RU2630663C9 - Antibodies for treatment of infection and diseases associated with clostridium difficile - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: isolated antibodies or their antigen-binding fragments, that specifically bind toxin B and/or A of C. difficile, are described. Also, compositions and kits containing such antibodies are provided. Hybridoma cell lines deposited under access numbers ATCC PTA-9693, ATCC PTA-9692, ATCC PTA-9694, ATCC PTA-9888, where hybridoma produces a monoclonal antibody that specifically binds the toxin of C. difficile, are presented.

EFFECT: invention expands the arsenal of tools for inhibition or neutralisation of toxicity caused by said toxin.

50 cl, 42 dwg, 8 tbl, 13 ex

Description

Related Applications

This application claims priority under US Code 35 §119 on provisional patent applications US No. 61/324503, filed April 15, 2010, and 61/381669, filed September 10, 2010, the full contents of each of which is incorporated into this document by using the link.

Technical field

The present invention, in General, relates to compositions and methods of treatment that can be used to treat infections of Clostridium difficile (C. difficile), as well as disease states and pathologies associated with C. difficile, for example, associated with C. diff. diarrhea (CDAD), which may result from infection with C. difficile bacteria. The present invention also relates to antibodies or antigen-binding fragments thereof that specifically bind to epitopes on C. difficile toxin A and / or B toxin, compositions containing such antibodies, and methods for using antibodies or compositions.

State of the art

C. difficile (or C. diff) is a gram-positive spore-forming anaerobic bacterium that represents a leading cause of hospitalized (hospital-acquired) diarrhea associated with the use of antibiotics and pseudomembranous colitis. C. difficile infection is estimated to total more than 750,000 cases per year in the United States and cause more deaths than all other intestinal infections combined (1). In many hospitals, C. difficile poses a greater risk to patients than methicillin-resistant Staphylococcus aureus (MRSA) or any other bacterium (2). The annual costs of organizing the prevention and treatment of Clostridium difficile-associated disease (CDAD) are estimated to exceed $ 3.2 billion in the United States (3). Recent outbreaks of C. difficile strains with increased virulence or antibiotic resistance have led to treatment failure, more frequent relapses, and increased mortality rates (4).

As a rule, CDAD is caused by a violation of the colon microflora due to the use of antibiotics such as clindamycin, cephalosporins and fluoroquinolones (3,8). This violation of the microenvironment of the colon, together with exposure to spores of C. difficile, leads to colonization. About one third of all colonized patients develop CDAD (9), which can lead to severe diarrhea, colon perforation, colectomy, and death (10). CDAD is the result of the acquisition and proliferation of C. difficile in the intestine, where the C. difficile bacterium produces toxin A and toxin B, two important CDAD virulence factors. C. difficile toxins A and B show significant sequence homology and structural homology. Both have a C-terminal receptor binding domain containing multiple repeating sequences, a central hydrophobic domain and an N-terminal glucosyl transferase domain. The receptor binding domain mediates the binding of toxins to intestinal epithelial cells through host recognition receptors that remain poorly defined in humans. After internalization through the endosomal pathway, the central hydrophobic domain is introduced into the endosome membrane. The acidic pH of the endosome triggers the formation of pores and the movement of amino-terminal toxin domains into the cytosol. Glycosylation of target cytosolic Rho OTPases leads to destruction of the cytoskeleton and cell death. Toxins A and B exhibit different pathology profiles with possible synergies in the disease being caused.

Recent outbreaks of hypervirulent C. difficile strains have led to elevated levels of severe illness, more frequent relapses, and increased mortality. One hypervirulent strain, BI / NAP 1/027 toxinotype III, was rare in the past, but is now epidemic. Hypervirulent strains, such as BI / NAP 1/027, produce several times more toxin A and toxin B than non-hypervirulent C. difficile strains, making these strains more difficult to treat after infection. Since the resistance of hypervirulent strains to commonly used antimicrobials and antibiotics is a growing problem that makes these strains more difficult to treat and contain, additional treatment approaches and stronger therapeutic agents are needed to combat the hypervirulence and relapse associated with C.difficile hypervirulent isolates diseases.

Current antibiotic treatments for C. difficile infections include the use of vancomycin and / or metronidazole; however, these antibiotics are limited by incomplete response levels and increased rates of reinfection and relapse. Significantly higher failure rates have been reported for metronidazole therapy since 2000 (23-25). High relapse rates after antibiotic treatment can occur as a result of prolonged disturbance of the normal microflora of the colon, which allows C. difficile to gain an advantage with little competition (26-28). The risk of relapse is increased in patients who have already had one relapse, increasing from about 20% after the first episode to more than 60% after two or more relapses (29, 30). This increased risk of relapse was associated with the inability to provide an appropriate humoral immune response with antibodies to the toxin (31). Indeed, patients with the highest titers of serum IgG to toxin at the end of antibiotic therapy had a reduced risk of relapse (32). In a separate study, serum toxin B antibody levels correlated with protection against recurrent CDAD (33).

The prevalence of C. difficile infection has steadily increased, especially in the elderly, who are often weakened. About a third of patients with C. difficile infection have relapses of their infection, usually within two months of the initial illness. Re-infections tend to be more severe than the initial disease; they are more often fatal. Older adults and people with weakened immune systems are particularly susceptible to recurrent infections. In the absence of immediate and correct treatment for complications of C. difficile infection, dehydration, renal failure, intestinal perforation, and toxic megacolon can result in rupture of the colon and death.

Although C. difficile infection is the most common infection acquired by hospitalized patients in the United States, it can also be acquired outside hospitals in the community. It is estimated that 20,000 C. difficile infections are detected in society in the United States every year. On an international scale, the incidence is highly variable and depends on many factors, including the frequency with which endoscopy is used to establish a diagnosis, the antimicrobial regimen and epidemiological features.

Thus, it is obvious that the disease caused by C. difficile infection puts the lives of people of all ages at risk, both in the hospital and in society. In the modern world, there is always a risk of infection with C. difficile for those who are faced with hospitalization, or for those who are on long-term inpatient treatment. Since there is a possibility of infection with C. difficile infection outside the hospital environment, the likelihood of infection by young children and infants is high. Additionally, there is the possibility that the antibiotic regimens currently used to treat C. difficile may be less optimal. Patients who suffer from C. difficile-associated disease require serious inpatient treatment and extended hospital stays. The costs associated with a high level of supportive care in a hospital setting and treatment that is necessary for patients with C. difficile-associated disease are high and require expensive resources, such as large numbers of doctors and nurses, laboratory tests and monitoring, concomitant medication therapy and additional supportive measures.

Therefore, there is a need for more effective drugs, drugs and therapeutic agents that target life-threatening diseases caused by C. difficile, and in particular the potent toxins produced by C. difficile, for the benefit of prevention and treatment. There is an unmet medical need for successful and sustainable treatments for C. difficile-associated diseases that provide a lower likelihood of developing resistance and a higher likelihood of a successful patient response and elimination of the disease leading to the eradication of the disease.

SUMMARY OF THE INVENTION

The present invention provides, at least in part, new antibody-based reagents and compositions comprising antibodies to C. difficile Toxin A and / or Toxin B. The present reagents and compositions may be useful in treating an increasing number of subjects affected by C. difficile-associated infection and disease, to provide improved quality of life, to eliminate CDAD and C. difficile infection, and to facilitate the survival of infected individuals.

In one aspect, an isolated antibody or antigen-binding fragment thereof is provided that specifically binds C. difficile toxin A and which cross-competes for binding to C. difficile toxin A with a monoclonal antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9692, PTA -9694 or PTA-9888. In an embodiment, the hybridoma cell line is deposited under accession number ATCC PTA-9692. In an embodiment, the hybridoma cell line is deposited under accession number ATCC PTA-9694. In an embodiment, the hybridoma cell line is deposited under accession number ATCC PTA-9888. In an embodiment, the monoclonal antibody or antigen binding fragment thereof is in a chimeric or humanized form.

In another aspect, an isolated antibody or antigen-binding fragment thereof is provided that specifically binds to an epitope of C. difficile toxin A defined by a monoclonal antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9692, PTA-9694 or PTA-9888. In an embodiment, the hybridoma cell line is deposited under accession number ATCC PTA-9692. In an embodiment, the hybridoma cell line is deposited under accession number ATCC PTA-9694. In an embodiment, the hybridoma cell line is deposited under accession number ATCC PTA-9888. In an embodiment, the monoclonal antibody or antigen binding fragment thereof is in a chimeric or humanized form.

In another aspect, there is provided an isolated antibody or antigen binding fragment thereof that specifically binds to C. difficile Toxin B and which cross-competes for binding to C. difficile Toxin B with a monoclonal antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9693 or PTA-9692. In an embodiment, the hybridoma cell line is deposited under accession number ATCC PTA-9693. In an embodiment, the hybridoma cell line is deposited under accession number ATCC PTA-9692. In an embodiment, the monoclonal antibody or antigen binding fragment thereof is in a chimeric or humanized form.

In another aspect, an isolated antibody or antigen-binding fragment thereof is provided that specifically binds to an epitope of the C. difficile Toxin B determined by a monoclonal antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9693 or PTA-9692. In an embodiment, the hybridoma cell line is deposited under accession number ATCC PTA-9693. In an embodiment, the hybridoma cell line is deposited under accession number ATCC PTA-9692. In an embodiment, the monoclonal antibody or antigen binding fragment thereof is in a chimeric or humanized form. In an embodiment, the isolated antibody or antigen binding fragment thereof neutralizes the in vivo toxicity of C. difficile Toxin B. In an embodiment, the antibody or antigen binding fragment thereof neutralizes the in vivo toxicity of C. difficile Toxin B in an amount of from 0.1 to 1000 μg.

