., 91 kDa, and/or -63-68 kDa, e.g., 63 kDa or 68 kDa in size.
- the epitope-containing fragments or portions of toxin A are -158-160 kDa; -90-95 kDa, e.g., 91 kDa, and/or -63-68 kDa, e.g., 68 kDa in size.
- the epitope-containing fragments or portions of toxin B are -100-105 kDa, e.g., 103 kDa, and/or -63-68 kDa, e.g., 63 kDa in size.
- Such portions, fragments, or peptides of the toxins when administered in the form of a vaccine or immunogen to a subject infected with C. difficile or afflicted with C. difficile- associated disease, may elicit a humoral response in the subject, i.e., antibodies having specificities for toxin A and/or toxin B, thereby allowing the subject to mount an immune response against the toxins and to neutralize, block, reduce, ameliorate, cure, or treat the C. difficile-associated disease, infection, or CDAD in the subject.
- another embodiment provides a method of neutralizing, blocking, reducing, ameliorating, curing, or treating C. difficile infection or a C.
- the subject elicits a humoral response to toxin A and/or toxin B of C. difficile, thereby neutralizing, blocking, reducing, ameliorating, curing, or treating C. difficile-associated disease, infection, or CDAD in the subject.
- the subject elicits a cellular immune response to toxin A and/or toxin B of C. difficile.
- the subject elicits both a humoral and a cellular immune response to toxin A and/or toxin B of C. difficile.
- the invention encompasses a method of neutralizing, inhibiting, or blocking toxin A and/or toxin B activity in or against a cell susceptible to C. difficile infection, comprising contacting the cell with an antibody, or antigen-binding fragment thereof, in accordance with the present invention, wherein the antibody, or antigen-binding fragment thereof, neutralizes, inhibits, or blocks the toxin A and/or toxin B activity in or against the cell by a competitive or a mixed competitive mechanism of action.
- the antibody is one or more of a monoclonal antibody, a humanized antibody, or a chimeric antibody.
- the cell is in a subject and the antibody, or antigen-binding fragment thereof, is administered in an effective amount to the subject.
- the cell is within the gastrointestinal tract, e.g., an intestinal epithelial cell, of the subject.
- the toxin is toxin A.
- the toxin is toxin B.
- the toxin is toxin A and the antibody's mechanism of action is a competitive inhibition mechanism of action.
- the toxin is toxin A and the antibody, or antigen binding fragment thereof, is PA-50 (ATCC Accession No.
- toxin is toxin A and the antibody's mechanism of action is a mixed-competitive inhibition mechanism of action.
- toxin is toxin A and the antibody, or antigen binding fragment thereof, is PA-39 (ATCC Accession No. PTA-9692), a humanized form thereof, or an antibody, or fragment thereof, which competes with PA-39 for neutralizing toxin A activity.
- the toxin is toxin B and the antibody's mechanism of action is a mixed competitive inhibition mechanism of action.
- the toxin is toxin B and the antibody, or antigen binding fragment thereof, is PA-41 (ATCC Accession No. PTA-9693), a humanized form thereof, or an antibody, or fragment thereof, which competes with PA-41 for neutralizing toxin B activity.
- PA-41 ATCC Accession No. PTA-9693
- the term "competitive inhibitor" of toxin refers to a toxin neutralization inhibitor, e.g., an antibody, agent, or small molecule or chemical entity, that displays a rightward EC 50 shift in the neutralization curve, without a change in the maximal percent neutralization as the concentration of toxin increases in the culture.
- a compeititive inhibitor is typically able to overcome the toxin's cytotoxic effect by addition of more inhibitor.
- non-competitive inhibitor" of a toxin refers to a toxin neutralization inhibitor that displays a decrease in the maximal percent neutralization without a change in concentration, producing a half-maximal response (EC 50 ) as the concentration of toxin increases in the culture.
- a non-competitive inhibitor is typically unable to overcome completely the toxin's cytotoxic effect by addition of more inhibitor.
- the term "mixed- competitive inhibitor" of toxin refers to a toxin neutralization inhibitor that displays some degree of both competitive and non-competititve inihibition as the concentration of toxin increases in the culture.
- a mixed-competitive inhibitor of toxin may bind to toxin and exert its effect by blocking the binding of toxin to a cell, as well as by blocking other cytotoxic effects of the toxin; thereby exerting a mixed-competitive mechanism of action.
- Neutralizing monoclonal antibodies directed against toxin A and/or toxin B of C. difficile were generated by immunizing mice with C. difficile toxin A toxoid (inactive form of toxin) and with active forms of toxin A and/or toxin B.
- the murine mAbs (PA-38: antitoxin A mAb, ATCC # PTA-9888; PA-39: anti-toxin A and B mAb, ATCC # PTA-9692; PA- 41: anti-toxin B mAb ATCC # PTA-9693; and PA-50: anti-toxin A mAb, ATCC # PTA- 9694) were generated by immunizing animals with toxoid A followed by immunizations with the active form of toxin A and/or toxin B.
- Toxin A toxoid, toxin A, toxin B (List Biological Laboratories Inc., Campbell, CA) and toxin A (Techlab Inc., Blacksburg, VA) were stored at 4°C until use.
- the toxins and toxoid were derived from strain VPI 10463, a commonly used reference strain of C. difficile.
- Quil A adjuvant (Accurate Chemical, Westbury, NY) was added to the required volume of toxoid or toxin and mixed. The mixture was prepared within 60 minutes of immunizations and stored on ice until ready to immunize. For the final boost prior to fusion, the required toxin was diluted in PBS and stored on ice until used for immunization.
- PA-38 For PA-38, one mouse received a boosting immunization every three weeks for a total of three boosts with toxin A (List Biological Laboratories Inc.), each boost escalating in dose from 500 ng to 2 ⁇ g, and a final boost of toxin A (8 ⁇ g) three days prior to splenectomy.
- PA-39 and PA-41 two mice received either three or five boosting immunizations, respectively, every three weeks with toxin B, each boost escalating in dose from 2 ⁇ g to 12.5 ⁇ g, and a final boost of toxin B (20 ⁇ g) three days prior to splenectomy.
- PA-50 For PA-50, one mouse received a boosting immunization every three weeks for a total of four boosts with toxin A (Techlab Inc.), each boost escalating in dose from 20 ng to 2.5 ⁇ g, and a final boost of toxin A (10 ⁇ g) three days prior to splenectomy.
- adjuvant e.g., Quil A (10 ⁇ g).
- Boosting with the active form of toxin A or toxin B served to identify animals that may have developed protective antibodies.
- Sera from the immunized animals were serially diluted and tested for neutralization of toxin A cytotoxic effect on CHO-K1 cells as described below. Animals with the highest titer of neutralizing antibodies were chosen for fusions and boosted with toxin without adjuvant.
- Hybridomas were suspended in selection medium, RPMI-1640, 10% FBS, 10% BM Condimed-Hl (Roche Applied Science, Indianapolis, IN) and beta mercaptoethanol (for PA-38 and PA-39) or Hybridoma- SFM and 10% FBS (for PA-41 and PA-50) which contained 100 ⁇ hypoxanthine, 1 ⁇ g/ml azaserine and 16 ⁇ thymidine for selective pressure.
- the hybridomas were plated into 96 well flat bottom tissue culture plates (BD Biosciences, San Jose, CA). The plates were incubated at 37°C for 3 days, followed by addition of HT growth medium (the selection media without azaserine). Incubation continued for an additional 4-7 days prior to screening the hybridoma supematants for neutralizing activity.
- a second assay examined inhibition of toxin-mediated agglutination of rabbit erythrocytes. From the screening procedure, four mAbs, which were designated PA-38 (anti-toxin A), PA-39 (anti-toxin A/B), PA-50 (anti-toxin A) and PA-41 (anti-toxin B) and which effectively inhibited or neutralized the C. difficile toxins in the screening assays, were isolated. Hybridoma cell lines that produced these mAbs were cloned twice by limiting dilution to generate clonal cell lines.
- the PA-38, PA-39, PA-41, and PA-50 mAbs were determined to be isotype IgG2a,K, IgGl,K, IgGl,K, and IgGI,K, respectively, using IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche Applied Science, Indianapolis, IN).
- the mAb-producing hybridoma cell lines are designated by the same name as the mAbs that they produce.
- Hybridoma supematants were screened for the ability to neutralize the cytotoxic effect of toxin A or toxin B on cells.
- a high throughput method was developed to process thousands of hybridoma supematants at one time.
- the cytoxicity assay used either CHO-Kl cells (for PA-38, PA-39 and PA-41) or T-84 cells (for PA-50).
- the cells were added to assay plates (96 well, white opaque wall, clear flat bottom plates; Perkin Elmer, Waltham, MA) using the Biomek FX robotic system (Beckman Coulter, Brea, CA). The assay plates were incubated for 4 hours at 37°C to allow the cells to attach to the wells.
- toxin A was diluted to 240 ng/ml.
- toxin A was diluted to 2 ⁇ g/ml or toxin B was diluted to 6 ng/ml.
- Diluted toxin was added to reagent dilution plates (96 well round bottom plates; BD, Franklin Lakes, NJ) manually in a Biosafety Cabinet (BSC).
- BSC Biosafety Cabinet
- Hybridoma supernatants were harvested manually and added to the wells of the reagent dilution plates using the Biomek FX system.
- the supernatant and toxin mixture was incubated at 37°C for 1 hour and added to assay plates containing the cells using the Biomek FX system.
- Clarified material was diluted into a final concentration of running buffer (60 mM glycine/3 M NaCl, pH 8.5) and loaded onto a protein A column equilibrated with running buffer. After washing the column, the PA-39 or PA-41 mAbs were eluted with 0.1 sodium acetate, pH 5.5, and neutralized to pH 7.0. Supernatants containing PA-38 (IgG2a,K) were clarified by centrifugation at 2000 rpm for 10 minutes and filtered. Clarified material was adjusted to a final concentration of 25 mM sodium phosphate buffer/100 mM NaCl, pH 7.0 and loaded onto a protein A column equilibrated with 50 mM sodium phosphate buffer/0.5 M NaCl. The column was washed; PA-38 mAb was eluted with 0.1 M sodium acetate, pH 3.0, and the eluted antibody was neutralized to pH 7.0.
- running buffer 60 mM glycine/3 M NaC
- hybridomas were inoculated into a WAVE Bioreactor (GE Healthcare, Piscataway, NJ) with an initial density of 2xl0 5 cells/ml of Hybridoma-SFM with 5% Ultra Low IgG FBS. Cell count and viability were monitored daily. By about day 6 or 7 when antibody production had plateaued, the culture was terminated. The culture was clarified and was then concentrated 10-20 fold by tangential flow filtration. The antibody was loaded on a protein A column equilibrated with 60 mM glycine 3 M NaCl at pH 8.5. The column was washed with the same buffer and the antibody was eluted with 50 mM acetate, pH 3.5. The pooled antibody was neutralized to pH 7.4 with 1 M Tris, concentrated to lOmg/mL, and diafiltered into PBS. Purified mAbs were sterile filtered and stored at -80°C.
- ELISA plates (BD Biosciences) were coated with 50 ng/well of toxin A (List Laboratories) or 25 ng/well of toxin B (List Laboratories) overnight at 4°C. After washing plates with PBS- (PBS without calcium or magnesium, 0.05% Tween 20), wells were blocked with 200 ⁇ of blocking buffer (PBS without calcium or magnesium, 0.1 % Tween 20, 2.5% non-fat milk) for one hour at 37 °C. The wash step was repeated, and hybridoma supernatants or purified mAb were added for one hour at 37°C.
- the plate was washed and incubated for one hour at 37°C with goat anti-mouse IgG-Fc, horseradish peroxidase (HRP)-conjugated (Jackson Immunoresearch, West Grove, PA).
- HRP horseradish peroxidase
- the plate was developed with ABTS peroxidase substrate system (KPL, Gaithersburg, MD), with ABTS peroxidase stop solution (KPL), and read on a SpectraMax plate reader (Molecular Devices) at 405 nm.
- Figs. 1A-C Titration data are shown in Figs. 1A-C.
- Fig. 1A demonstrates that PA-38 binds to toxin A and not to toxin B.
- Fig. IB demonstrates that PA-39 can bind to both toxin A and toxin B.
- Fig. 1C demonstrates that PA-41 binds to toxin B and not to toxin A.
- a Biacore 3000 instrument (GE Healthcare) was used to determine the binding specificity of mAbs of the invention to toxin A and/or toxin B.
- MAbs were immobilized at approximately 10,000 resonance units (RU) to CM5 sensor chips (GE Healthcare) according to the manufacturer's instructions for amine coupling.
- a reference surface of isotype-matched antibody of irrelevant specificity (Southern Biotech) was used as a control.
- Binding experiments were performed at 25°C in HEPES-based HPS-EP buffer (GE Healthcare). Purified toxin A or toxin B (30 nM; List Biological Laboratories) was passed over control and test flow cells at a rate of 5 ⁇ 7 ⁇ . Where indicated, additional mAb (100 nM) was then passed over the flow cell at 5 ⁇ to examine multivalent or competitive binding.
- mAb PA-38 (Fig. 2A) and mAb PA-50 (Fig. 2C) specifically bound to toxin A
- mAb PA-41 (Fig. 2D) specifically bound to toxin B
- mAb PA-39 (Fig. 2B) bound preferentially to toxin A, but also demonstrated binding to toxin B.
- Results from these data are consistent with the ELISA data (Figs. 1A-C) and demonstrate the binding specificities of mAbs of the invention to toxin A and/or toxin B.
- Biacore analysis was also used to determine the binding avidity of mAbs of the invention to their respective toxins.
- the mAb was captured using a CM5 sensor chip prepared with Biacore' s mouse antibody capture kit. Toxin was then passed through the flow cells at varying concentrations (0.4 -100 nM, two-fold escalation). All toxin concentrations were tested in duplicate and the chip surface was regenerated after each run using the conditions specified in the kits. The changes in RU were recorded and analyzed using Bia Evaluation Software 1 : 1 (Langmuir) binding model which calculated the KD of the mAb for the toxin. The association and dissociation data and the fitting are illustrated in Figs. 3A-E.
- the K D of the mAbs for toxin A was determined by Biacore analysis to be 1.0 nM for PA-38, 0.16 nM for PA-39, and 0.16 nM for PA-50.
- the K D of the mAbs for toxin B was determined to be 2.4 nM for PA-39 and 0.59 nM for PA-41.
- CHO-K1 cells were seeded (2,000 cells in 50 ⁇ ) in assay plates (96 well, white opaque wall, clear flat bottom plates (Perkin Elmer)). Cells were allowed to attach for 4 hours prior to treatment. Equal volumes (35 iL) of 2 ⁇ g/mL toxin A (List Biological Laboratories) and serially diluted mAbs were mixed in reagent dilution plates (96-well round bottom plates (Falcon)) for 1 hour at 37°C, and then 50 ⁇ of the mixture was added to each well of the plates. After incubating for 72 hours, 20 ⁇ CellTiter-Blue (Promega) was added to each well.
- mAb PA-39 fully neutralized toxin A activity on CHO-Kl cells with an EC 50 of 93 pM.
- a CHO-Kl cytotoxicity assay was used to evaluate the neutralization activity of anti-toxin B specific mAbs. Similar to the evaluation of anti-toxin A mAbs, toxin B (8 pg/mL, TechLab) was incubated for 1 hr at 37°C with serially diluted mAbs prior to addition to CHO-Kl (2,000 cells/well) in a 96-well plate. After 72 hours, 20 ⁇ / ⁇ CellTiter-Blue (Promega) was added to each well. Plates were incubated for an additional 4 hours, and then read on a SpectraMax M5 Plate Reader (Molecular Devices) using an excitation wavelength of 560 nm and an emission wavelength of 590 nm.
- the present invention encompasses a novel mAb having the dual ability to bind both toxin A and toxin B and also to neutralize the cytotoxicity of a C. difficile toxin, i.e., toxin A.
- T-84 cytotoxicity assay was used to evaluate the neutralization activity of the described anti-toxin A mAbs.
- T-84 cells were seeded (15,000 cells in 50 ⁇ ) in assay plates (96 well, white opaque wall, clear flat bottom plates (Perkin Elmer)). Cells were allowed to attach for 4 hours prior to treatment. Equal volumes (35 ⁇ ) of 240 ng/mL toxin A (Techlab) and serially diluted mAbs were mixed in reagent dilution plates (96- well round bottom plates (Falcon)) for 1 hour at 37°C, and then 50 ⁇ of mixture was added to each well of the assay plates.
- mAbs PA-38 and PA-50 fully neutralized toxin A activity on T-84 cells with an EC 50 of 175 pM and 146 pM, respectively.
- mAb PA-39 demonstrated minimal activity against toxin A and PA-41 was not active.
- mAbs of the invention to block the binding of toxin A to cellular receptors was assessed using a hemagglutination assay.
- assay equal volumes (30 of toxin A (8 ⁇ g/mL; TechLab) and serially-diluted mAbs were mixed in reagent dilution plates (96- well round bottom plates (Falcon)) for 1 hour at 4°C.
- Rabbit red blood cells (RBC), (Colorado Serum Co., Denver, CO) were washed with PBS three times and resuspended in PBS.
- mAb PA-38 filled squares
- mAb PA-50 filled triangles
- PA-38 and PA-50 appear to neutralize toxin A by blocking the binding of toxin A to its receptor.
- mAbs PA-39 and PA-41 were found to be inactive in the assay.
- Caco-2 cells were seeded (25,000 cells in 75 in the upper chamber of 96-well Multiscreen Caco-2 plates (Millipore Billerica, MA) with 250 ⁇ ⁇ of media added to the lower chamber. Cells were allowed to grow for 10 days with regular changes of medium every 3-4 days. After an incubation of 10-14 days, the formation of a tight monolayer was confirmed by measuring transepithelial electrical resistance (TEER) using an epithelial voltohmmeter (model: EVOMX, World Precision Instruments, Sarasota, FL).
- TEER transepithelial electrical resistance
- PA- 38 and PA-50 appear to represent one class of anti-toxin A mAbs
- PA-39 represents another class of anti-toxin A mAbs
- mAbs PA-38 and PA-50 appear to bind a toxin A epitope important for receptor binding
- mAb PA-39 appears to bind toxin in a manner that more directly blocks the cytotoxic effects of toxin A in vitro.
- An in vivo mouse model was used to measure the ability of the mAbs described herein to neutralize circulating C. difficile toxins in an animal.
- the in vivo neutralizing activities of mAbs PA-38, PA-39, PA-41, or PA-50, administered alone or in combinations, were tested against the effects of combined, systemic C. difficile toxin A and toxin B (Techlab) in test animals.
- mice/group Female Swiss Webster 4-6 mice/group (age: -6-8 weeks at start of study; Charles River Laboratories) were used in the experiments. The mice were acclimated in the facility for a minimum of 4 days, and the health of the animals was checked before use. Animal experiments were conducted under IACUC approved protocol.
- mice Animals were dosed at 0, 20, 100, 500, 2500 ng toxin/animal intraperitoneally (i.p.) and a dose that was lethal to animals was selected for use in subsequent antibody neutralization experiments.
- the control mice injected with PBS were unaffected.
- a dose of 100 ng of toxin A (TechLab) was selected for the neutralization experiments, as it was the lowest dose level found to be lethal to 100% of the mice within 24 hours after the injection.
- a dose of 100 ng of toxin B (TechLab) was selected for neutralization experiments, as it was the lowest dose level found to be lethal to 100% of the mice within 24 hours after the injection.
- mice mice (5 per group) on Day 0, followed by i.p. administration of 100 ng/200 ⁇ of toxin A or toxin B on Day 1. Animals were observed daily for three days and then weekly for up to 21 days following toxin administration. Survival of animals was the primary endpoint of the study.
- PA-38 and PA-50 were found to neutralize a 100 ng dose of toxin A and enabled 100% survival at dose levels as low as 2 ⁇ g of mAb per animal as shown in Figs. 9A and B.
- a comparator human anti-toxin A monoclonal antibody WO/2006/121422 and US2005/0287150
- CDA-1 comparator mAb at 5 ⁇ g per animal, did not protect animals from toxin-related death as did mAbs of the invention (Fig. 9C).
- PA-41 Doses of PA-41 ranging from 0.5 ⁇ g to 250 ⁇ g were evaluated, and a single dose of 5 ⁇ g of mAb per animal was found to neutralize completely a 100 ng dose of toxin B toxicity in animals as shown in Fig. 10.
- MAb PA-39 100 ⁇ g per animal was not observed to provide a delay of toxin-related death of the mice for either toxin A or B.
- the CDAD model in hamsters reproduces the key aspects of CDAD disease in humans. Upon treatment with antibiotics, normal colonic flora is eradicated and the hamsters become readily susceptible to infection by C. difficile. Infection results in severe diarrhea, pseudomembranous colitis and death.
- the hamster CDAD model was utilized to evaluate the potential efficacy of mAbs of the invention to prevent disease and death associated with challenge of animals from live C. difficile bacteria. These experiments were conducted under IACUC approved protocols.
- the mAb concentration in the sera was determined via ELISA.
- Ninety-six well ELISA plates (BD Biosciences) were coated overnight with toxin A (Techlab) or toxin B (Techlab) at 250 ng/well at 4°C. Plates were washed three times with PBS/0.05% Tween-20® (PBS-T) and blocked with 200 ⁇ of blocking buffer (PBS without calcium or magnesium, 0.1% Tween 20®, 2.5% non-fat milk) for one hour at room temperature.
- the antibody reference standard (purified mAb PA-38 or mAb PA-41) was diluted in 1% pooled naive hamster serum to generate a standard curve with a range of 0.3-1000 ng/ml. Diluted test samples and standards were incubated for one hour at room temperature.
- Plates were washed (as above) and incubated for one hour at room temperature with HRP- conjugated goat anti-mouse IgG, Fey specific (Jackson Immunoresearch). Plates were developed with ABTS peroxidase substrate system (KPL), stopped with ABTS peroxidase stop solution (KPL) and read on a SpectraMax plate reader (Molecular Devices) at 405 nm. mAb concentration in each hamster at different time points was calculated using the standard curves. Approximately 10% of the samples did not have antibody titer, probably due to missed injection or no absorption; these samples were not included in the PK parameter calculation.
- KPL ABTS peroxidase substrate system
- KPL ABTS peroxidase stop solution
- Noncompartmental pharmacokinetic analysis was performed using WinNonLin, Version 4.0 (Pharsight Corp., Mountain View, CA) and the data are illustrated in Table 1 and in Figs. 12A and B. As indicated, Cmax and area under curve (AUC) were dose dependent. Each of the antibodies demonstrated a terminal half life of greater than 6 days, which ensured antibody retention in the efficiacy studies described hereinbelow.
- hamsters in the relevant test groups received an oral dose (1 x 10 CFU in 0.5 mL) of a suspension of C. difficile (ATCC 43596 strain).
- Strain 43596 has been previously used in hamster models for evaluating neutralizing antibodies. Animals were weighed weekly and monitored daily for health status and survival.
- test antibodies comprised combinations of the murine mAbs of the invention, i.e., a combination of mAbs PA-38 and PA-41 or a combination of mAbs PA-39 and PA-41.
- Goat anti-C. difficile toxin A and toxin B polyclonal antibodies (Techlab) were included as a positive control.
- the mAbs and control reagents were administered as described in Table 2.
- the hamsters in Group 1 received no treatment throughout the study.
- the hamsters in Groups 2-7 were pretreated with a single subcutaneous dose of clindamycin phosphate at 50 mg/kg (Day -1).
- the hamsters in Groups 5-7 were dosed with the polyclonal goat antibodies (Group 5) or with the mAb combinations (Groups 6 and 7) as set forth in Table 2 by i.p. administration immediately after the clindamycin treatment.