In another aspect, a RA-39 monoclonal antibody (accession number ATCC 9692) or an antigen-binding fragment thereof is provided. In another aspect, a RA-50 monoclonal antibody (accession number ATCC PTA-9694) or an antigen binding fragment thereof is provided. In another aspect, a RA-38 monoclonal antibody (accession number ATCC PTA-9888) or an antigen binding fragment thereof is provided. In another aspect, a RA-41 monoclonal antibody (accession number ATCC PTA-9693) or an antigen-binding fragment thereof is provided. In an embodiment, the monoclonal antibody or antigen binding fragment thereof is in a chimeric or humanized form.

In yet another aspect, an expression vector is provided comprising at least one nucleic acid molecule encoding antibodies or antigen binding fragments thereof, as described above and herein. In yet another aspect, an expression vector is provided comprising a nucleic acid molecule encoding a heavy chain or part of an antibody or antigen binding fragments thereof, as described above or herein. In yet another aspect, an expression vector is provided comprising a nucleic acid molecule encoding a light chain or part of an antibody or antigen binding fragments thereof, as described above or herein. In yet another aspect, an expression vector is provided comprising at least one nucleic acid molecule encoding a heavy chain or part thereof and a light chain or part of antibodies or antigen binding fragments thereof, as described above or herein.

In another aspect, host cells are transformed or transfected with any of the expression vectors described above and herein. In another aspect, a plasmid is provided that encodes any of the antibodies or antigen binding fragments thereof, as described above and herein.

In another aspect, an isolated C. difficile toxin A antibody or antigen binding fragment is provided, as described above and herein, wherein the antibody or antigen binding fragment neutralizes the in vivo toxicity of C. difficile toxin A. In an embodiment, the antibody or antigen binding fragment neutralizes the in vivo toxicity of C. difficile Toxin A in an amount of 0.1 μg to 1000 μg or 1 μg / kg to 100000 μg / kg. In another embodiment, the isolated antibody or antigen binding fragment neutralizes the in vivo toxicity of C. difficile Toxin A in an amount selected from 0.5 μg to 1000 μg, or 5 μg to 250 μg, or 10 mg / kg to 50 mg / kg In an embodiment, the antibody is a RA-39 monoclonal antibody (Access No. ATCC 9692) or an antigen-binding fragment thereof. In an embodiment, the antibody is a RA-50 monoclonal antibody (access no. ATCC PTA-9694) or an antigen binding fragment thereof. In an embodiment, the antibody is a RA-38 monoclonal antibody (access no. ATCC PTA-9888) or an antigen binding fragment thereof. In an embodiment, the monoclonal antibody or antigen binding fragment thereof is in a chimeric or humanized form.

In another aspect, an isolated C. difficile toxin B antibody or antigen binding fragment is provided, as described above and herein, wherein the antibody or antigen binding fragment neutralizes the in vivo toxicity of C. difficile B toxin. In this embodiment, the isolated antibody or antigen binding fragment thereof neutralizes the in vivo toxicity of C. difficile Toxin B in an amount selected from 0.5 μg to 1000 μg, 0.5 μg, 5 μg, 40 μg, 50 μg, 100 μg, 200 mcg, 1000 mcg or 10 mg / kg to 50 mg / kg. In an embodiment, the antibody is a RA-39 monoclonal antibody (Access No. ATCC 9692) or an antigen-binding fragment thereof. In an embodiment, the antibody is a RA-41 monoclonal antibody (access no. ATCC PTA-9693) or an antigen binding fragment thereof. In an embodiment, the monoclonal antibody or antigen binding fragment thereof is in a chimeric or humanized form.

In another aspect, an isolated C. difficile toxin A antibody or antigen binding fragment is provided as described above and herein, wherein the antibody or antigen binding fragment is administered to a subject infected with C. difficile in combination with an isolated antibody or antigen binding fragment that is specific binds and / or neutralizes C. difficile Toxin B, treats CDAD and / or enhances subject survival. In an embodiment, antibodies to toxin A and toxin B or fragments thereof are administered simultaneously. In an embodiment, antibodies to toxin A and toxin B or fragments thereof are administered at different points in time. In an embodiment, antibodies to toxin A and toxin B or fragments thereof are administered sequentially. In an embodiment, an isolated antibody or antigen binding fragment that specifically binds C. difficile toxin A is an antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9692, PTA-9694 or PTA-9888, its antigen binding fragment, its humanized form or a monoclonal antibody that cross-reacts with it to bind toxin A. In an embodiment, an isolated antibody or antigen-binding fragment that specifically binds C. difficile pre-toxin B ulation an antibody produced by hybridoma cell line deposited under the access № ATCC PTA-9693 or PTA-9692, antigen binding fragment, or its humanized form monoclonal antibody that cross reacts with them when bound toxin B.

In another aspect, an isolated C. difficile toxin B antibody or antigen binding fragment is provided, as described above and herein, wherein the antibody or antigen binding fragment is administered to a subject infected with C. difficile in combination with an isolated antibody or antigen binding fragment which is specific binds and / or neutralizes C. difficile toxin A, treats CDAD and / or enhances subject survival. In an embodiment, antibodies to toxin A and toxin B or fragments thereof are administered simultaneously. In an embodiment, antibodies to toxin A and toxin B or fragments thereof are administered at different points in time. In an embodiment, antibodies to toxin A and toxin B or fragments thereof are administered sequentially. In an embodiment, an isolated antibody or antigen binding fragment that specifically binds C. difficile toxin A is an antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9692, PTA-9694 or PTA-9888, its antigen binding fragment, its humanized form or a monoclonal antibody that crosshairs react with upon binding of toxin A. In an embodiment, an isolated antibody or antigen binding fragment that specifically binds toxin B of C. difficile pre ulation an antibody produced by hybridoma cell line deposited under the access № ATCC PTA-9693 or PTA-9692, antigen binding fragment, or its humanized form monoclonal antibody that cross reacts with them when bound toxin B.

In another aspect, an isolated C. difficile toxin A antibody or antigen binding fragment is provided as described above and herein, wherein the antibody or antigen binding fragment is administered to a subject infected with C. difficile in combination with an isolated antibody or antigen binding fragment that is specific binds C. difficile Toxin B, treats CDAD and / or enhances subject survival. In an embodiment, the anti-toxin A antibody or antigen binding fragment thereof is administered in an amount of 1 μg to 1000 μg, or 1 μg to 250 μg, or 5 μg to 100 μg, and a dose of the anti-toxin B antibody or antigen binding fragment thereof is administered in an amount from 0.1 μg to 1000 μg, or from 1 μg to 250 μg, or from 5 μg to 100 μg. In an embodiment, the isolated antibody or antigen binding fragment that specifically binds C. difficile toxin A is an antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9692, PTA-9694 or PTA-9888, its antigen binding fragment, and its humanization a form or monoclonal antibody that cross-reacts with it to bind toxin A. In an embodiment, an isolated antibody or antigen binding fragment that specifically binds toxin B of C. difficile pre is an antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9693 or PTA-9692, its antigen-binding fragment, its humanized form or monoclonal antibody that cross-reacts with it when toxin B is bound.

In another aspect, an isolated C. difficile toxin A antibody or antigen binding fragment is provided as described above and herein, wherein the antibody or antigen binding fragment is administered to a subject infected with C. difficile in combination with an isolated antibody or antigen binding fragment that is specific binds C. difficile Toxin B, treats CDAD and / or enhances subject survival. In an embodiment, an anti-toxin A antibody or antigen binding fragment thereof is administered in an amount of 50 mg / kg, an anti-toxin B antibody or antigen binding fragment thereof is administered in an amount of 50 mg / kg. In an embodiment, the isolated antibody or antigen binding fragment that specifically binds C. difficile toxin A is an antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9692, PTA-9694 or PTA-9888, its antigen binding fragment, and its humanization a form or monoclonal antibody that cross-reacts with it to bind toxin A. In an embodiment, an isolated antibody or antigen binding fragment that specifically binds C. difficile Toxin B, pre nent an antibody produced by hybridoma cell line deposited under the access № ATCC PTA-9693 or PTA-9692, antigen binding fragment, or its humanized form monoclonal antibody that cross reacts with them when bound toxin B.

In another aspect, an isolated C. difficile toxin A antibody or antigen binding fragment is provided as described above and herein, wherein the antibody or antigen binding fragment is administered to a subject infected with C. difficile in combination with an isolated antibody or antigen binding fragment that is specific binds toxin B C. difficile, provides a cure rate or survival rate of 50%, 60%, 70%, 80%, 90% or 100%. In an embodiment, the antibody or antigen binding fragment is administered q2d × 4 at a dose of 40-50 mg / kg. In an embodiment, the isolated antibody or antigen binding fragment that specifically binds C. difficile toxin A is an antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9692, PTA-9694 or PTA-9888, its antigen binding fragment, and its humanization a form or monoclonal antibody that cross-competes for the binding of toxin A with one or more monoclonal antibodies deposited under accession number ATCC PTA-9692, PTA-9694 or PTA-9888. In an embodiment, the isolated antibody or antigen binding fragment that specifically binds C. difficile Toxin B is an antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9693 or PTA-9692, its antigen binding fragment, its humanized form, or monoclonal which cross-competes for the binding of toxin B with one or more monoclonal antibodies deposited under accession number ATCC PTA-9692 or PTA-9693.