- each hamster in Groups 3-7 was inoculated with 0.5 mL of the appropriate suspension of C. difficile ATCC 43596 (10 6 -10 7 CFU/mL) via oral gavage (day 0).
- Vancomycin (20 mg/kg BID) was administered via oral gavage to the animals in Group 4 twice daily, approximately 6 hours apart on Days 1-5.
- the administration of vancomycin (Group 4 animals) began approximately 20-24 hours after the animals had been inoculated with C. difficile.
- the body weights of animals in the polyclonal- and mAb-treated groups and in the control groups are illustrated in Fig. 14.
- Hamsters in the uninfected control group (Group 1) gained weight steadily, ranging from 13-29 g over the course of the study. All infected control animals died prior to the first post-inoculation weight measurement.
- the hamsters in Groups 1-7 were pretreated with a single subcutaneous dose of clindamycin phosphate at 50 mg/kg (day -1). Animals in groups 3-7 were dosed with mAbs by i.p. administration immediately after the clindamycin treatment. After 24 hours, each hamster in Groups 1-7 was inoculated with 0.5 mL of the suspension of C. difficile via oral gavage (day 0), as described in Section B, supra. The test mAb treatments were administered to the animals in Groups 3-7 in a single dose on days 1, 3, and 5. Vancomycin was administered to animals twice daily on days 1-5 (Group 2). The hamsters were observed twice daily for viability. Body weights were recorded once per week. A necropsy was performed on animals that were found dead or that were euthanized during the study. At the end of the study (40 days post inoculation), a terminal necropsy was performed on all remaining hamsters.
- Fig 16A Survival of mAb-treated and control animal groups is depicted in Fig 16A, and the day of death for the hamsters in all groups is summarized in Table 5.
- the PA-39 + PA-41 mAb combination treatment (Group 3) was very effective in protecting infected hamsters in this study. Six of the seven hamsters in Group 3 survived to the end of the study. One hamster was found dead on day 12 of the study. The postmortem examination indicated that this hamster had an inflamed GI tract typical of C. difficile infection.
- mAb PA-39 alone (Group 6) exhibited some protective activity in treated animals. Hamsters in this group were found dead on days 2 through 12. In the groups treated with the individual mAbs, PA-41 (Group 4), PA-38 (Group 5), or PA-50 (Group 7), the hamsters were found dead on days 2 and 3. At terminal necropsy, all of the hamsters in these groups had inflamed GI tracts, which is indicative of C. difficile infection. In contrast, all of the treated hamsters that survived had normal GI tracts.
- Example 5C At terminal necropsy from group 3 in Example 5C, the cecum of each hamster was exposed and each appeared normal. No inflammation or redness was observed and the contents of the ceca were relatively firm in consistency.
- the wall of each cecum was cut open with a sterile, disposable scalpel. A new scalpel was used for each hamster to avoid cross-contamination.
- a small amount of feces was removed from the cecum with a sterile swab and placed in a sterile test tube.
- a ⁇ - ⁇ . inoculating loop was used to collect a sample of the feces from the tube and to streak the sample across an agar plate containing CCFA with horse blood medium
- test antibodies comprised combinations of the humanized mAbs of the invention, i.e., a combination of humanized anti-toxin A mAb PA-50 and humanized anti-toxin B mAb PA-41 (hPA-41 + hPA-50), or a combination of comparator human anti-toxin A mAb referred to as CDA-1 comparator mAb and comparator human anti-toxin B mAb referred to as CDB-1 comparator mAb (CDA-1 + CDB-1) in the amounts indicated in Table 5 A.
- the comparator mAbs were synthesized (DNA2.0) based on the published heavy and light chain regions of 3D8 and 124, (WO2006/121422 and US2005/0287150), cloned into full-length IgGl expression vectors (pCON-gamma and pCON-kappa), expressed in CHO-KSV1 cells and purified using methods described herein.
- the mAb combinations and control treatments were administered as described in Table 5A. The treatment methods were essentially as described for Part B of this Example, supra. Briefly, Golden Syrian hamsters (Charles River Laboratories, Stone Ridge, NY, 50 days old) were utilized in this Example 5E study. The hamsters in the control Group 1 were uninfected (and untreated).
- the animals in Groups 2-7 were pretreated with a single subcutaneous dose of clindamycin phosphate at 50 mg/kg to disrupt the normal colonic flora (Day -1).
- the Groups 4-7 animals were dosed by IP administration immediately after the clindamycin treatment. After 24 hours, each animal in Groups 2-7 was inoculated with 0.5 mL of C. difficile (ATCC 43596, strain 545) suspension via oral gavage (Day 0), (i.e., oral dose). Additional administrations of the test treatments were given to the animals in Groups 4-7 in a single dose on Days 1, 3 and 5. Vancomycin was administered to the animals of Group 3 twice daily, approximately 6 hours apart, on Days 1-5.
- Fig. 16B-1 Survival results for the mAb-treated and control groups are illustrated in Fig. 16B-1. Mortality data for the study are presented in Table 5B below. A summary of hamster survival is presented in Table 5C below. Table 5B. Mortality data and day of hamster death in each group
- comparator mAb groups nine of ten animals dosed with 50 mg/kg (Group 6), died between days 5 and 14; one animal died by day 28 of the study. All ten of the hamsters treated with 20 mg/kg of the comparator mAb combination (Group 7) died between days 5 and 14 of the study.
- these findings support a mechanism of action by which the humanized mAb combinations protected the animals from C. difficile disease, and/or allowed the animals to mount a response against the C. difficile infection, for a time long enough to allow outgrowth and repopulation of the normal gut flora in the humanized mAb-treated animals, thus affording therapeutic protection to the infected animals, effective resolution of C. difficile-associated disease and restoration to gastrointestinal health and survival.
- C. difficile toxin A and toxin B were approximately 300 kDa and share considerable sequence and structural homology. Both have a C-terminal receptor-binding domain that contains clostridial repetitive oligopeptides (CROPs), a central hydrophobic domain that is believed cause pore formation and mediate the insertion of the toxin into the membrane of the endosome and a proteolytic domain that cleaves the N-terminal enzymatic domain, thereby allowing the glucosyltransferase to enter the cytosol. Nucleic acid sequences encoding the toxins of C.
- CROPs clostridial repetitive oligopeptides
- NCBI National Center for Biotechnology Information
- Full length C. difficile toxin B consists of three major domains: an N-terminal enzyme domain processing glucosyltransferase (GT) activity (63 kDa) and a C-terminal cell receptor binding (59 kDa), which are on either end of a putative translocation domain (148 kDa) (Figs. 17A and C).
- GT glucosyltransferase
- 59 kDa C-terminal cell receptor binding
- 148 kDa putative translocation domain
- toxin B Following treatment of toxin B with capase 1 (enzyme/toxin ratio ⁇ 1 U ⁇ g toxin) at 37°C for 96 hours, four major fragments were produced, including two C-terminal containing fragments (193 and 167 kDa) and two N-terminal containing fragments (103 and 63 kDa) as detected via SDS-PAGE (Fig. 17B). Other smaller fragments, such as 26 and 14 kDa, also appear to be generated, but are not detectable in 3-8% Tris-Acetate gel analysis.
- PA-39 bound the 167 kDa fragment of caspase 1-treated toxin B (Fig. 18C, right lane), as well as a 63 kDa protein in the untreated toxin B preparation (Fig. 18C, left lane), thus suggesting that PA-39 binds an epitope in the translocation domain of toxin B.
- mAbs PA-41 and PA-39 were observed to interact differently with toxin B.
- Murine mAbs PA-39 and PA-41 were observed to bind to a single site or epitope on toxin B; these mAbs were not found to bind to the C-terminal cell receptor binding (CRB) domain.
- CRB C-terminal cell receptor binding
- Full length toxin A of C. difficile has a molecular weight of 310 kDa (Fig. 20A) and contains three major domains: an N-terminal enzyme processing domain having glucosyltransferase (GT) activity ( ⁇ 63kDa) and a C-terminal CRB domain (-101 kDa), which are at either end of a hydrophobic domain (-144 kDa).
- GT glucosyltransferase
- toxin A fragments were generated through enzymatic cleavage of the full length toxin using the enzyme enterokinase (EK). Following treatment of toxin A with enterokinase (enzyme/toxin ratio: about 3 mU ⁇ g toxin) at 25°C for 48 hours, nine major fragments were produced, including four C-terminal fragments (-223, -158-160, -91, and -68 kDa) and three N-terminal fragments (-195, -181, and -127 kDa). Smaller fragments (-53 and -42 kDa) were also observed. (Figs. 20 B and C).
- mAb PA-39 bound C-terminal containing fragments (-223 and -158-160 kDa), as well as an -181 kDa N-terminal containing fragment (Fig. 21C), thus indicating that mAb PA-39 binds an epitope in a region outside of the receptor binding domain of toxin A.
- Multiple binding sites (at least two binding sites) were also identified in a mAb PA-50 and toxin A interaction study using a Biacore assay (Fig. 22A-1).
- Biacore assay Fig. 22A-1
- toxin A was able to bind additional PA-50 and, subsequently, comparator anti-toxin A mAb CDA-1 (WO/2006/121422; US2005/0287150), (Fig. 22A-2). Additionally, toxin A captured by comparator anti-toxin A mAb CDA-1 on the Biacore chip further bound additional CDA-1 and PA-50 mAb, indicating that comparator mAb CDA-1 binds to multiple repeats on toxin A, which are different from the PA-50 mAb binding epitopes on toxin A (Fig. 22B).
- the PA-50 mAb epitope is present in multiple copies on toxin A and does not overlap with the epitope for CDA-1.
- the PA-39 mAb bound epitope(s) on toxin A different from the toxin A epitope(s) bound by the comparator CDA-1 mAb (Fig. 22C).
- MAbs PA-39 and PA-50 were found to bind different epitopes on toxin A (Fig. 22D).
- Western blot analysis showed that PA-39 and comparator mAb CDA-1 have different binding patterns to EK-treated toxin A, thus indicating different binding domains and different epitopes on toxin A (Fig. 22E).
- Western blot analysis showed that PA-50 and comparator mAb CDA-1 bind to the same domain of EK-treated toxin A (Fig. 22F), but to different epitopes (Fig. 22B).
- Murine mAb PA-50 recognized full-length toxin A and several enterokinase cleavage products, including a large 223 kDa fragment and carboxy- terminal fragments of 68, 91 and 160 kDa in size (Fig. 22F). N-terminal sequencing confirmed that the 68 kDa fragment corresponds to the carboxy-terminal domain of toxin A. The same fragments were recognized by CDA-1 comparator mAb.
- Murine mAb PA-41 bound full-length toxin B as well as the 63 and 103 kDa amino-terminal fragments generated by caspase-1 digestion (Fig. 18B), while CDB-1 comparator mAb recognized a distinct set of caspase-1 cleavage products. N-terminal sequencing confirmed that the 63 kDa fragment corresponds to the amino-terminal domain of toxin B.
- CHO-Kl assay was used to evaluate the neutralization potency of anti-toxin A and anti-toxin B mAbs (PA-39 and PA-41).
- CHO-Kl cells were seeded (2,000 cells/well) in 96- well plates. Cells were allowed to attach for 4 hours prior to treatment.
- Different concentrations (60, 30, 15, or 6 ng/mL) of C. difficile toxin (strain VPI 10463) were incubated with serially-diluted mAbs, mixed in 96-well round bottom plates for 1 hr at 37°C and the mixtures were then added to the cell culture plates. After incubation for 72 hrs, 20 CellTiter-Blue was added; the mixtures were incubated for an additional 4 hours; and the percent cell survival compared to controls was measured.
- T-84 cytotoxicity assay was also used to evaluate the neutralization potency of anti-toxin A mAbs.
- T-84 cells human colon carcinoma cell line
- Cells were allowed to attach for 4 hours prior to treatment.
- Different concentrations 240, 120, 60, or 30 ng/mL
- C. difficile toxin strain VPI 10463
- strain VPI 10463 C. difficile toxin
- each test antibody (PA-39, PA-50, comparator anti-toxin A mAb CDA-1 and an anti-toxin A goat polyclonal antibody control) was mixed and incubated for 1 hour at lOOx its EC 90 value with a CC 90 concentration of toxin A for Vero cells to insure complete neutralization at a highly cytotoxic concentration of toxin A.
- the mixture was then incubated with Vero cells at 37°C for 15 minutes. The cells were then washed with PBS, fixed and permeabilized.
- chemiluminescence detection uses an enzyme to catalyze a reaction (i.e., the catalyzed oxidation of luminol by peroxide) between the HRP enzyme and its substrate in the presence of peroxide that results in the generation of visible light.
- Oxidized luminol emits light as it decays to its ground state. Once the substrate is catalyzed by HRP, the light signal is quantified by a luminometer (Analyst GT). C. Results of neutralization activity and MOA studies
- Anti-toxin A mAbs In the cellular cytotoxicity assay used to evaluate neutralization activity and mechanism of action for the anti-toxin A antibodies, toxin A was added in increasing concentrations to cells, as described supra in Section A of this Example. The results of these experiments in which toxin A neutralization by the anti-toxin A mAbs PA-39, PA-50 and comparator mAb CDA-1 was assessed are shown in Figs. 31B-D and in the below Table A.
- the in vitro activity of mAb PA-39 in the toxin potency assay shows shifts in both EC 50 and the maximum percent inhibition as more toxin A is added to the culture, indicating a mixed-competitive mechanism of inhibition for PA-39.
- ELISA detection of toxin A after protection by 100-fold excess of PA-39 confirmed that inhibition of toxin by PA-39 occurs by preventing toxin internalization and cytocellular toxin effect.
- the in vitro activity of mAb PA-50 in the toxin potency assay shows a shift in EC 50 as more toxin A is added to the culture, indicating a competitive mechanism of inhibition for PA-50.
- ELISA detection of toxin A after protection by 100-fold excess of PA-50 confirmed that inhibition of toxin by PA-50 occurs by preventing toxin internalization.
- an inhibitor that has the capacity to neutralize completely increasing concentrations of toxin A or toxin B simply by the addition of greater concentrations of inhibitor would display a shift in EC 50 as more antibody binds and neutralizes the higher concentrations of toxin and thus would be considered to be a competitive inhibitor.
- An inhibitor thai is unable to overcome the toxic effects of increasing concentrations of toxin would display a lowered maximal percent effect at higher concentrations of inhibitor, but would not display a shift in EC 5 0 and thus would be considered to be a non-competitive inhibitor.
- an inhibitor that displays both a shift in EC 50 and a lowered maximal percent effect as a result of increasing concentrations of inhibitor at higher concentrations of toxin would be considered to be a mixed-competitive inhibitor.
- the evaluation of the mechanism of action using cellular cytoxicity assays must be performed under consideration of the error involved in assay repeatability and the background cytoxicity observed in control wells containing no inhibitor, which affects the plateau of the maximal percent inhibition. Consequently, a slight rightward shift in the EC 50 values may be observed as toxin concentrations are increased due to these effects.
- the PA-39 mAb is considered a mixed-competitive inhibitor due to the shift in EC 50 and lowered plateau obse.rved for the PA-39 cytoxicity curves (Fig. 31B), as well as in the lowered maximal percent effect calculated in Table A.
- Fig. 31H show a degree of cytotoxic effect at high concentrations of PA-39, thus indicating that at least some of PA-39 's MOA occurs intracellularly.
- the PA-50 mAb is observed to be a competitive inhibitor as evidenced by the rightward shift in EC 50 values observed in the cytoxicity assay curves (Fig. 31C) and by the data presented in Table A showing minimal change in maximal percent inhibition.
- Fig, 311 show complete inhibition of toxin A binding and internalization at high concentrations of PA-50.
- comparator mAb CDA-1 shows a minimal shift in EC 50 , but considerable lowering of the maximal percent effect as toxin is increased.
- the CDA- 1 comparator mAb demonstrates a non-competitive mechanism of action, as all of its activity is observed outside of the cell, Steric hindrance of toxin binding and cellular internalization are likely to be responsible for yielding the data plots in Fig. 31D, thereby supporting a non-competitive MOA for the CDA-1 comparator mAb.
- the PA-41 mAb is considered to exhibit a mixed-competitive mechanism of action due to the rightward shift of EC5 0 values and the lowered maximal percent effect observed both in Fig. 31E and in the data presented in Table B.
- C. difficile anti-toxin A and anti-B mAbs of the invention To evaluate the ability of C. difficile anti-toxin A and anti-B mAbs of the invention to neutralize toxins from a broad range of relevant isolates of C. difficile, the neutralization activity of the mAbs was tested against a collection of twenty toxigenic, clinical C. difficile isolates or strains, including hypervirulent BI/NAP1/027 isolates. Since C. difficile exhibits considerable interstrain heterogeneity in the genes encoding toxins A and B, these studies were undertaken to examine the breadth of toxin neutralization by the described mAbs, and in particular, mAbs PA-50 and PA-41.
- a panel of toxigenic, clinical C. difficile isolates (Table 6) was selected for geographic and genetic diversity based on toxinotype, ribotype and restriction endonuclease analyses from an international collection of C. difficile isolates or strains maintained at TechLab (Blacksburg, VA).
- the strains of C. difficile include 3 reference strains (VPI 10463 (ATCC 43255), 630 (ATCC BAA-1382) and 545 (ATCC 43596), six hospital-derived BI/NAP1/027 strains (CCL678, HMC553, Pitt45, CD196, 5, 7.1), two toxin A-/ toxin B+ (tcdA-tcdB+) strains (F1470, 8864), three outpatient isolates (MH5, CCL13820 and CCL14402) and other frequent clinical isolates (Pitt2, CCL14137, UVA17, UVA30/TL42, Pittl02, Pitt7).
- isolates 13 (CCL13820) and 19 (Pitt 102), which are categorized as an "outpatient isolate” and a "frequent clinical isolate (other than Ribotype 027)", respectively, in Table 6 are also toxA-/toxB+ strains.
- Culture supernatants containing these C. difficile toxins were produced at TechLab, filter- sterilized and stored at 4°C. The presence of the toxins in the culture supernatants was confirmed using the C. difficile Toxin/ Antitoxin Kit (TechLab) and cytotoxicity assay.
- the cytotoxic activity of each toxin-containing culture supernatant for CHO-K1 cells (used to determine toxin B activity of culture supernatants) and T-84 cells (used to determine toxin A activity of culture supernatants) was assessed by treating cells with titrated culture supernatants.
- toxin-containing culture supematants were used at the maximum dilution that resulted in >95% loss in cell viability.
- Toxin supematants were premixed with various concentrations of the mAbs for lh and then added to cells for incubation at 37°C for 72h. Cell viability was measured using Cell-Titer Blue (Promega). The percent survival of treated wells was compared to that of untreated control wells and graphed to calculate in vitro neutralization activities (EC 50 ) of the mAbs.
- PA-41 potently (EC 50 range from 1. U M to 6.5 "10 M) neutralized supematants of all toxigenic strains of C. difficile, including all hypervirulent strains, with the exception of three toxinA-/toxin B+ strains. It has been reported that there are significant sequence differences in the enzymatic domains of toxin A- /toxin B+ from conventional strains of C. difficile.
- PA-41 binds to the enzymatic domain of toxin B, the sequence diversity in this domain may explain the less effective neutralizing activity of PA-41 against toxin B from the two toxin A-/toxin B+ strains - I l l -
- hPA-41 and CDB-1 comparator mAb for the 2 reference strains (VPI 10463 and ATCC 43596) and 6 BI/027/027 strains (CCL678, HMC553, Pitt 45, CD196, Montreal 5.1 and Montreal 7.1) is illustrated in Fig 23B.
- hPA-41 was found to be inactive against three Ribotype 017 isolates (Table 6) which are toxin A-/B+, although the hPA-41 anti-toxin B mAb exhibited significantly greater neutralization activity than the comparator mAb against other strains in the panel.
- PA-50 in neutralizing toxin A-containing supernatants of C. difficile cultures using T-84 cells ranged from 2.6 "12 M to 7.7 "U M as shown in Fig. 24A.
- PA-50 fully neutralized supernatants from all available strains which produce toxin A, including hypervirulent strains.
- PA-50 did not neutralize the four toxin A-/toxin B+ strains (F1470, 8864, CCL 13820, CCL 14402), since these strains do not produce any toxin A.
- hPA-50 was also significantly more effective in neutralizing the activity of the remaining strains in the panel.
- hPA-41 showed high levels of neutralization activity against the supernatants from all 6 BI/NAP1/027 strains, while the comparator mAb CDB-1 showed minimal activity.
- Humanized PA-41 was seen to have a neutralization activity >l,000-fold greater than that of comparator mAb CDB-1 in neutralizing the hypervirulent BI/NAP1/027 strains of C. difficile.
- the neutralization activities of hPA-41 and CDB-1 in these studies against the 2 reference strains (VPI 10463 and ATCC 43596) and 6 BI/027/027 strains (CCL678, HMC553, Pitt 45, CD196, Montreal 5.1 and Montreal 7.1) are illustrated in Fig 23B.
- hPA-41 showed significantly higher neutralization activity in these studies compared with comparator mAb CDB-1 against the other strains in the panel, with the exception of three Ribotype 017 isolates, which are toxin A-/B+. Similar experiments were performed to assess the neutralization activity of hPA-39 and that of comparator human antitoxin A mAb CDA-1 on T-84 cells. The results showed that neutralization of all 6 BI/NAP1/027 strains by hPA-39 was >100-fold greater than that of comparator mAb CDA-1 (Fig 25B). Humanized PA-39 also showed significantly higher neutralization activity in the studies than comparator mAb CDA-1 against the remaining strains in the panel.
- hPA-41 and hPA-39 mAbs displayed high levels of anti-C. difficile hypervirulant strain neutralization activities against all strains tested. This indicates that the epitopes recognized by these humanized mAbs are highly conserved across diverse strains.
- Table 7 presents the results of the above-described in vitro C. difficile toxin neutralization experiments, showing the panel of toxigenic C. difficile strains isolated from North America and Europe and the resulting EC 50 values generated by the humanized anti-toxin mAbs and comparator mAbs CDA-1 and CDB-1.
- the panel includes ribotypes 001, 002, 003, 012, 014, 017, 027 and 078 in approximate proportion to the rates observed clinically (94, 95), with the exception of ribotype 017 tcdA ' tcdB + strains, which are over- represented in the panel.
- PA-41 potently inhibited each of the tcdA + tcdB + strains with a median EC 50 value of 23 pM (range: 7.7 to 129 pM, Table 7), and essentially complete neutralization was observed at higher concentrations (Fig. 25D).
- CDB1 but not PA-41, was effective in neutralizing toxin B from ribotype 017 tcdA ' tcdB + strains.
- PA-41 and PA-50 exhibited similar activities against crude and purified forms of toxin from the reference strain VPI 10463 (Table 7 and Figs. 25C and 25D).
- humanized mAbs PA-50 and PA-41 showed a high level of neutralization activity against C. difficile toxins A and B, respectively, from genetically diverse strains representative of the current CDI epidemic.
- the breadth of activity of these mAbs is notable in light of the genotypic and phenotypic variation within the toxins.
- PA-50 and PA-41 neutralized toxins produced by the hypervirulent, antibiotic resistant 027 strains with picomolar activity, while the comparator mAbs were observed to have nanomolar activity. This result may reflect the reduced binding of CDA-1 to toxin A from 027 strains as previously reported (90).
- Toxin B from 027 strains exhibits marked sequence divergence relative to other C. difficile strains.
- CDI is typically caused by strains of C. difficile that produce both toxins A and B.
- tcdA ' tcdB + strains also have been linked with disease.
- Clinically relevant tcdA ' tcdB + strains are predominantly ribotype 017.
- Ribotype 017 strains have been reported to exhibit reduced pathogenicity in hamsters and encode an atypical tcdB whose amino-terminal region bears 70-80% sequence identity with both tcdB from VPI 10463 and lethal toxin (tcsL) from C. sordellii.