In another aspect, an isolated C. difficile toxin A antibody, or C. difficile toxin B antibody, or an antigen binding fragment thereof, as described herein, is provided, wherein the antibody or antigen binding fragment is in the form or derived from one or more of a monoclonal antibody , a humanized antibody, a human antibody, or a chimeric antibody.

In another aspect, an isolated C. difficile toxin A antibody or C. difficile toxin B antibody or antigen binding fragment thereof, as described herein, is provided, wherein the antibody or antigen binding fragment thereof is in the form or derived from a bispecific and bifunctional antibody.

In another aspect, a bispecific antibody or antigen binding fragment thereof is provided which comprises (i) a monoclonal antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9692, PTA-9694 or PTA-9888, an antigen binding fragment thereof, humanized antibody an antigen binding fragment, an antibody heavy chain variable domain or an antigen binding fragment thereof and / or an antibody light chain variable domain or an antigen binding fragment; and (ii) a monoclonal antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9693 or PTA-9692, an antigen binding fragment thereof, a humanized variant of an antibody or antigen binding fragment thereof, a heavy chain variable domain of an antibody or antigen binding or variable binding fragment thereof light chain domain of an antibody or antigen binding fragment thereof.

In another aspect, a bispecific antibody or antigen binding fragment thereof is provided, wherein the antibody contains (1) a monoclonal antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9692, its antigen binding fragment, a humanized variant of the antibody, or its antigen binding domain fragment an antibody or antigen binding fragment thereof and / or a light chain variable domain of an antibody or antigen binding fragment; and (ii) an isolated monoclonal antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9693 or PTA-9692, an antigen binding fragment thereof, a humanized variant of an antibody or antigen binding fragment thereof, an antibody heavy chain variable domain or an antigen binding or variable binding domain or antigen binding fragment light chain domain of an antibody or antigen binding fragment thereof.

In various embodiments, an antibody or antigen binding fragment thereof is provided as described above and herein, wherein the antigen binding fragment is selected from a Fab fragment, a P (ab ') ₂ fragment, and an Fv fragment. In another aspect, an isolated antibody or antigen binding fragment thereof is provided as described above and herein, wherein the antibody or antigen binding fragment thereof is or comprises a single chain antibody.

In another aspect, a composition is provided comprising one or more antibodies or antigen binding fragments thereof according to the present invention, as described above and herein, and a pharmaceutically acceptable carrier, excipient, medium or diluent. In an embodiment, the composition comprises at least one anti-toxin A antibody of the present 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 antibody In the present invention, for example, mAb PTA-9692, mAb PTA-9693, an antigen binding fragment thereof or a humanized form thereof. In an embodiment, the composition comprises one anti-toxin A antibody of the present invention, for example, a PTA-9888 mAb, its antigen binding fragment or its humanized form, and one anti-toxin B antibody of the present invention, for example mAb 9693, its antigen binding fragment or its humanized form. In an embodiment, the composition comprises one anti-toxin A antibody of the present invention, for example, the PTA-9694 mAb, its antigen binding fragment or its humanized form, and one anti-toxin B antibody of the present invention, for example the mAb 9693, its antigen binding fragment or its humanized form. In an embodiment, each mAb is present in the composition in the same amount. In an embodiment, 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 varying amounts. In an embodiment, each mAb is present in the composition in ratios other than 1: 1 by weight relative to each other, the ratios being as provided herein. In an embodiment, the composition further comprises an additional therapeutic agent. In an embodiment, the additional therapeutic agent is an antibiotic, antibacterial, bactericidal, bacteriostatic agent, or a combination thereof. In an embodiment, the additional therapeutic agent is metronidazole, vancomycin, fidaxomycin, or a combination thereof.

In another aspect, there is provided a composition comprising an expression vector according to the present invention, as described above and herein, and a pharmaceutically acceptable carrier, excipient, medium or diluent. In another aspect, a composition is provided comprising host cells carrying an expression vector of the present invention as described above and herein, and a pharmaceutically acceptable carrier, excipient, medium or diluent. In another aspect, a composition is provided comprising a plasmid according to the present invention, as described above and herein, and a pharmaceutically acceptable carrier, excipient, medium or diluent.

In another aspect, a binding protein is provided comprising at least two polypeptide chains containing binding sites for the binding of C. difficile toxin A and toxin B, wherein at least one polypeptide chain comprises a first variable domain of a heavy chain, a second variable domain of a heavy chain, and a constant heavy chain domain or part thereof; and at least one polypeptide chain contains a first variable domain of the light chain, a second variable domain of the light chain and a constant domain of the light chain or part thereof, where the variable domains containing the polypeptide chains form functional binding sites for toxin A and C. difficile B toxin. In an embodiment, the first variable domain of the heavy chain and the first variable domain of the light chain of the binding protein form a functional binding site for toxin A C. difficile, and the second variable domain of the heavy chain and the second variable domain of the light chain of the binding protein form a functional binding site for toxin B C. difficile In an embodiment, the first variable domain of the heavy chain and the first variable domain of the light chain of the binding protein form a functional binding site for the C. difficile bacterium, and the second variable domain of the heavy chain and the second variable domain of the light chain of the binding protein form a functional binding site for toxin A C . difficile. In an embodiment, the binding protein comprises an Fc region. In an embodiment, the binding protein neutralizes the toxicity of C. difficile Toxin A and Toxin B. In various embodiments, the binding protein has an association rate constant (Kon) with toxin A or toxin B selected from at least 10 ² M ⁻¹ s ⁻¹ ; at least 10 ³ M ^-1 s ^-1 ; at least 10 ⁴ M ^-1 s ^-1 ; at least 10 ⁵ M ^-1 s ^-1 ; at least 10 ⁶ M ^-1 s ^-1 ; or at least 10 ⁷ M ^-1 s ^-1 , as measured by surface plasma resonance. In various embodiments, the binding protein has a dissociation rate constant (Koff) with toxin A or toxin B, selected from not more than 10 ⁻³ s ⁻¹ ; not more than 10 ^-4 s ^-1 ; not more than 10 ^-5 s ^-1 ; or not more than 10 ⁻⁶ s ⁻¹ , as measured by surface plasma resonance. In various embodiments, the binder protein has a dissociation constant (K _D ) with toxin A or toxin B, selected from not more than 10 ⁻⁷ M; no more than 10 ^-8 M; no more than 10 ^-9 M; no more than 10 ^-10 M; no more than 10 ^-11 M; no more than 10 ^-12 M; or no more than 10 ^-13 M.

In another aspect, there is provided a composition comprising a binding protein, as described above and herein, a pharmaceutically acceptable carrier, excipient, medium or diluent. In an embodiment, the composition further comprises an additional therapeutic agent. In an embodiment, the additional therapeutic agent of the composition is an antibiotic, antibacterial, bactericidal, bacteriostatic agent, or a combination thereof. In an embodiment, the additional therapeutic agent of the composition is metronidazole, vancomycin, fidaxomycin, nitazoxanide, rifaximin, ramoplanin, or a combination thereof.

In another aspect, hybridoma cell lines are deposited under accession number ATCC PTA-9692, PTA-9693, PTA-9494 or PTA-9888.

In another aspect, there is provided a method of treating a subject with a C. difficile infection or a C. difficile associated disease, comprising administering to the subject at least one composition as described herein. In an embodiment, the compositions comprise one or more antibodies of the present invention, preferably in a humanized form. In an embodiment, the compositions comprise at least one anti-toxin A antibody provided herein in a humanized form or an antigen binding fragment thereof and at least one anti-toxin B antibody according to the present invention in a humanized form or an antigen binding fragment thereof. In various embodiments, one or more therapeutic reagents, drugs, compounds, or ingredients may be included in the compositions. In an embodiment, the compositions further comprise a pharmaceutically acceptable carrier, diluent, medium or excipient. In an embodiment, the compositions are administered in an amount effective to treat C. difficile infection or a C. difficile associated disease, for example C. difficile associated diarrhea (CDAD). In an embodiment, the two compositions are administered to a subject in an amount effective to treat a C. difficile infection or a C. difficile associated disease. In an embodiment, two compositions are administered simultaneously. In another embodiment, the two compositions are administered at different points in time.

In another aspect, there is provided a method for suppressing or neutralizing the toxicity of C. difficile Toxin A and Toxin B against a cell, which comprises exposing the cell to an effective inhibiting or neutralizing C. difficile Toxin A dose of a monoclonal anti-Toxin A antibody of the present invention or an antigen binding fragment thereof and an effective suppressing or neutralizing toxin B C. difficile dose of a monoclonal antibody to toxin B according to the present invention or its antigen-binding fragment. In an embodiment, the anti-toxin A antibody and the anti-toxin B antibody are in humanized form. In an embodiment, the anti-toxin A antibody and the anti-toxin B antibody are in chimeric form. In an embodiment, antibodies or antigen binding fragments thereof are administered simultaneously. In an embodiment, antibodies or antigen binding fragments thereof are administered at different points in time. In an embodiment of the method, a cell is present in the subject, and the antibodies or antigen binding fragments thereof are administered to the subject in an amount effective to suppress or neutralize C. difficile Toxin A and Toxin B.

In another aspect, there is provided a method of suppressing and neutralizing the toxicity of a C. difficile toxin in relation to a cell, which comprises exposing the cell to an effective inhibiting or neutralizing C. difficile toxin dose of at least one of the compositions of the present invention as described herein. In an embodiment of the method, the cell is exposed to an effective inhibitory or neutralizing C. difficile toxin dose of two compositions, one of which contains an anti-toxin A antibody or antigen-binding fragment thereof, and the other contains an anti-toxin B antibody or antigen-binding fragment thereof. In an embodiment, the antibodies are humanized. In an embodiment, the antibodies are chimeric. In embodiments, the two compositions are administered simultaneously or at different points in time. In an embodiment, the cell is present in the subject, and at least one composition is administered to the subject in an amount effective to suppress or neutralize the C. difficile toxin.

In another aspect, there is provided a method for suppressing and neutralizing toxins produced by a hypervirulent C. difficile strain, which comprises administering to a subject in need thereof (i) an antibody or antigen binding fragment thereof according to the present invention, wherein the antibody binds and neutralizes C. difficile toxin A, and (ii) an antibody or antigen-binding fragment thereof according to the present invention, wherein the antibody binds and neutralizes C. difficile Toxin B, in an amount effective to neutralize toxins produced by the hypervirulent strain. In an embodiment, antibodies (i) and (ii) are humanized antibodies. In an embodiment, antibodies (i) and (ii) are chimeric antibodies. In embodiments, antibodies or antigen binding fragments thereof are administered simultaneously or at different points in time. In an embodiment, the toxins of the hypervirulent strain are Toxin A and Toxin B. In an embodiment, the hypervirulent C. difficile strain is one or more of BI / NAP1 / 027, CCL676, HMC553, Pitt45, CD196, montreal 5 or montreal 7.1. In an embodiment, the anti-toxin A antibody or antigen binding fragment thereof has a neutralizing activity against the toxin A produced by the hypervirulent C. difficile strains, as determined by an EC ₅₀ value of from 2.6 ^-12 M to 7.7 ^-11 M or from 7, 7 ^-12 M to 4.8 ^-8 M. In an embodiment, the anti-toxin B antibody or antigen binding fragment thereof has a neutralizing activity against the toxin B produced by the hypervirulent C. difficile strains, as determined by an EC ₅₀ value of from 1.1 ^-11 M to ^6.5-10 M.

In another aspect, a kit is provided comprising an antibody or antigen binding fragment thereof according to the present invention, and as described herein, especially in a humanized form, and instructions for use. In an embodiment, antibodies or antigen binding fragments are contained in the same container in the kit. In an embodiment, antibodies or antigen binding fragments are contained in separate containers in a kit. In an embodiment, the kit comprises a linker for conjugating antibodies or antigen binding fragments thereof. In an embodiment, the kit comprises an additional therapeutic agent, which may be an antibiotic, antibacterial, bactericidal or bacteriostatic agent. In an embodiment, the additional therapeutic agent is metronidazole, vancomycin, fidaxomycin, nitazoxanide, rifaximin, ramoplanin, or a combination thereof.

In another aspect, a monoclonal antibody or antigen binding fragment thereof is provided, especially in a humanized form, which neutralizes toxin A or toxin B of the hypervirulent C. difficile strain. In an embodiment, the monoclonal antibody is identified by an ATCC accession number PTA-9692, PTA-9694, PTA-9888 or PTA-9693 and is produced by a hybridoma cell line deposited under accession number ATCC PTA-9692, PTA-9694, PTA-9888 or PTA-9693 , respectively. In an embodiment, an antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9692, PTA-9694, PTA-9888, PTA-9693 or PTA-9692 was humanized or in chimeric form.

In an embodiment, the hypervirulent C. difficile strain is one or more of BI / NAP1 / 027, CCL676, HMS553, Pitt45, CD196, montreal 5 and montreal 7.1.

In another aspect, there is provided a method of treating a subject who is symptom-free, but which is susceptible to or at risk of contracting C. difficile infection, which method comprises administering to the subject (i) an antibody to C. difficile toxin A or an antigen-binding fragment thereof, given and described herein, and (ii) antibodies to C. difficile Toxin B, or an antigen binding fragment thereof, shown and described herein, in an amount effective to treat a subject. In an embodiment of the method, the subject is hospitalized.

In another aspect, there is provided a humanized monoclonal antibody produced against C. difficile toxin A. In an embodiment, such a C. difficile toxin A antibody consists of two heavy chain polypeptides, where each heavy chain contains a VH region containing the amino acid sequence as set forth in SEQ ID NO: 1, and a human CH region, and two light chains where each light chain contains a VL region containing the amino acid sequence as set forth in SEQ ID NO: 3, and a human CL region. In an embodiment, such a C. difficile toxin A antibody consists of two heavy chain polypeptides, where each heavy chain contains a VH region containing the amino acid sequence as set forth in SEQ ID N0: 2, and a human CH region, and two lung polypeptides chains, where each light chain contains a VL region containing the amino acid sequence as set forth in SEQ ID NO: 3, and a human CL region. In an embodiment, such a C. difficile toxin A antibody consists of two heavy chain polypeptides, where each heavy chain contains a VH region containing the amino acid sequence as set forth in SEQ ID NO: 1, and a human CH region, and two lung polypeptides chains, where each light chain contains a VL region containing the amino acid sequence as set forth in SEQ ID NO: 4, and a human CL region. In an embodiment, such a C. difficile toxin A antibody consists of two heavy chain polypeptides, where each heavy chain contains a VH region containing the amino acid sequence as set forth in SEQ ID NO: 2, and a human CH region, and two lung polypeptides chains, where each light chain contains a VL region containing the amino acid sequence as set forth in SEQ ID NO: 4, and a human CL region. In an embodiment, such a C. difficile toxin A antibody consists of two heavy chain polypeptides, where each heavy chain contains a VH region containing the amino acid sequence as set forth in SEQ ID NO: 5, and a human CH region, and two lung polypeptides chains, where each light chain contains a VL region containing the amino acid sequence as set forth in SEQ ID NO: 7, and a human CL region. In an embodiment, such a C. difficile toxin A antibody consists of two heavy chain polypeptides, where each heavy chain contains a VH region containing the amino acid sequence as set forth in SEQ ID NO: 6, and a human CH region, and two lung polypeptides chains, where each light chain contains a VL region containing the amino acid sequence as set forth in SEQ ID NO: 7, and a human CL region.

In another aspect, a humanized monoclonal antibody to toxin B of C. difficile is provided. In an embodiment, such an antibody to C. difficile Toxin B consists of two heavy chain polypeptides, where each heavy chain contains a VH region containing the amino acid sequence as set forth in SEQ ID NO: 8, and a human CH region, and two lung polypeptides chains, where each light chain contains a VL region containing the amino acid sequence as set forth in SEQ ID NO: 10, and a human CL region. In an embodiment, such an anti-Toxin B antibody C. difficile consists of two heavy chain polypeptides, where each heavy chain contains a VH region containing the amino acid sequence as set forth in SEQ ID NO: 9, and a human CH region, and two light chains where each light chain contains a VL region containing the amino acid sequence as set forth in SEQ ID NO: 10, and a human CL region.

In another aspect, there is provided a monoclonal antibody or fragment thereof obtained against C. difficile toxin A, wherein the antibody consists of two heavy chain polypeptides, each heavy chain comprising a human VH region and a CH region, and two light chain polypeptides, each light the chain contains the VL region and the CL region of a person. The nucleic acid (or cDNA) sequence 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).

In another aspect, there is provided a monoclonal antibody or fragment thereof obtained against C. difficile toxin A, wherein the antibody consists of two heavy chain polypeptides, each heavy chain comprising a human VH region and a CH region, and two light chain polypeptides, each light the chain contains the VL region and the CL region of a person. The nucleic acid (or cDNA) sequence 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).

In another aspect, there is provided a monoclonal antibody or fragment thereof obtained against C. difficile Toxin B, wherein the antibody consists of two heavy chain polypeptides, each heavy chain comprising a human VH region and a CH region, and two light chain polypeptides, each light the chain contains the VL region and the CL region of a person. The nucleic acid (or cDNA) sequence 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. 40A).

In various embodiments directed to any of the aforementioned humanized monoclonal antibodies of the present invention, the CH region of the monoclonal antibody is selected from IgGI, IgG2a, IgG2b, IgG3, IgG4, IgA, IgE or IgM. In an embodiment, the CH region is IgGI. In an embodiment, the CL region is selected from isotype k or λ. In an embodiment, the CL region is an isotype of k. In other embodiments, a CDR, i.e. CDR1, CDR2 and / or CDR3 of humanized antibodies or antigen-binding fragments thereof, as described herein, are included to bind and / or neutralize C. difficile Toxin A and / or Toxin B in the products and methods of the present invention.

In another aspect, an anti-C. difficile toxin A antibody or fragment thereof is provided, wherein the V region of the L chain contains a sequence selected from one or more of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 7. Also provided is an antibody to C. difficile Toxin B or a fragment thereof, wherein the L chain V region contains a sequence as set forth in SEQ ID NO: 10. An anti-C. difficile toxin A antibody or fragment thereof is also provided, wherein the H chain V region contains a sequence selected from one or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 5 : 6. An anti-C. C. difficile toxin B antibody or fragment thereof is also provided, wherein the H chain V region contains a sequence selected from one or more of SEQ ID NO: 8 or SEQ ID NO: 9.

In another aspect, an isolated antibody or antigen-binding fragment thereof is provided that (i) specifically binds C. difficile toxin A and which cross-competes for binding to C. difficile toxin A with a monoclonal antibody produced by a hybridoma cell line deposited under accession number ATCC PTA- 9692, or which (ii) specifically binds to an epitope of C. difficile toxin A, defined by a monoclonal antibody produced by a hybridoma cell line, deposited under accession number ATCC PTA-9692, where the epitope is determined by a monocl a single antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9692 contains an area outside the receptor binding domain, for example, a translocation domain of C. difficile toxin A. In an embodiment, the antibody is in humanized form. In an embodiment, the antibody is in chimeric form.