- ribotype 017 tcdB has hybrid characteristics and exhibits the receptor-binding properties and glucosylating specificities of typical tcdB and tcsL toxins, respectively.
- the atypical amino-terminal region of 017 tcdB provides a likely explanation for why this toxin was not neutralized by PA-41 in this study.
- 017 strains can be regionally prevalent and cause local outbreaks of CDI, overall they were determined to comprise ⁇ 2% of the strains encountered in recent international phase 3 clinical studies of investigational therapies for treating CDI (94, 95).
- Chimeric monoclonal antibodies that comprise the variable region of the parent mouse mAb and the constant region of human IgGl were produced and characterized. Chimeric mAbs were generated to ensure that murine variable regions having the correct anti-toxin activity and binding specificity were cloned and that construction of the murine variable regions with a human constant region did not significantly alter the binding and neutralization properties of each cloned mAb. Chimeric mAbs typically exhibit the same binding activity as the parent mouse mAbs. MAbs PA-38, PA-39, PA-41 and PA-50 all contain kappa light chains.
- the nucleic acid sequences encoding the variable regions of the heavy and light chains were inserted into suitable expression vectors, such as, but not limited to, pCON Gammal and pCON kappa, respectively (Lonza Biologies, Berkshire, UK).
- suitable expression vectors such as, but not limited to, pCON Gammal and pCON kappa, respectively (Lonza Biologies, Berkshire, UK).
- Suitable plasmids encode either the constant region of the human kappa light chain or the constant region of the human IgGl heavy chain.
- the variable region of the heavy chain of each mAb was cloned into the pCON gammal plasmid.
- the complete heavy chain gene was subcloned into the plasmid containing the light chain gene to create a single plasmid that encoded both the heavy and light chain genes.
- 293F cells were transiently transfected with this expression vector using Effectene (Qiagen, Valencia, CA) according to the manufacturer's suggested protocol.
- Cell supernatant containing secreted chimeric mAb was collected seven days following transfection and purified using Protein A chromatography. The potencies and activities of the chimeric mAb(s) were compared with those of the murine mAbs in cytotoxicity and hemagglutination assays.
- chimeric mAbs (cPA-39 and cPA-41) were generated based on the parental mouse PA-39 and PA-41 mAbs.
- concentrations of these chimeric mAbs in crude cell supernatants ranged from about 2-11 ⁇ g/mL.
- crude supernatant from a transfected 293F cell culture producing cPA-39 contained 10.6 ⁇ g/mL of the chimeric mAb
- crude supernatants from several transfected 293F cell cultures producing cPA-41 contained from 9.6-10.9 ⁇ g/mL of the chimeric mAb.
- chimeric mAbs were compared with their respective parental mouse mAbs for the ability to neutralize C. difficile toxins in vitro by conducting cytotoxicity assays (cPA-39: CHO-K1 cells, cPA-41: CHO-Klcells and cPA-50: T-84 cells) as previously described (Example 3). All chimeric mAbs were found to be equally effective when compared to their murine parental mAb as shown in Fig. Fig. 26 (PA-41), Fig. 27 (PA-39) and Fig. 28 (PA-50). These results demonstrate the success of chimerization and the production of functional chimeric mAbs having toxin neutralization potencies equivalent to those of the parental mouse mAbs.
- Humanized mAbs have been generated by methods known in the art. Examples and descriptions of various humanized mAbs include, for example, Zenapax (65,66) Synagis (67- 69), Herceptin (70-72), Mylotarg (73,74), Xolair (75-77), Raptiva (78-80), Avastin (81,82), and Tysabri (83). Humanized monoclonal antibodies that are effective in minimizing immunogenicity can be generated, such that mAb activity in humans is not adversely affected. (84-87). Preferably, the humanized mAbs demonstrate toxin-neutralizing activity that is within two-fold of the parent mouse mAbs. Further, humanized mAbs optimally demonstrate potent efficacy in the hamster model of C. difficile infection.
- CDR complementarity-determining region
- TRIzol reagent Invitrogen
- the resulting cDNA was treated with RNAse H to remove the RNA template and was then purified using a QIAquick PCR purification kit (Qiagen) to remove free nucleotides and primers.
- a tail of guanidine nucleotides was added to the 3' end of the cDNA using the enzyme terminal transferase (NEB) in the presence of dGTP according to the manufacturer's suggested protocol.
- NEB enzyme terminal transferase
- the resulting tailed cDNA was then subjected to PCR using one primer that annealed to the constant region of either the heavy or light chain and one universal primer that annealed to the guanosine tail on the cDNA.
- the universal primer 5'TATATCTAGAATTCCCCCCCCCCCCCCCCCCC3' SEQ ID NO: 11 was used. To amplify the light chain, the primer
- the framework amino acid residues important for the CDR structure were first identified.
- human V H and V L sequences having high homology to the murine V H and V L , respectively, were selected from among known human immunoglobulin sequences.
- CDR sequences from the murine mAb, together with any framework amino acid residues important for maintaining the structure of the CDRs, if necessary, were grafted into the selected human framework sequences.
- human framework amino acid residues that were found to be atypical in the corresponding V region subgroup were substituted with the typical residues in an effort to reduce potential immunogenicity of the resulting humanized mAb.
- V H and V L regions were cloned into expression vectors, such as, but not limited to, pCON Gammal and pCON kappa (Lonza Biologies, Berkshire, UK), respectively. These vectors encode the constant region(s) of the human immunoglobulin heavy and light chain genes.
- 293F cells were transiently transfected with these expression vectors using the Effectene system (Qiagen, Valencia, CA). Cell supernatants containing secreted humanized mAb were collected seven days following transfection and purified using Protein A chromatography.
- CHO Kl SV cells can be used from Lonza Biologies (Berkshire, UK) using a glutamine synthetase selection and amplification system (GS) to generate stably transfected CHO cells.
- GS glutamine synthetase selection and amplification system
- the Lonza GS system typically yields high-production CHO cell lines which can produce sizable quantities of the humanized mAbs.
- CHO Kl SV cells were expanded in CD CHO cell culture medium (Invitrogen) supplemented with IX glutamine (Invitrogen) and IX H/T Supplement (Invitrogen). 1 x 10 viable cells were electroporated at 290 V, infinite resistance and 960 uF with 40 ⁇ g of linearized plasmid DNA resuspended in 100 ⁇ of sterile TE buffer. The cells were transferred to a T-150 flask containing 50 ml of complete CD CHO medium and incubated for approximately 48 hours at 37°C and 8.0% C0 2 .
- the cells were centrifuged and resuspended to a final density of 3.3 x 10 5 cells/ml in GS selection medium (CD CHO + IX GS Supplement (JRH Biosciences) + IX H/T Supplement) containing MSX (Sigma) at 100 ⁇ , plated out at 5000 viable cells/well in 96 well plates (Corning) and incubated for approximately 3-4 weeks until primary cell colonies (clones of transfected cells) began to appear. Approximately 300 cell colonies (clones) were sampled for recombinant mAb production by carefully removing 20 ⁇ of supernatant and performing a ELISA assay in a 96-well format.
- GS selection medium CD CHO + IX GS Supplement (JRH Biosciences) + IX H/T Supplement
- MSX Sigma
- 96-well plates were coated with a capture antibody (goat anti-human antibody) and then supernatants from cloned CHO transfectants (diluted 1 :800) were added to allow binding to the capture antibody bound to the plate wells. After washing, a secondary antibody (goat anti-human antibody conjugated to alkaline phosphatase) was added to the plate and allowed to bind to the human antibody in the sample before being washed to remove non-specific binding. The plate was then assayed for alkaline phosphatase activity using a 1-Step PNPP kit (Thermo, Rockford, IL) to identify the clones producing the greatest amount of secreted antibody.
- a capture antibody goat anti-human antibody
- supernatants from cloned CHO transfectants diluted 1 :800
- Clones producing high amounts of mAb were expanded in CD CHO cell culture medium supplemented with IX glutamine and IX H/T Supplement. Cell supernatants containing secreted humanized mAb were collected and purified using Protein A. The clones exhibiting the best production were subcloned by limiting dilution and scaled up to produce gram quantities of recombinant, humanized, monoclonal antibody.
- the molecularly cloned, humanized mAbs were isolated as described above and characterized (see Section E below).
- the light (L) chain constant (CL) region of each of the humanized antibodies is of the kappa ( ⁇ ) class; the heavy (H) chain constant region (CH) of each of the humanized antibodies is of the IgGl isotype.
- the humanized mAbs containing unique variable (V) regions were found to bind and neutralize the activity of either toxin A or toxin B of C. difficile.
- V regions of the L and H chains of the humanized mAbs may form a part of a complete immunoglobulin (Ig) or antibody molecule composed of two H chain polypeptides and two L chain polypeptides, typically linked by disulfide bonding, or they may be discrete portions or fragments of the antibody, in particular, antibody portions or fragments that bind toxin A and/or toxin B and/or that neutralize toxin activity.
- suitable V-region-containing immunoglobulin fragments or portions include F(ab), F(ab'), or F(ab') 2 fragments.
- Humanized anti-C. difficile toxin A and toxin B mAbs were produced according to the above- described procedures. The humanization process yielded several anti-C. difficile toxin A humanized mAbs (hmAbs) that bound toxin A and neutralized toxin A activity on susceptible cells. Examples of such hmAbs include humanized anti-C. difficile toxin A mAb comprising a H chain polypeptide sequence comprising a VH region of SEQ ID NO:l (Fig. 32A) and a human IgGl C region and a L chain polypeptide sequence comprising a VL region of SEQ ID NO:3 (Fig. 33A) and a human ⁇ C region; anti-C.
- hmAbs humanized anti-C. difficile toxin A mAb comprising a H chain polypeptide sequence comprising a VH region of SEQ ID NO:l (Fig. 32A) and a human IgGl C region and a L chain polypeptide sequence comprising
- a hmAb comprising a H chain polypeptide sequence comprising a VH region of SEQ ID NO:2 (Fig. 32B) and a human IgGl C region and a L chain polypeptide sequence comprising a VL region of SEQ ID NO:3 (Fig. 33A) and a human ⁇ C region; anti-C. difficile toxin A hmAb comprising a H chain polypeptide sequence comprising a VH region of SEQ ID NO:l (Fig. 32A) and a human IgGl C region and a L chain polypeptide comprising a VL region of SEQ ID NO:4 (Fig. 33B) and a human ⁇ C region; and anti-C.
- Such humanized anti-C. difficile toxin A mAbs embrace a PA-39 hmAb (hPA-39) of the invention.
- Complete hPA-39 immunoglobulin having two L chains and two H chains can be produced in a host cell which co-expresses and secretes a hPA-39 H chain polypeptide composed of a VH region of the invention (e.g., SEQ ID NO:l; SEQ ID NO:2) and a suitable CH region, e.g., of the IgGl isotype, such as is contained within Genbank Accession No.
- VH region of the invention e.g., SEQ ID NO:l; SEQ ID NO:2
- suitable CH region e.g., of the IgGl isotype, such as is contained within Genbank Accession No.
- hPA-39 L chain polypeptide composed of a hPA-39 VL region of the invention (e.g., SEQ ID NO:3; SEQ ID NO:4), and a suitable CL region, e.g., of the ⁇ subtype, such as is contained within GenBank Accession No. NW_001838785.
- humanized anti-C. difficile toxin A mAbs of the invention include humanized anti-C. difficile toxin A mAb comprising a H chain polypeptide sequence comprising a VH region of SEQ ID NO: 5 (Fig. 34A) and a human IgGl C region and a L chain polypeptide sequence comprising a VL region of SEQ ID NO:7 (Fig. 35) and a human K C region; and humanized anti-C. difficile toxin A mAb comprising a H chain polypeptide sequence comprising a VH region of SEQ ID NO:6 (Fig. 34A) and a human IgGl C region and a L chain polypeptide sequence comprising a VL region of SEQ ID NO:7 (Fig. 35) and a human K C region; and humanized anti-C. difficile toxin A mAb comprising a H chain polypeptide sequence comprising a VH region of SEQ ID NO:6 (Fig.