In another aspect, an isolated antibody or antigen-binding fragment thereof is provided that (i) specifically binds C. difficile toxin A and which cross-competes for binding to C. difficile toxin A with a monoclonal antibody produced by a hybridoma cell line deposited under accession number ATCC PTA- 9694, or which (ii) specifically binds to an epitope of C. difficile toxin A, determined by a monoclonal antibody produced by a hybridoma cell line, deposited under accession number ATCC PTA-9694, where the epitope is determined by a monocl a single antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9694 contains at least two sites in the receptor binding domain, for example C-terminal receptor binding epitopes, C. difficile toxin A. In an embodiment, the antibody is in humanized form. In an embodiment, the antibody is in chimeric form.

In another aspect, an isolated antibody or antigen-binding fragment thereof is provided that (i) specifically binds C. difficile toxin A and which cross-hair competes for binding to C. difficile toxin A with a monoclonal antibody produced by a hybridoma cell line deposited under accession number ATCC PTA- 9888, or which (ii) specifically binds to an epitope of C. difficile toxin A, defined by a monoclonal antibody produced by a hybridoma cell line, deposited under accession number ATCC PTA-9888, where the epitope determined by the monocle a single antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9888 contains C-terminal receptor-binding epitopes of toxin A. C. difficile. In an embodiment, the antibody is in humanized form. In an embodiment, the antibody is in chimeric form.

In another aspect, an isolated antibody or antigen-binding fragment thereof is provided that (i) specifically binds C. difficile Toxin B and crosshairs competes for binding to C. difficile Toxin B with a monoclonal antibody produced by a hybridoma cell line deposited under accession number ATCC PTA- 9693, or which (ii) specifically binds to an epitope of C. difficile Toxin B, defined by a monoclonal antibody produced by a hybridoma cell line, deposited under accession number ATCC PTA-9693, where the epitope is determined by a monocl a single antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9693 contains the N-terminal enzymatic domain of C. difficile toxin B. In an embodiment, the epitope determined by the monoclonal antibody produced by the hybridoma cell line deposited under accession number ATCC PTA-9693 contains a 63 kDa fragment obtained by treating toxin B with caspase 1 containing the N-terminal enzymatic domain of C. difficile toxin B. In an embodiment, an epitope defined by a monoclonal antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9692 contains a translational domain of C. difficile Toxin B.

In an embodiment, an epitope defined by a monoclonal antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9692 contains a 167 kDa fragment obtained by treating toxin B with caspase 1 and a 63 kDa protein that contains untreated toxin B. In an embodiment, the antibody is in humanized form. In an embodiment, the antibody is in chimeric form.

In another aspect, there is provided a method for producing a monoclonal antibody that binds and neutralizes toxin A or C. difficile toxin B, comprising immunizing one or more animal recipients with an inactive toxoid A at a certain frequency; booster immunization of animals with increasing amounts of active toxin A or active toxin B at regular intervals; obtaining hybridoma cells from immune cells of an immunized and booster-immunized animal fused to a suitable immortalized cell line, where the hybridoma cells produce and secrete antibodies to toxin A that bind and neutralize C. difficile toxin A, or antibodies to toxin B that bind and neutralize toxin B C. difficile. In an embodiment, neutralizing monoclonal antibodies to C. difficile Toxin A and / or neutralizing monoclonal antibodies to C. difficile Toxin B are isolated. In embodiments of the method, the steps of immunization and booster immunization include the use of an adjuvant. In an embodiment, the adjuvant is Quil A. In other embodiments of the method, the steps of immunization and booster immunization are carried out at intervals of three weeks. In other embodiments, animal recipients are immunized with two or three doses of toxoid A, followed by three to five booster immunizations with increasing doses of either active toxin A or active toxin B.

In another aspect, an isolated antibody or antigen binding fragment thereof is provided that inhibits, blocks or prevents the toxicity of C. difficile Toxin A by suppressing, blocking, or preventing the internalization and cytotoxicity of Toxin A. In an embodiment, the antibody is a monoclonal antibody. In an embodiment, the antibody is a humanized or chimeric antibody. In an embodiment, the antibody is RA-39 (access no. ATCC PTA-9692) or humanized RA-39. In an embodiment, the antibody is RA-50 (access no. ATCC PTA-964) or humanized RA-50. In other embodiments, the antibody competes with PA-39, humanized PA-39, PA-50 or humanized PA-50 for binding to toxin A. In an embodiment, the antibody binds to one site in the region of toxin A outside the receptor-binding domain of toxin A. In an embodiment the implementation of the antibody competes with RA-39 or its humanized form by binding one site in the region of toxin A outside the receptor-binding domain of toxin A. In an embodiment, the antibody binds to at least two sites in etseptorsvyazyvayuschem domain of toxin A. In the embodiment, the antibody competes with the PA-50 or a humanized form by binding to at least two sites in the receptor-binding domain of toxin A. In the embodiment, the antibody inhibits the toxicity of toxin A by mixed competitive mechanism of action. In an embodiment, the antibody suppresses the toxicity of toxin A through a competitive mechanism of action. All of the above embodiments are intended to include an antigen binding fragment of an antibody.

In another aspect, an isolated antibody or antigen-binding fragment thereof is provided that suppresses, blocks or prevents the toxicity of C. difficile Toxin B by binding to an epitope site in the N-terminal enzymatic region of Toxin B. In an embodiment, the antibody is a monoclonal antibody. In an embodiment, the antibody is a humanized or chimeric antibody. In an embodiment, the antibody is RA-41 (accession number ATCC PTA-9693) or a humanized form of RA-41. In an embodiment, the antibody competes with PA-41 or humanized RA-41 for binding to the C. terminal difficile N-terminal enzyme region of C. difficile. In an embodiment, the antibody competes with PA-41 or humanized PA-41 for binding to a single site in the N-terminal enzymatic region of C. difficile Toxin B. In an embodiment, the antibody suppresses the toxicity of toxin B through a mixed competitive mechanism of action.

In another aspect, there is provided a vaccine or immunogen comprising parts, fragments, or peptides of C. difficile Toxin A and / or Toxin B, containing etitope regions recognized and / or linked by one or more of the RA-39 monoclonal antibody (Accession No. ATCC PTA-9692 ), a humanized form of RA-39, a monoclonal antibody PA-50 (access number ATCC PTA-9694), a humanized form of RA-51, a monoclonal antibody PA-41 (accession number ATCC PTA-9693), a humanized form of RA-41, antibodies, which competes for the binding of toxin A to monoclonal antibody P -39 or humanized form of antibody that competes for binding to toxin A with a monoclonal antibody RA-50 or its humanised form, or an antibody that competes for binding to toxin B with a monoclonal antibody RA-41 or a humanized form. In an embodiment, the vaccine or immunogen contains parts, fragments, or peptides of C. difficile toxin A and / or toxin B, containing etitope regions recognized and / or linked by one or more of the RA-39 monoclonal antibody (Access No. ATCC PTA-9692), a humanized form of RA-39 or an antibody that competes for the binding of toxin A and toxin B to the monoclonal antibody RA-39 or its humanized form. In an embodiment, the epitope-containing portions, fragments or peptides of the toxin A and / or toxin B of the vaccine or immunogen are derived from a protein of the toxin A or toxin B by proteolytic cleavage. In an embodiment, fragments, parts or peptides of the vaccine or immunogen toxin A are obtained by proteolytic cleavage by enterokinase. In an embodiment, fragments, parts or peptides of the vaccine or immunogen toxin B are obtained by proteolytic cleavage of caspase (caspase 1). In an embodiment, epitope-containing portions or fragments of a vaccine or immunogen are chemically synthesized or recombinantly produced peptides of a toxin A or toxin B protein. In an embodiment, fragments, portions or peptides of a vaccine or immunogen containing one or more etitope regions of toxin A and / or toxin B that are recognized and bound by an antibody are derived from one or more amino terminus of toxin A; amino end of toxin B; the carboxy terminus of toxin A; carboxy terminus of toxin B; toxin A receptor binding domain; areas outside the toxin A receptor binding domain; toxin B receptor binding domain; N-terminal enzymatic region of toxin B; toxin A glucosyltransferase domain; glucosyltransferase domain of toxin B; the proteolytic domain of toxin A; the proteolytic domain of toxin B; hydrophobic pore-forming domain of toxin A; hydrophobic pore-forming domain of toxin B. In an embodiment, the sizes of the epitope-containing fragments or parts of toxin A or toxin B are <300 kDa, ~ 158-160 kDa, ~ 100-105 kDa, for example 103 kDa, ~ 90-95 kDa, for example 91 kDa , and / or ~ 63-68 kDa, for example 63 kDa or 68 kDa. In an embodiment, the sizes of the epitope-containing fragments or portions of toxin A are ~ 158-160 kDa, ~ 90-95 kDa, for example 91 kDa, and / or ~ 63-68 kDa, for example 68 kDa. In an embodiment, the sizes of the epitope-containing fragments or portions of the toxin B are ~ 100-105 kDa, for example 103 kDa, and / or ~ 63-68 kDa, for example 63 kDa. In any of the vaccine or immunogen embodiments, the toxin A or toxin B, or a fragment, part or peptide thereof, is one of any of the strains described herein.

In another aspect, neutralizing, suppressing, blocking, reducing, decreasing the intensity, curing or treating a C. difficile infection or a C. difficile associated disease in a subject in need thereof is provided, comprising administering to the subject an effective amount of the above vaccine or immunogen. In an embodiment of the method, the subject is given a humoral response to C. difficile toxin A and / or toxin B after administration of a vaccine or immunogen, thereby producing antibodies to toxin A and / or toxin B, which can specifically neutralize, suppress, block, reduce, reduce the intensity, cure or treat C. difficile-associated disease or CDAD, including mild-severe diarrhea and, in some cases, associated with severe life-threatening complications, such as pseudomembranous colitis, toxic megacol he, intestinal perforation, sepsis and death. In an embodiment of the method, antibodies generated by a humoral response include antibodies having specificities and mechanisms of action similar to or identical to the mAbs of the present invention or antibodies that compete with the mAbs of the present invention to neutralize toxin A and / or toxin B C .difficile or which compete with the mAb of the present invention in a mechanism of action involved in neutralizing C. difficile toxin A and / or toxin B.

In another aspect, there is provided a method of neutralizing, inhibiting or blocking the activity of toxin A and / or toxin B in or with respect to a cell susceptible to C. difficile infection, comprising contacting the cell in an antibody or antigen binding fragment thereof in accordance with the present invention, wherein the antibody or its antigen-binding fragment neutralizes, inhibits or blocks the activity of toxin A and / or toxin B in or in relation to the cell using a competitive or mixed competitive mechanism of action. In an embodiment of the method, the antibody is one or more of a monoclonal antibody, a humanized antibody, or a chimeric antibody. In an embodiment of the method, a cell, for example, an intestinal epithelial cell, is in the subject, and the antibody or antigen binding fragment thereof is administered to the subject in an effective amount. In an embodiment of the method, the toxin is toxin A. In an embodiment of the method, the toxin is toxin B. In an embodiment of the method, the toxin is toxin A, and the mechanism of action is the mechanism of action of competitive suppression. In an embodiment of the method, the antibody or antigen-binding fragment thereof is RA-50 (accession number ATCC PTA-9694), its humanized form or antibody, or fragment thereof, which competes with RA-50 to neutralize the activity of toxin A. In an embodiment of the method, the toxin is is toxin A, and the mechanism of action is the mechanism of action of mixed competitive suppression. In an embodiment of the method, the antibody or antigen binding fragment thereof is RA-39 (accession number ATCC PTA-9692), its humanized form or antibody or fragment thereof, which competes with RA-39 to neutralize the activity of toxin A. In an embodiment of the method, the toxin is toxin B, and the mechanism of action is the mechanism of action of mixed competitive suppression. In an embodiment, the antibody or antigen binding fragment thereof is RA-41 (accession number ATCC PTA-9693), its humanized form or antibody or fragment thereof that competes with RA-41 to neutralize toxin B activity.

These and other aspects of the present invention will be described in more detail in the detailed description of the present invention.

A brief description of the graphic materials

1A-1C show the specificity of the mAb for the C. difficile toxin of the present invention with respect to toxin A and / or toxin B by ELISA. ELISA plates were coated with Toxin A (filled circles) or Toxin B (empty squares) overnight at 4 ° C. Then the plates were washed and blocked, the mouse mAbs PA-38 (A), PA-39 (B) or PA-41 (C) were titrated and introduced into the tablets. Binding of monoclonal antibodies was determined using HRP-conjugated goat antibodies to mouse IgG-Fc. OD was measured on a SpectraMax M5 plate reader (Molecular Devices).

On figa-2D presents the results of the analysis of the characteristics of the binding of Biacore using murine mAb PA-38, PA-39, PA-41 and PA-50. Binding specificity was determined using a Biacore 3000 device (GE Healthcare). mAb (PA-38 (2A), PA-39 (2B), PA-50 (2C), PA-41 (2D) or non-specific mAb as a control) were covalently immobilized on the surface of a CM5 sensor chip (GE Healthcare) at approximately 10,000 resonance units (RU) according to the manufacturer's instructions for amine coupling. Binding experiments were carried out at 25 ° C. in PBS. Purified Toxin A or Toxin B (List Biological Laboratories) at 30 nM was passed over control (non-specific mAb) and test flow cells at a flow rate of 5 μl / min with an association phase (600 s for PA-38, PA-39 and PA-41 ; and 300 s for RA-50) and a dissociation phase (300 s for RA-38, PA-39 and PA-41; and 600 s for RA-50). Graphs are presented in RU with respect to time.

3A-3E and 3F-3H show the results of the kinetics of antibody attachment to a toxin as determined by Biacore. For FIGS. 3A-3E, mouse mAbs were captured using a CM5 sensor chip prepared with a mouse antibody capture kit from Biacore. The toxin was then passed through flow cells at various concentrations (0.4-100 nM, double increase) at a flow rate of 30 μl / min. All mAb concentrations were tested in duplicate, and the chip surface was regenerated after each experiment using the conditions indicated in the kit. Changes in RU were recorded and analyzed using the 1: 1 binding model (Langmuir) in the Bia Evaluation Software, which calculates kd mAb for toxin. Figa: binding of RA-38 to toxin A; figv: binding of RA-50 with toxin A; figs: binding of PA-39 with toxin A; 3D: binding of PA-39 to toxin B; and FIG. 3E: binding of PA-41 to toxin B. For FIG. 3F-3H, as above, murine mAbs, for example, mPA-50, mPA-41 or mPA-39, were covalently coupled to the CM5 sensor chip by amine coupling . Toxin A (line indicated by "(red)") or toxin B (line indicated by "(blue)") at 30 nM was passed over test flow cells (mPA-50, mPA-41 or mPA-39) at a flow rate of 5 μl / min 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 exhibits cross-reactivity to toxin B (FIG. 3H).

Figure 4 shows the in vitro neutralization activity of the toxin A activity using purified murine PA-39 mAb on CHO-K1 cells. For cytotoxicity measurements, toxin A was incubated with various concentrations of RA-39 for 1 hour at 37 ° C (Example 3A). The mAb-toxin mixtures were then added to CHO-K1 cells grown in 96-well plates at 2000 cells / well and incubated for 72 hours. Cell survival was compared in untreated and treated cultures and the concentration of mAb required for 50% neutralization of cytotoxicity (EC ₅₀ ) was calculated. Cell survival was determined by CellTiter-Blue; baseline data was normalized relative to the untreated control wells. Values were plotted using Prism, and the curves were calculated using the dose-response sigmoidal curve model (variable slope). The curve was then used to determine the EC ₅₀ mAb. Data points represent the average of three wells on one tablet.

Figure 5 shows the in vitro activity of neutralizing the activity of toxin B using purified murine PA-41 mAb on CHO-K1 cells. For cytotoxicity measurements, toxin B was incubated with various concentrations of RA-41 for 1 hour at 37 ° C (Example 3B). The mAb-toxin mixtures were then added to CHO-K1 cells grown in 96-well plates at 2000 cells / well and incubated for 72 hours. Cell survival was compared in untreated and treated cultures and the concentration of mAb required for 50% neutralization of cytotoxicity (EC ₅₀ ) was calculated. Cell survival was determined using CellTiter-Blue; baseline data was normalized relative to the untreated control wells. Values were plotted using Prism, and the curves were calculated using the dose-response sigmoidal curve model (variable slope). The curve was then used to determine the EC ₅₀ mAb. Data points represent the average of three wells on one tablet.

Figure 6 shows the in vitro activity of neutralizing the activity of toxin A using purified murine PA-38 and PA-50 mAbs on T-84 cells (Example 3C). T-84 cells were seeded (15,000 cells / well) in 96-well plates and treated with a combination of a soaked mAb (PA-38 (■) or PA-50 (▲)) and toxin A (60 ng / ml). After incubation (72 hours), cell survival in the treated and untreated cultures was compared, and the concentration of mAbs required for 50% neutralization of cytotoxicity (EC ₅₀ ) was calculated. Cell survival was determined by CellTiter-Blue; baseline data was normalized relative to the untreated control wells. Values were plotted using Prism, and the curves were calculated using the dose-response sigmoidal curve model (variable slope). The curve was then used to determine the EC ₅₀ mAb. Data points represent the average of three wells on one tablet.

7 shows the results of testing mouse PAb-38 (■) or PA-50 (▲) mAbs with respect to their ability to block or prevent toxin A-induced rabbit red blood cell (RBC) hemagglutination. Toxin A (2 μg / ml) was combined with various dilutions of PA-38 or RA-50, and the mixture was added to tablets containing 50 μl of rabbit erythrocytes. The plates were incubated at 4 ° C for 4 hours. Hemagglutination was quantified as color intensity using ImageQuant 400 dot matrix analysis (GE Healthcare). Data were presented as% control, with 100% representing no hemagglutination. Data points represent the average of three wells analyzed on a single plate.

Figure 8 shows the mAb activity of the C. difficile toxin of the present invention in preventing the destruction of the monolayer of Caco-2 cells 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 incubation for 10-14 days, the formation of a dense monolayer was confirmed by measuring transepithelial electrical resistance (TEER) using an epithelial voltmeter (World Precision Instruments). After the monolayer integrity was determined and determined, toxin A (25 ng / ml) and serially diluted murine mAb (PA-38 (■) or PA-50 (▲)) were added to the upper chamber of the tablet for analysis. The plates were incubated for 18-24 hours, and the TEER value was measured using a multimeter. Monolayer integrity was compared in untreated and toxin-treated wells. Inhibition data were approximated with a sigmoidal dose-response curve with non-linear regression using GraphPad Prism software to determine the mAb concentration required for 50% toxin suppression (EC ₅₀ ).