- Such humanized anti-C. difficile toxin A mAbs embrace a humanized PA-50 (hPA-50) mAb of the invention.
- Complete hPA-50 immunoglobulin having two L chains and two H chains can be produced in a suitable host cell which co-expresses and secretes a hPA-50 H chain polypeptide composed of a VH region of the invention (e.g., SEQ ID NO:5; SEQ ID NO:6) and a suitable CH region, e.g., of the IgGl isotype, such as is contained within Genbank Accession No. NW_001838121, and a hPA-50 L chain polypeptide composed of a VL region of the invention (SEQ ID NO:7) and a CL region, e.g., of the K subtype, such as is contained within GenBank Accession No. NW_001838785.
- a VH region of the invention e.g., SEQ ID NO:5; SEQ ID NO:6
- a suitable CH region e.g., of the IgGl isotype, such as is contained within Genbank Accession No. NW_001838121
- the humanization process further yielded anti-C. difficile toxin B humanized mAbs that bound toxin B and neutralized toxin B activity on susceptible cells in vitro.
- hmAbs include humanized anti-C. difficile toxin B mAb comprising a H chain polypeptide sequence comprising a VH region of SEQ ID NO: 8 (Fig. 36A) and a human IgGl C region and a L chain polypeptide sequence comprising a VL region of SEQ ID NO: 10 (Fig. 37) and a human ⁇ C region; and humanized anti-C. difficile toxin B mAb comprising a H chain polypeptide sequence comprising a VH region of SEQ ID NO: 9 (Fig.
- Such humanized anti-C. difficile toxin B mAbs embrace a humanized PA-41 (hPA-41) mAb of the invention.
- Complete hPA- 41 immunoglobulin having two L chains and two H chains can be produced in a suitable host cell which co-expresses and secretes a hPA-41 H chain polypeptide composed of a hPA-41 VH region of the invention (e.g., SEQ ID NO:8; SEQ ID NO:9) and a suitable CH region, e.g., of the IgGl isotype, such as is contained within Genbank Accession No.
- a suitable host cell which co-expresses and secretes a hPA-41 H chain polypeptide composed of a hPA-41 VH region of the invention (e.g., SEQ ID NO:8; SEQ ID NO:9) and a suitable CH region, e.g., of the IgGl isotype, such as is contained within Genbank Accession No.
- hPA-41 L chain polypeptide composed of a hPA-41 VL region of the invention (e.g., SEQ ID NO: 10) and a CL region, e.g., of the ⁇ subtype, such as is contained within GenBank Accession No. NW_001838785.
- Anti-C. difficile toxin A hmAb PA-50 is composed of two heavy chain polypeptides, each heavy chain containing a VH region and a human CH region and two light chain polypeptides, each light chain containing a VL region and a human CL region.
- the nucleic acid sequence (or cDNA) encoding the amino acid sequence of the hPA-50 heavy chain polypeptide of SEQ ID NO: 14 is set forth in SEQ ID NO: 15, (Fig.
- the nucleic acid sequence (or cDNA) encoding the amino acid sequence of the hPA-50 light chain polypeptide of SEQ ID NO: 16 is set forth in SEQ ID NO: 17.
- Fig. 38A Anti-C. difficile toxin A hmAb PA-39 is composed of two heavy chain polypeptides, each heavy chain containing a VH region and a human CH region and two light chain polypeptides, each light chain containing a VL region and a human CL region.
- the nucleic acid sequence (or cDNA) encoding the amino acid sequence of the hPA-39 heavy chain polypeptide of SEQ ID NO: 18 is set forth in SEQ ID NO:19, (Fig.
- Anti-C. difficile toxin B hmAb PA-41 is composed of two heavy chain polypeptides, each heavy chain containing a VH region and a human CH region and two light chain polypeptides, each light chain containing a VL region and a human CL region.
- the nucleic acid sequence (or cDNA) encoding the amino acid sequence of the hPA-41 heavy chain polypeptide of SEQ ID NO:22 is set forth in SEQ ID NO:23 (Fig. 40B); the nucleic acid sequence (or cDNA) encoding the amino acid sequence of the hPA-41 light chain polypeptide of SEQ ID NO:24 is set forth in SEQ ID NO:25 (Fig. 40A).
- the monoclonal antibodies CDA-1 and CDBl (7, 89) were prepared for use as comparator mAbs.
- DNA sequences encoding the Ig heavy and light chain variable regions of 3D8 and 124 (WO2006/121422 and US2005/0287150) were synthesized (DNA2.0) and cloned into vectors pCON-gammal and pCON-kappa.
- Full-length IgGl,K mAbs were expressed in stably transfected CHO-Kl SV cells and purified as described above. When tested for binding affinity to toxins A and B by Biacore, inhibition of toxin-mediated cytopathic effects, and hemagglutination according to published methods (7), the CDA1 and CDBl preparations exhibited the expected levels of activity.
- PA-50 exhibited a distinct dose-response neutralization curve with Hill coefficients that typically were greater than two, indicating cooperative inhibition. Cooperative interactions are common in nature and often are characterized by steep dose-response curves and Hill coefficients of >1. Drugs that display cooperative activity have been associated with enhanced clinical activity in treating viral infections. In addition, PA-50 binds toxin A in a multivalent fashion, a condition that is often necessary, but not sufficient for cooperativity.
- Fab fragmentation was performed using a Mouse IgGl Fab and F(ab')2 Preparation Kit (Pierce) according to manufacturer's instructions and reagents supplied with the kit. The same protocol for fragmentation was used for all mAbs; PA-39, PA-41, and PA-50. Briefly, immobilized ficin slurry (750 ⁇ ) was washed with digestion buffer (75mM cysteine, pH 5.6) before approximately 3 mg of mAb was added and the mixture was incubated at 37 °C for four hours with constant end-over-end rotation. Once digestion was completed, the slurry was centrifuged and the digest product was collected.
- digestion buffer 75mM cysteine, pH 5.6
- the slurry was washed three times with Protein A binding buffer and the wash material was added to the completed digest.
- the NAb Protein A column was equilibrated with Protein A binding buffer and the digested antibody sample was added.
- the column and sample were incubated at room temperature for 10 minutes.
- the column was centrifuged at lOOOg for one minute to collect the flow through which contained the Fab fragments.
- the column was washed three times with Protein A binding buffer. The flow through was collected, buffer-exchanged into PBS- and concentrated.
- Samples were analyzed via SDS-PAGE using the Novex gel system (Invitrogen) and all reagents listed below were from Invitrogen unless noted otherwise. Samples were mixed with NuPage sample buffer and reduced with DTT. Reduced and non-reduced samples were incubated at 100°C for 10 minutes. After loading samples (4 ⁇ g) into a 4-12% Bis Tris NuPage gel, electrophoresis was performed with MOPS running buffer at 180V for 60 minutes. After electrophoresis, the gel was incubated with fixative (40% methanol, 10% acetic acid) for 20 minutes, rinsed with water, and stained with Simply Blue Stain overnight with constant rotation.
- fixative 50% methanol, 10% acetic acid
- CHO-K1 cells (EC 50 of 88 pM and EC 50 of 80 pM, respectively), (Fig. 41B).
- the PA-50 Fab had an EC 50 value of 1.8 nM compared with an EC 50 value of 100 pM of the whole PA-50 mAb in neutralizing toxin A on T-84 cells (Fig. 41C).
- IHC immunohistochemistry
- Humanized mAbs PA-41 and PA-50 were biotinylated to determine an immunohistochemical binding pattern in a selection of frozen human tissues, which included adrenal, bladder, bone marrow, breast, cerebellum, cerebral cortex, cervix, colon, esophagus, eye, fallopian tube, heart, ileum, jejunum, kidney, liver, lung, lymph node, muscle, ovary, peripheral nerve, pancreas, parathyroid, pituitary, placenta, prostate, skin, small intestine, spinal cord, spleen, stomach, testis, thymus, thyroid, ureter, uterus and white blood cells.
- Working IHC assays were developed for both antibodies.
- An irrelevant human IgGl ,K isotype control antibody was included for all samples.
- OCT Compound Optimal Cutting Temperature embedding compound; Sakura, Torrance, CA
- the IHC staining methods and conditions for each antibody and tissue specimen were developed, tested and optimized.
- a direct biotinylated IHC procedure was performed using freshly-cut frozen unfixed tissue sections. Slides were removed from the cryostat, allowed to air-dry for 10 minutes at room temperature, fixed in 95% ethanol for 5 minutes at room temperature and then washed in three sequential baths of Tris Buffered Saline/0.1% Tween-20 wash buffer (TBST; DakoCytomation) for 3 minutes. All subsequent washes were performed in this manner.
- TST Tris Buffered Saline/0.1% Tween-20 wash buffer
- Endogenous peroxidase activity was blocked with a 5 minute incubation of ready-to-use Peroxidase Block at room temperature. After a buffer wash, endogenous biotin activity was then blocked with 15 minute incubations each of avidin followed by biotin with each step followed by buffer washes.
- For PA-41 slides were then incubated with Background Sniper protein blocking reagent for 10 minutes at room temperature with no buffer wash to follow. Slides were incubated with the test article or negative control reagent (1.25 g/ml for PA-41 and 10 g/ml for PA-50) for 30 minutes at room temperature.
- the PA-50 primary antibody was diluted at 1:350 in Dako Diluent while the PA-41 primary antibody was diluted at 1:3520 in Dako diluent with proline (250mM, 0.576g, Genzyme, CA) and histidine (15mM, 0.046g, Genzyme, CA) added to 20 ml of diluent (pH 7.7).
- proline 250mM, 0.576g, Genzyme, CA
- histidine 15mM, 0.046g, Genzyme, CA
- washes in TBST ABC detection reagent (1:50 in TBST) was applied to the tissue sections for both antibody assays and incubated for 30 minutes at room temperature followed by buffer washes.
- the immunoreaction was visualized by incubating with a 3,3'-diaminobenzidine tetrahydrochloride (DAB) solution for 5 minutes at room temperature.
- DAI deionized
- DI deionized
- DI deionized
- a modified Mayers hematoxylin DakoCytomation
- blued in 0.2% ammonia dehydrated through graded alcohols, cleared in xylene, and coverslipped.
- the interpretation of stained slides was performed by microscopic examination. In general, a morphologic review of the tissue on the slide after antibody staining determined whether an adequate amount of tissue was present, and whether the designated normal tissue elements were appropriately represented.
- the scoring system included a semi-quantitative analysis of staining intensity.
- the staining intensity of the test article was judged relative to the intensity of the tissue control slide containing an adjacent section stained with a negative control antibody. Staining of the section labeled with the negative reagent control was considered "background” staining. A score of "0" indicated no staining relative to background; indicated weak staining; "2+” indicated moderate staining; and "3+” indicated strong staining. In keeping with standard pathology practice, staining intensity was reported at the highest level of intensity observed in all tissue elements.
- PA-41 For PA-41, no true positive staining was exhibited in the 37 human tissues tested, however, weak (1+) positive staining was seen as a highest staining intensity in normal liver (due to lipochrome pigment), normal lung (pulmonary macrophages with a foreign body) and normal muscle (reaction consistent with artifact staining). Such weak staining values for PA-41 were deemed to be inconsequential relative to all controls and in view of minimal staining variation within the assay.
- PK pharmacokinetic
- Table 8 presents the PK study format, showing that each mAb (at a 10 mg/kg concentration) was administered intravenously at two dose levels to non-naive animls.
- the mAb concentration in the sera was determined via ELISA.
- Ninety-six well ELISA plates (Thermo Fisher Scientific, Rochester, NY) were coated overnight with toxin A (Techlab) or toxin B (Techlab) at 100 ng/well at 4°C. Plates were washed three times with PBS/0.05% Tween-20® (PBS-T) and blocked with 200 ⁇ of blocking buffer (PBS without calcium or magnesium, 0.1% Tween 20®, 1% casein) for one hour at room temperature.