On figa-9C demonstrates the ability of the mAb to toxin A PA-38 (9A) and RA-50 (9B) to neutralize the activity of toxin A in vivo. Female Swiss Webster mice (6-8 weeks old, 5 mice / group) were injected (ip) with PA-38 mouse mAb or PA-50 mouse mAb in the indicated amounts or PBS (200 μl) on day 0. Neutralizing activity of the monoclonal reference antibody to toxin A, indicated herein as CDA-1, was evaluated at the indicated amounts of antibody (9C). comparison mAbs for toxin A, CDA-1, were prepared by synthesizing (DNA2.0) nucleic acids encoding the variable regions of the heavy and light chain 3D8 (W02006 / 121422 and US2005 / 0287150), which were cloned into human IgGI expression vectors (pCON gamma-1 and pCON-kappa). CDA-1 comparison mAbs were expressed and produced in CHO-KSV1 cells and purified as described in the Examples section of this document. The mice were then injected with 100 ng of toxin A (200 μl) on day 1, and they were observed daily for the first 72 hours and weekly thereafter. The results show that both PAb-38 and PA-50 mAbs are able to completely suppress toxin A-associated toxicity after a single dose of 2 μg mAb / animal, whereas CDA-1 comparison mAbs (5 μg / animal) could not completely suppress the toxin associated A C. difficile toxicity.

Figure 10 shows the ability of the PAb-41 mAb to neutralize toxin B activity in vivo. Female Swiss Webster mice (6-8 weeks old, 5 mice / group) were injected (ip) or PA-41 mouse mAb in the indicated amounts or PBS (200 μl) on day 0. Then, 100 ng of toxin B (200 μl) was injected into the mice. ) on day 1, and they were observed daily for the first 72 hours and weekly thereafter. The results of this experiment show that PAb-41 mAb completely suppresses C. difficile Toxin B associated toxicity after a single dose of 5 μg mAb / animal. A similar experiment was performed using a monoclonal reference antibody to toxin B, referred to herein as a CDB-1 comparison mAb. Comparison mAb to Toxin CDB-1 was prepared by synthesizing (DNA2.0) nucleic acids encoding the variable regions of the heavy and light chains 124 (WO2006 / 121422 and US2005 / 0287150), which were cloned into human IgGI expression vectors (pCON-gamma1 and pCON-kappa). CDB-1 comparison mAbs were expressed and produced in CHO-KSV1 cells and purified as described in the Examples section of this document. The results of these experiments showed a lack of neutralization activity against toxin B in the CDB-1 comparison mAb, even at 250 μg.

Figure 11 shows the possibility of combining the murine mAb RA-38 and RA-41 (RA-38 + RA-41) of the present invention to neutralize the activity of toxin A and toxin B in vivo. Female Swiss Webster mice (6-8 weeks of age, 5 mice / group) were injected (ip) with a combination of mAb PA-38 + PA-41 or PBS (200 μl) per day 0. Then, mice were injected with 100 ng of a combination of toxin A and toxin In (200 μl) on day 1, and they were observed daily for the first 72 hours and weekly thereafter. Graphs for the toxin, only RA-38 (empty circles) and only RA-41 (filled diamonds) are superimposed on the general schedule. The results show that only mAb RA-38 (empty circles), only mAb RA-41 (filled diamonds) were not sufficient to suppress the effects of both toxins, and they did not protect animals from C. difficile infection. In contrast, the combination of RA-38 and RA-41 (RA-38 + RA-41), at 50 μg each (free, inverted triangles), was able to protect infected animals and prevent death associated with the toxin in 4 of 5 test animals. The combination of RA-38 and RA-41 (RA-38 + RA-41) of 5 μg each (filled circles) provided some protection against toxicity of C. difficile toxins A and B in infected test animals.

12A and 12B show pharmacokinetic results (PK) on hamsters for mouse mAb RA-38 and RA-41. Hamsters i.p. doses of 2 mg / kg (○) or 10 mg / kg (■) mAb RA-38 (12A) or PA41 (12V) were given. Animals were bled at certain times and serum was assayed using HPR-conjugated goat anti-mouse IgG plates and goat anti-mouse antibodies to detect ELISA. The resulting curves illustrate the dose-dependent response in the cohorts of 2 mg / kg and 10 mg / kg for each antibody. WinNonLin analysis was performed on each curve. Both monoclonal antibodies have a final half-life of more than 6 days.

Figure 13 illustrates the survival results from the hamster study described in Example 5 B. In this study, hamsters were treated with clindamycin, infected with C. difficile (■ infected control, group 3), and then treated with vancomycin (◆ 20 mg / kg, group 4), with the combination of mAb PA-38 + PA-41 (Δ 50, 50 mg / kg, group 6) or with the combination of mAb PA-39 + PA-41 (○ 50, 40 mg / kg, group 7). All animals in the uninfected control (group 1) and the control treated with goat polyclonal Ab (group 5) survived. Animals treated with a combination of a mAb to toxin A and toxin B of the present invention survived and were protected against C. difficile toxicity for the duration of the study.

On Fig shows the average body weight (in grams) of the hamsters from the study described in example 5B. The experimental groups of animals are as follows: uninfected control (◆, group 1); control treated with vancomycin (■, group 4); a group treated with a combination of murine mAb PA-38 + PA-41 (x, group 6), or a group treated with a combination of mouse mAbs PA-39 + PA-41 (●, group 7). Animals in the infected control group (group 3) did not survive for 5 days, so for group 3 measurements of body weight after infection were not possible.

On figa-15D shows the results of a post mortem examination from the study on the hamsters described in example 5B. Typical animals from each respective group from the study were evaluated: (A) group 1, uninfected control; (B) group 3, infected control; (C) group 6, a group treated with a combination of murine mAb PA-38 + PA-41, and (D) group 7, a group treated with a combination of mouse mAb PA-39 + PA-41. Arrows indicate the cecum of each hamster. In the infected control group 3 (B), the cecum was noticeably reddened and inflamed. In contrast, the hamsters cecum in group 6 (C) and group 7 (D) were similar to the cecum in healthy, uninfected control animals of group 1 (A).

Figa and 16B-1 and B-2. On figa illustrates the results of survival in the study on the hamsters described in example 5C. In this study, hamsters were treated with clindamycin, infected with C. difficile (■ infected control, group 1), and then various groups of animals were treated with vancomycin (◆ 20 mg / kg, group 2), a combination of murine mAb PA-39 + PA-41 (▲ 50 + 50 mg / kg, group 3), only mouse mAb PA-41 (○ 50 mg / kg, group 4), only mouse mAb PA-38 (∇ 50 mg / kg, group 5), only mouse mAb PA- 39 (□ 50 mg / kg, group 6) or only mouse PAb-50 mAb (◇ 50 mg / kg, group 7). All animals in uninfected control (group 1) survived the continuation of the study. 16 B-1 illustrates the results of animal survival in a hamster study described in Example 5E. Kaplan-Meer survival curves are shown for animals treated with clindamycin and then infected with C. difficile (■ infected control, group 2); treated with vancomycin ( ◇ , 20 mg / kg, group 3); treated with a 1: 1 combination of humanized mAb PA-50 + PA-41 ((hPA-50 + hPA-41), Δ, 50 + 50 mg / kg, group 4); treated with a 1: 1 combination of humanized mAb PA-50 + PA-41 ((hPA-50 + hPA-41), ○ , 20 + 20 mg / kg, group 5); treated with a 1: 1 combination mAb comparing CDA-1 + CDB-1 preparation ((CDA-1 + CDB-1), ∇, 50 + 50 mg / kg, group 6) or treated with a 1: 1 combination mAb comparing CDA-1 + CDB-1 ((CDA-1 + CDB-1), □, 20 + 20 mg / kg, group 7). FIG. 16 B-2 illustrates the results of weight change in a hamster study described in Example 5E. The average (± SD) of the animal body weight over time in the groups with different treatments described for FIG. 16 B-1 was compared with uninfected control animals.

On figa-17C shows the treatment of toxin b. Difficile caspase 1 (a): toxin b. Difficile full length 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 noted: 193, 167, 103 and 63 kDa. (C): possible fragments formed after caspase 1 treatment of toxin B.

FIGS. 18A-18C show SDS-PAGE (A) and Western blotting (B, C) detection of C. difficile toxin B fragments using a mAb to toxin B. (A): SDS-PAGE analysis of toxin B fragments generated by caspase 1 treatment (same as in FIG. 17B.); (B): Western blot detection of toxin B fragments using mAb RA-41. (C) Western blot detection of toxin B fragments using mouse RA-39 mAb.

On figa-19E shows the characteristic of mouse mAb toxin C. difficile using Biacore binding. Competitive binding of mAb to toxin was evaluated. (A): mAb RA-41 binds one epitope on toxin B. (B): mAb RA-39 binds one epitope on toxin A. (C): mAb RA-39 and RA-41 bind to different epitopes on toxin B. The binding of mAb RA-41 to toxin B is epitopically different from the binding of a reference antibody to toxin B of CDB-1. (D) mPA-41 immobilized on a CM5 chip captures toxin B but is unable to bind additional tA-41, which indicates that there is only one mPA-41 binding epitope. The addition of CDB-1 mAb comparisons gave an increased signal, demonstrating that mPA-41 and CDB-1 mAb comparisons bind different epitopes on toxin B. (E) Western blot analysis using toxin B, either untreated or caspase 1 treated, demonstrate that mPA-41 and CDB-1 comparison mAbs have different binding patterns and bind different epitopes on toxin B.