- PBS-T PBS/0.05% Tween-20®
- blocking buffer PBS without calcium or magnesium, 0.1% Tween 20®, 1% casein
- the antibody reference standard (purified mAb PA-41 or mAb PA-50) was diluted in 1% pooled naive cynomolgus serum (Bioreclamation) to generate a standard curve with a range of 0.3 - 4000 ng/ml. Diluted test samples and standards were tested in triplicate and were incubated for one hour at room temperature.
- Serum anti-toxin B antibody correlates with protection from recurrent Clostridium difficile associated diarrhea (CD AD).
- CD AD Clostridium difficile associated diarrhea
- Daclizumab a review of its use in the management of organ transplantation. BioDrugs 15:745-773.
- Omalizumab an effective anti-IgE treatment for allergic asthma and rhinitis.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Gastroenterology & Hepatology (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Radiology & Medical Imaging (AREA)
- Toxicology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Biotechnology (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Description
Claims
Priority Applications (12)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA2795953A CA2795953C (en) | 2010-04-15 | 2011-04-15 | Antibodies for the treatment of clostridium difficile-associated infection and disease |
MX2012011926A MX339253B (en) | 2010-04-15 | 2011-04-15 | Antibodies for the treatment of clostridium difficile-associated infection and disease. |
ES11769680T ES2757675T3 (en) | 2010-04-15 | 2011-04-15 | Antibodies for the treatment of Clostridium difficile infection and disease |
US13/641,315 US8986697B2 (en) | 2010-04-15 | 2011-04-15 | Antibodies for the treatment of Clostridium difficile-associated infection and disease |
CN201180024312.0A CN102947334B (en) | 2010-04-15 | 2011-04-15 | For treating the antibody of clostridium difficile infections relating and disease |
EP11769680.7A EP2558493B1 (en) | 2010-04-15 | 2011-04-15 | Antibodies for the treatment of clostridium difficile-associated infection and disease |
RU2012143837A RU2630663C9 (en) | 2010-04-15 | 2011-04-15 | Antibodies for treatment of infection and diseases associated with clostridium difficile |
BR112012026021-9A BR112012026021B1 (en) | 2010-04-15 | 2011-04-15 | Antibodies, their antigen-binding fragments and their uses, compositions and use thereof, hybridoma cell line, kit, nucleic acid, and expression vector |
KR1020127029208A KR101820987B1 (en) | 2010-04-15 | 2011-04-15 | Antibodies for the treatment of clostridium difficile-associated infection and disease |
JP2013505183A JP5965389B2 (en) | 2010-04-15 | 2011-04-15 | Antibodies for the treatment of Clostridium difficile-related infections and diseases |
AU2011239470A AU2011239470B2 (en) | 2010-04-15 | 2011-04-15 | Antibodies for the treatment of Clostridium difficile-associated infection and disease |
US14/642,342 US20150175681A1 (en) | 2010-04-15 | 2015-03-09 | Antibodies for the treatment of clostridium difficile-associated infection and disease |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US32450310P | 2010-04-15 | 2010-04-15 | |
US61/324,503 | 2010-04-15 | ||
US38166910P | 2010-09-10 | 2010-09-10 | |
US61/381,669 | 2010-09-10 |
Related Child Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US13/641,315 A-371-Of-International US8986697B2 (en) | 2010-04-15 | 2011-04-15 | Antibodies for the treatment of Clostridium difficile-associated infection and disease |
US14/642,342 Continuation US20150175681A1 (en) | 2010-04-15 | 2015-03-09 | Antibodies for the treatment of clostridium difficile-associated infection and disease |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2011130650A2 true WO2011130650A2 (en) | 2011-10-20 |
WO2011130650A3 WO2011130650A3 (en) | 2012-04-12 |
Family
ID=44799348
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2011/032713 WO2011130650A2 (en) | 2010-04-15 | 2011-04-15 | Antibodies for the treatment of clostridium difficile-associated infection and disease |
Country Status (11)
Country | Link |
---|---|
US (2) | US8986697B2 (en) |
EP (1) | EP2558493B1 (en) |
JP (2) | JP5965389B2 (en) |
KR (1) | KR101820987B1 (en) |
CN (1) | CN102947334B (en) |
AU (1) | AU2011239470B2 (en) |
BR (1) | BR112012026021B1 (en) |
CA (1) | CA2795953C (en) |
ES (1) | ES2757675T3 (en) |
MX (1) | MX339253B (en) |
WO (1) | WO2011130650A2 (en) |
Cited By (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102590515A (en) * | 2012-01-13 | 2012-07-18 | 张春华 | Clostridium difficile exotoxin A test kit and monoclonal antibodies therein |
WO2013130981A1 (en) | 2012-03-02 | 2013-09-06 | Regeneron Pharmaceuticals, Inc. | Human antibodies to clostridium difficile toxins |
WO2014061783A1 (en) | 2012-10-19 | 2014-04-24 | 株式会社イーベック | Human antibody specific to toxin produced from clostridium difficile, or antigen-binding fragment thereof |
WO2014176276A1 (en) * | 2013-04-22 | 2014-10-30 | Board Of Regents Of The University Of Oklahoma | Clostridium difficile vaccine and methods of use |
WO2014144292A3 (en) * | 2013-03-15 | 2014-11-13 | Sanofi Pasteur Biologics , Llc | Antibodies against clostridium difficile toxins and methods of using the same |
JP2015500827A (en) * | 2011-12-08 | 2015-01-08 | ノバルティス アーゲー | Clostridium difficile toxin-based vaccine |
JP2015505540A (en) * | 2012-01-23 | 2015-02-23 | サンタリス ファーマシューティカルズ インコーポレイテッド | Sandalwood oil and its use in connection with Clostridium infection |
US8986697B2 (en) | 2010-04-15 | 2015-03-24 | Progenics Pharmaceuticals, Inc. | Antibodies for the treatment of Clostridium difficile-associated infection and disease |
US20150290319A1 (en) * | 2012-11-28 | 2015-10-15 | Cnj Holdings, Inc, | Antibodies against clostridium difficile |
US9315555B2 (en) | 2012-04-04 | 2016-04-19 | The Secretary Of State For Health | Clostridium difficile antigens |
WO2016156474A1 (en) | 2015-03-31 | 2016-10-06 | Vhsquared Limited | Polypeptide comprising an immunoglobulin chain variable domain which binds to clostridium difficile toxin a |
WO2016156475A1 (en) | 2015-03-31 | 2016-10-06 | Vhsquared Limited | Polypeptide comprising an immunoglobulin chain variable domain which binds to clostridium difficile toxin b |
US9505847B2 (en) | 2011-08-22 | 2016-11-29 | Cnj Holdings Inc | Clostridium difficile antibodies |
WO2018148537A1 (en) * | 2017-02-10 | 2018-08-16 | Stc. Unm | Recombinant expression systems and products |
US10709758B2 (en) | 2015-09-03 | 2020-07-14 | The Board Of Regents Of The University Of Oklahoma | Peptide inhibitors of clostridium difficile toxin B (TcdB) toxin |
Families Citing this family (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3167899B1 (en) | 2010-09-03 | 2021-03-24 | Valneva Austria GmbH | Isolated polypeptide of the toxin a and toxin b proteins of c.difficile and uses thereof |
MX2014002769A (en) * | 2011-09-16 | 2014-06-11 | Ucb Pharma Sa | Neutralising antibodies to the major exotoxins tcda and tcdb of clostridium difficile. |
CN107880126B (en) | 2012-12-21 | 2021-03-30 | 西雅图基因公司 | anti-NTB-A antibodies and related compositions and methods |
ES2940827T3 (en) * | 2013-04-19 | 2023-05-11 | Immuron Ltd | Methods and compositions for the treatment and/or prophylaxis of the disease associated with Clostridium difficile |
US10513552B2 (en) | 2014-06-20 | 2019-12-24 | Immunimed Inc. | Use of polyclonal antibodies against clostridium difficile for treatment of inflammatory bowel disease |
EP2957570B1 (en) | 2014-06-20 | 2019-04-17 | Immunimed Inc. | Polyclonal antibodies against clostridium difficile and uses thereof |
JP2018517670A (en) * | 2015-04-15 | 2018-07-05 | メディミューン,エルエルシー | Method for treating Clostridium difficile infection and related diseases |
GB201509326D0 (en) * | 2015-05-29 | 2015-07-15 | Antibio Tx Aps | Novel use |
CN109152789A (en) * | 2016-03-24 | 2019-01-04 | 帕拉特克药品公司 | Method for treating and preventing clostridium difficile infection |
US11185555B2 (en) | 2016-04-11 | 2021-11-30 | Noah James Harrison | Method to kill pathogenic microbes in a patient |
WO2017189964A2 (en) * | 2016-04-29 | 2017-11-02 | Voyager Therapeutics, Inc. | Compositions for the treatment of disease |
US11326182B2 (en) | 2016-04-29 | 2022-05-10 | Voyager Therapeutics, Inc. | Compositions for the treatment of disease |
JP7339262B2 (en) * | 2018-01-12 | 2023-09-05 | アムジェン インコーポレイテッド | PAC1 antibody and uses thereof |
GB201807367D0 (en) * | 2018-05-04 | 2018-06-20 | Univ Newcastle | Biomarker |
RU2694549C1 (en) * | 2018-08-16 | 2019-07-16 | Федеральное государственное автономное учреждение "Национальный медицинский исследовательский центр здоровья детей" Министерства здравоохранения Российской Федерации (ФГАУ "НМИЦ здоровья детей" Минздрава России) | Method of conducting children with hirsperfly disease in perioperative period |
WO2021219786A1 (en) | 2020-04-30 | 2021-11-04 | Gottfried Himmler | Anti-toxin secretory iga2 preparation |
CN111499738B (en) * | 2020-06-03 | 2022-04-05 | 郑州师范学院 | Antibody for resisting clostridium difficile enterotoxin A |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20050287150A1 (en) | 2004-02-06 | 2005-12-29 | University Of Massachusetts | Antibodies against Clostridium difficile toxins and uses thereof |
Family Cites Families (20)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5292668A (en) | 1981-12-21 | 1994-03-08 | Boston Biomedical Research Institute, Inc. | Bispecific antibody determinants |
US4879218A (en) * | 1982-09-13 | 1989-11-07 | Virginia Tech Intellectual Properties, Inc. | Antibody for C.difficile |
US6071517A (en) | 1986-07-07 | 2000-06-06 | Medarex, Inc. | Bispecific heteroantibodies with dual effector functions |
GB8823869D0 (en) | 1988-10-12 | 1988-11-16 | Medical Res Council | Production of antibodies |
US5175384A (en) | 1988-12-05 | 1992-12-29 | Genpharm International | Transgenic mice depleted in mature t-cells and methods for making transgenic mice |
US5530101A (en) | 1988-12-28 | 1996-06-25 | Protein Design Labs, Inc. | Humanized immunoglobulins |
US6150584A (en) | 1990-01-12 | 2000-11-21 | Abgenix, Inc. | Human antibodies derived from immunized xenomice |
US5231003A (en) * | 1990-05-11 | 1993-07-27 | Cambridge Bioscience Corporation | Monoclonal antibodies specific for toxin b of clostridium difficile |
US5545806A (en) | 1990-08-29 | 1996-08-13 | Genpharm International, Inc. | Ransgenic non-human animals for producing heterologous antibodies |
US5932448A (en) | 1991-11-29 | 1999-08-03 | Protein Design Labs., Inc. | Bispecific antibody heterodimers |
EP0690452A3 (en) | 1994-06-28 | 1999-01-07 | Advanced Micro Devices, Inc. | Electrically erasable memory and method of erasure |
US20100081792A1 (en) | 2001-06-28 | 2010-04-01 | Smithkline Beecham Corporation | Ligand |
US7235641B2 (en) | 2003-12-22 | 2007-06-26 | Micromet Ag | Bispecific antibodies |
EA011879B1 (en) | 2004-09-24 | 2009-06-30 | Эмджин Инк. | MODIFIED Fc MOLECULES |
EP1833510A4 (en) | 2004-12-27 | 2010-02-10 | Progenics Pharmaceuticals Neva | Orally deliverable and anti-toxin antibodies and methods for making and using them |
US9963510B2 (en) | 2005-04-15 | 2018-05-08 | Macrogenics, Inc. | Covalent diabodies and uses thereof |
US7612181B2 (en) | 2005-08-19 | 2009-11-03 | Abbott Laboratories | Dual variable domain immunoglobulin and uses thereof |
EP1820513A1 (en) | 2006-02-15 | 2007-08-22 | Trion Pharma Gmbh | Destruction of tumor cells expressing low to medium levels of tumor associated target antigens by trifunctional bispecific antibodies |
EP2033657A1 (en) | 2007-09-04 | 2009-03-11 | Trion Pharma Gmbh | Intraoperative trifunctional antibody application for prophylatic intraperitonal tumour cell dissemination |
US8986697B2 (en) | 2010-04-15 | 2015-03-24 | Progenics Pharmaceuticals, Inc. | Antibodies for the treatment of Clostridium difficile-associated infection and disease |
-
2011
- 2011-04-15 US US13/641,315 patent/US8986697B2/en active Active
- 2011-04-15 MX MX2012011926A patent/MX339253B/en active IP Right Grant
- 2011-04-15 KR KR1020127029208A patent/KR101820987B1/en active IP Right Grant
- 2011-04-15 JP JP2013505183A patent/JP5965389B2/en active Active
- 2011-04-15 AU AU2011239470A patent/AU2011239470B2/en active Active
- 2011-04-15 CN CN201180024312.0A patent/CN102947334B/en active Active
- 2011-04-15 WO PCT/US2011/032713 patent/WO2011130650A2/en active Application Filing
- 2011-04-15 ES ES11769680T patent/ES2757675T3/en active Active
- 2011-04-15 BR BR112012026021-9A patent/BR112012026021B1/en active IP Right Grant
- 2011-04-15 CA CA2795953A patent/CA2795953C/en active Active
- 2011-04-15 EP EP11769680.7A patent/EP2558493B1/en active Active
-
2015
- 2015-03-09 US US14/642,342 patent/US20150175681A1/en not_active Abandoned
-
2016
- 2016-07-01 JP JP2016131227A patent/JP6159854B2/en active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20050287150A1 (en) | 2004-02-06 | 2005-12-29 | University Of Massachusetts | Antibodies against Clostridium difficile toxins and uses thereof |
WO2006121422A2 (en) | 2004-02-06 | 2006-11-16 | University Of Massachusetts | Antibodies against clostridium difficile toxins and uses thereof |
Non-Patent Citations (2)
Title |
---|
"INFECTION AND IMMUNITY", vol. 74, 1 November 2006, AMERICAN SOCIETY FOR MACROBIOLOGY, USA, article "Human monoclonal antibodies directed against toxins A and B prevent Clostridium difficile-induced mortality in hamsters", pages: 6339 - 6347 |
See also references of EP2558493A4 |
Cited By (35)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8986697B2 (en) | 2010-04-15 | 2015-03-24 | Progenics Pharmaceuticals, Inc. | Antibodies for the treatment of Clostridium difficile-associated infection and disease |
US9994630B2 (en) | 2011-08-22 | 2018-06-12 | Cangene Corporation | Clostridium difficile antibodies |
US9505847B2 (en) | 2011-08-22 | 2016-11-29 | Cnj Holdings Inc | Clostridium difficile antibodies |
JP2015500827A (en) * | 2011-12-08 | 2015-01-08 | ノバルティス アーゲー | Clostridium difficile toxin-based vaccine |
CN102590515B (en) * | 2012-01-13 | 2014-07-30 | 张春华 | Clostridium difficile exotoxin A test kit and monoclonal antibodies therein |
CN102590515A (en) * | 2012-01-13 | 2012-07-18 | 张春华 | Clostridium difficile exotoxin A test kit and monoclonal antibodies therein |
JP2015505540A (en) * | 2012-01-23 | 2015-02-23 | サンタリス ファーマシューティカルズ インコーポレイテッド | Sandalwood oil and its use in connection with Clostridium infection |
US10322154B2 (en) | 2012-01-23 | 2019-06-18 | Santalis Pharmaceuticals, Inc. | Sandalwood oil and its uses related to clostridium infections |
US9579354B2 (en) | 2012-01-23 | 2017-02-28 | Santalis Pharmaceuticals, Inc. | Sandalwood oil and its uses related to clostridium infections |
US9399674B2 (en) | 2012-03-02 | 2016-07-26 | Regeneron Pharmaceuticals, Inc. | Human antibodies to Clostridium difficile toxins |
WO2013130981A1 (en) | 2012-03-02 | 2013-09-06 | Regeneron Pharmaceuticals, Inc. | Human antibodies to clostridium difficile toxins |
JP2015509962A (en) * | 2012-03-02 | 2015-04-02 | リジェネロン・ファーマシューティカルズ・インコーポレイテッドRegeneron Pharmaceuticals, Inc. | Human antibody against Clostridium difficile toxin |
CN104203976A (en) * | 2012-03-02 | 2014-12-10 | 瑞泽恩制药公司 | Human antibodies to clostridium difficile toxins |
AU2013203942B2 (en) * | 2012-03-02 | 2016-03-10 | Regeneron Pharmaceuticals, Inc. | Human antibodies to Clostridium difficile toxins |
AU2013203942A8 (en) * | 2012-03-02 | 2016-08-11 | Regeneron Pharmaceuticals, Inc. | Human antibodies to Clostridium difficile toxins |
US9896514B2 (en) | 2012-04-04 | 2018-02-20 | The Secretary Of State For Health | Clostridium difficile antigens |
US9315555B2 (en) | 2012-04-04 | 2016-04-19 | The Secretary Of State For Health | Clostridium difficile antigens |
WO2014061783A1 (en) | 2012-10-19 | 2014-04-24 | 株式会社イーベック | Human antibody specific to toxin produced from clostridium difficile, or antigen-binding fragment thereof |
CN109776676A (en) * | 2012-11-28 | 2019-05-21 | Cnj控股公司 | For the antibody of clostridium difficile |
CN105025926A (en) * | 2012-11-28 | 2015-11-04 | Cnj控股公司 | Antibodies against Clostridium difficile |
EP2925362A4 (en) * | 2012-11-28 | 2016-07-20 | Cnj Holdings Inc | Antibodies against clostridium difficile |
JP2016501877A (en) * | 2012-11-28 | 2016-01-21 | シーエヌジェー ホールディングス,インコーポレイテッド | Antibodies against Clostridium difficile |
US10117933B2 (en) | 2012-11-28 | 2018-11-06 | Emergent Biosolutions Canada Inc. | Antibodies against Clostridium difficile |
US20150290319A1 (en) * | 2012-11-28 | 2015-10-15 | Cnj Holdings, Inc, | Antibodies against clostridium difficile |
AU2013352034B2 (en) * | 2012-11-28 | 2018-08-02 | Emergent Biosolutions Canada Inc. | Antibodies against Clostridium difficile |
US10160797B2 (en) | 2013-03-15 | 2018-12-25 | Sanofi Pasteur Biologics, Llc | Antibodies against Clostridium difficile toxins and methods of using the same |
WO2014144292A3 (en) * | 2013-03-15 | 2014-11-13 | Sanofi Pasteur Biologics , Llc | Antibodies against clostridium difficile toxins and methods of using the same |
US11220537B2 (en) | 2013-03-15 | 2022-01-11 | Sanofi Pasteur Biologics, Llc | Antibodies against Clostridium difficile toxins and methods of using the same |
EP4079760A2 (en) | 2013-03-15 | 2022-10-26 | Sanofi Pasteur Inc. | Antibodies against clostridium difficile toxins and methods of using the same |
WO2014176276A1 (en) * | 2013-04-22 | 2014-10-30 | Board Of Regents Of The University Of Oklahoma | Clostridium difficile vaccine and methods of use |
US10226523B2 (en) | 2013-04-22 | 2019-03-12 | The Board Of Regents Of The University Of Oklahoma | Clostridium difficile vaccine and methods of use |
WO2016156475A1 (en) | 2015-03-31 | 2016-10-06 | Vhsquared Limited | Polypeptide comprising an immunoglobulin chain variable domain which binds to clostridium difficile toxin b |
WO2016156474A1 (en) | 2015-03-31 | 2016-10-06 | Vhsquared Limited | Polypeptide comprising an immunoglobulin chain variable domain which binds to clostridium difficile toxin a |
US10709758B2 (en) | 2015-09-03 | 2020-07-14 | The Board Of Regents Of The University Of Oklahoma | Peptide inhibitors of clostridium difficile toxin B (TcdB) toxin |
WO2018148537A1 (en) * | 2017-02-10 | 2018-08-16 | Stc. Unm | Recombinant expression systems and products |
Also Published As
Publication number | Publication date |
---|---|
US20150175681A1 (en) | 2015-06-25 |
EP2558493B1 (en) | 2019-09-18 |
JP2013523190A (en) | 2013-06-17 |
AU2011239470B2 (en) | 2015-09-10 |
EP2558493A2 (en) | 2013-02-20 |
JP5965389B2 (en) | 2016-08-03 |
KR101820987B1 (en) | 2018-01-22 |
CN102947334A (en) | 2013-02-27 |
US20130202618A1 (en) | 2013-08-08 |
AU2011239470A1 (en) | 2012-11-01 |
KR20130062279A (en) | 2013-06-12 |
MX2012011926A (en) | 2013-01-29 |
CA2795953C (en) | 2021-09-14 |
CN102947334B (en) | 2017-12-26 |
BR112012026021B1 (en) | 2022-05-03 |
EP2558493A4 (en) | 2013-11-27 |
MX339253B (en) | 2016-05-18 |
JP2017012166A (en) | 2017-01-19 |
ES2757675T3 (en) | 2020-04-29 |
CA2795953A1 (en) | 2011-10-20 |
BR112012026021A2 (en) | 2020-04-14 |
WO2011130650A3 (en) | 2012-04-12 |
US8986697B2 (en) | 2015-03-24 |
JP6159854B2 (en) | 2017-07-05 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU2011239470B2 (en) | Antibodies for the treatment of Clostridium difficile-associated infection and disease | |
US9994630B2 (en) | Clostridium difficile antibodies | |
AU2008333985B2 (en) | Antibodies to the PcrV antigen of pseudomonas aeruginosa | |
JP2018029625A (en) | Human antibody to clostridium difficile toxin | |
US20130164308A1 (en) | Staphylococcus aureus specific antibodies and uses thereof | |
KR20160135830A (en) | Anti-influenza b virus hemagglutinin antibodies and methods of use | |
US11970527B2 (en) | Antibody directed against S. aureus clumping factor A (ClfA) | |
AU2013352034A1 (en) | Antibodies against Clostridium difficile | |
RU2630663C2 (en) | Antibodies for treatment of infection and disease associated with clostridium difficile | |
NZ622798B2 (en) | Clostridium difficile antibodies |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
WWE | Wipo information: entry into national phase |
Ref document number: 201180024312.0 Country of ref document: CN |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 11769680 Country of ref document: EP Kind code of ref document: A2 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2011769680 Country of ref document: EP |
|
ENP | Entry into the national phase |
Ref document number: 2795953 Country of ref document: CA |
|
WWE | Wipo information: entry into national phase |
Ref document number: MX/A/2012/011926 Country of ref document: MX |
|
ENP | Entry into the national phase |
Ref document number: 2013505183 Country of ref document: JP Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 8998/DELNP/2012 Country of ref document: IN |
|
ENP | Entry into the national phase |
Ref document number: 2011239470 Country of ref document: AU Date of ref document: 20110415 Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 20127029208 Country of ref document: KR Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 2012143837 Country of ref document: RU Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 13641315 Country of ref document: US |
|
REG | Reference to national code |
Ref country code: BR Ref legal event code: B01A Ref document number: 112012026021 Country of ref document: BR |
|
ENP | Entry into the national phase |
Ref document number: 112012026021 Country of ref document: BR Kind code of ref document: A2 Effective date: 20121010 |