On figa-20C shows the cleavage of toxin A. C. difficile using enterokinase (EK). (A): C. difficile toxin A of full length and its domains. (B): 3-8% Tris-acetate SDS-PAGE (reducing) analysis of toxin A (TcdA) and toxin A treated with EK. (C): possible fragments formed after treatment of the EK toxin A at 25 ° C. for 48 hours.

21A-21C show Coomassie blue staining (SDS-PAGE), (A), and Western blotting (B, C) for detection of C. difficile toxin A fragments using mouse mAb to toxin A. (A): SDS- PAGE analysis of toxin A fragments formed by EK treatment (same as in FIG. 20B). (B): Western blot detection of toxin A fragments using mAb PA-50. (C): Western blot detection of toxin A fragments using mAb RA-39. On figv and 21C, the band (kDa) is ~ 158 kDa and can be considered as a component of ~ 158-160 kDa.

Figa-1 and A-2-22F. On figa-1 and A-2-22D shows the binding characteristics of the mouse mAb to toxin A toxin C. difficile using a Biacore binding assay. FIGS. 22A-1: PAb-50 mAb was noted to bind multiple sites on toxin A. FIGS. 22A-2: PA-50 mAb was immobilized on a sensor chip and then sequentially contacted with purified toxin A, additional PA-50 and CDA-1 comparison mAbs (WO / 2006/121422; US2005 / 0287150). Fig. 22B: Toxin A captured by the CDA-1 comparison mAb on a Biacore chip further binds additional CDA-1 and PA-50 mAb, showing differences in the toxin A epitopes associated with antibodies. Fig. 22C: PAb 39 mAb binding epitope on toxin A is different from CDA-1 mAb toxin A binding epitope. Fig. 22D: PA-50 and PA-39 murine mAb binding to toxin A were competitive using Biacore. The PA5 mAb immobilized on a CM5 chip captures toxin A, which can bind additional mPA-50 and also mPA-39, demonstrating that there are multiple copies of epitopes for mPA-50 on toxin A, and that mPA-50 and mPA- 39 bind to excellent epitopes of toxin A. FIGS. 22E and 22F: Biacore results were confirmed by Western blot analysis using toxin A, which was not treated and was treated with enterokinase (EK) enzyme. (E): mPA-39 and CDA-1 comparison mAbs show different binding patterns with EK-treated toxin A (lane: TcdA / EK), thus marking different binding domains and epitopes of toxin A. (F): mPA-50 and mAb CDA-1 comparisons bind to the same toxin A domain, but with different epitopes.

On figa and 23B shows the neutralizing activity of PA-41 in vitro against a panel of twenty different toxicogenic clinical isolates of C. difficile, including 6 BI / NAP 1/027 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. On figa shows the neutralizing activity of the mouse PAb-41 mAb on CHO-K1 cells against supernatants obtained from cultures of various clinical C. difficile isolates shown in example 8, table 6. The purified PA-41 mAb was diluted sequentially and then mixed with a supernatant with a predetermined dilution ratio that can cause> 90% cell death. The mixture was incubated for 1 hour at 37 ° C and then introduced into CHO-K1 cells. Cells were incubated for 72 hours, and cell viability was measured using Cell-Titer Blue. 23B illustrates the neutralizing activity of a humanized hPA-41 mAb, as well as a CDB-1 mAb for comparison of 2 C. difficile reference strains (VPI 10463 and ATCC 43596) and 6 BI / 027/027 C. difficile strains (CCL678, HMS553, Pitt 45, CD196, Montreal 5.1 and Montreal 7.1). The neutralizing activity of mAb hPA-41 (filled squares) and mAb comparisons of CDB-1 (filled triangles) on CHO-K1 cells against supernatant from reference strains (VPI 10483 and ATCC 43596) and BI / NAP 1/027 strains (CCL678 is shown) NMS553, Pitt 45, CD196, Montreal 5.1 and Montreal 7.1).

On figa and 24B shows the neutralizing activity of in vitro mAb PA-50 in relation to the toxicogenic clinical isolates of C. difficile described for figa and B. Figa shows the neutralizing activity of murine mAb PA-50 on T-84 cells with respect to the supernatant obtained from cultures of various C. difficile clinical isolates shown in Example 8, Table 6. The purified PA-50 mAbs were serially diluted and then mixed with a predetermined dilution ratio that could cause> 90% cell death. The mixture was incubated for 1 hour at 37 ° C and then introduced into the T-84 cells. Cells were incubated for 72 hours, and cell viability was measured using Cell-Titer Blue. On figv shows the results of similar experiments conducted using the humanized mAb hPA-50 and mAb comparison CDA-1 on T-84 cells. The neutralizing activity of hPA-50 (filled squares) and comparisons of CDA-1 (filled triangle) on T-84 cells against supernatants obtained from six BI / NAP1 / 027 strains (CCL678, HMC553, Pin 45, CD 196, Montreal 5.1 are shown). and Montreal 7.1). For figa: *: N / A: Not applicable; toxin A was not produced toxin a- / toxin B + with strains F1470, 8864, CCL13820 and CCL14402; ": The toxin A titer was very low; there was no measurable cytotoxicity on T-84 using the supernatant; ^: Not applicable; toxin A was not produced by the toxin A- / toxin B + strains or the concentration was too low.

25A-25D demonstrate the neutralization of toxins produced by various C. difficile strains. On figa and 25B shows the neutralizing activity of murine mAb PA-39 on T-84 cells in relation to supernatants obtained from cultures of various clinical isolates of C. difficile presented in example 8, table 6. mAb PA-39 (hybridoma supernatant) serially diluted, and a dilution factor for each supernatant is shown. On figv shows the results of similar experiments conducted using the mouse mAb PA-39 and mAb comparison CDA-1 on T-84 cells. The neutralizing activity of PA-39 (filled squares) and mAb comparisons of CDA-1 (filled triangles) on T-84 cells against supernatants obtained from six BI / NAP1 / 027 strains (CCL678, HMS553, Pitt45, CD196, Montreal 5.1 and Montreal 7.1). For figa: *: N / A: Not applicable; toxin A was not produced toxin a- / toxin B + with strains F1470, 8864, CCL13820 and CCL14402; ": the toxin A titer was very low; there was no measurable cytotoxicity on T-84 using the supernatant; ^: Not applicable; toxin A was not produced by the toxin A- / toxin B + strains or the concentration was too low. In Fig. 25C, humanized mAb to toxin A RA-50 and mAb comparison to toxin A CDA-1 to neutralize the cytotoxicity of the culture supernatant of C. difficile in relation to T-84 cells. (Example 8, table 7). In Fig.25D, a humanized mAb to toxin B was tested. RA-41 and mAb comparisons to CDB-1 toxin A on neutralizing culture cytotoxicity opernatant C. difficile in relation to T-84 cells (example 8, table 7).

Figure 26 shows that the chimeric PAb-41 mAbs (CPA-41 mAbs) effectively neutralize the toxicity of C. difficile Toxin B at CHO-K1 in comparison with similar murine PAb-41 mAbs. Received two chimeric mAb PA-41; in one of which the glycosylation site in the VL region was deleted, designated as cPA-41 (NG), and in one of which the glycosylation site was not removed, indicated as cPA-41 (G) in the figure. Both cPA-41 (NG) and cPA-41 (G) showed similar levels of neutralization relative to toxin B (2 pg / ml, TechLab) on CHO-K1 cells, and both chimeric mAb neutralized toxin B at a level similar to that of the original mouse mAb (mPA-41).

27 shows that the chimeric PAb-39 mAb (CPA-39) effectively neutralizes the toxicity of C. difficile Toxin A (1 pg / ml, Listlab) on CHO-K1 cells compared to the original murine PAb-39 mAb (mPA- 39).

Figure 28 shows that the chimeric PAb-50 mAb (CPA-50) effectively neutralizes the toxicity of C. difficile Toxin A (60 pg / ml, TechLab) on T-84 cells compared to the original murine PAb-50 mAb (mPA- fifty).

Figure 29 shows the in vitro neutralizing activity of murine PAb-41 mAb (TPA-41) and humanized PA-41 mAb (hPA-41) against C. difficile Toxin B. The percentage of cell survival compared to 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 EC ₅₀ - 6 pM, and was virtually equivalent to the original murine monoclonal antibody (mPA-41).

30 shows the in vitro neutralizing activity of murine PAb-39 mAb (mPA-39) and humanized PA-39 mAb (hPA-39) against C. difficile toxin A. hPA-39 mAb effectively neutralizes the toxicity of toxin A (20 pg / ml, TechLab) on CHO-K1 cells with EC ₅₀ - 50 pM, and was virtually equivalent to the original murine monoclonal antibody (mPA-39).

On figa-31H shows the results of studies of neutralizing activity in vitro and the mechanism of action (MOA) using the described mAb. Cell-based assays using CHO-K1 cells and T-84 cells were performed as described in Examples 1, 3 and 7. FIG. 31A shows that humanized PA-50 mAb (hPA-50) effectively neutralizes the toxicity of Toxin A (60 pg / ml, TechLab) on T-84 cells in comparison with the original murine mAb PA-50 (mPA-50). On figv-D shows the neutralizing activity of the mAb to toxin A PA-39 and PA-50 in comparison with that mAb comparison to toxin A CDA-1. The values of the EU ₅₀ and the maximum percent suppression are as shown in table A of example 7C. FIGS. 31E and F show the neutralizing activity of the mAb to toxin B of RA-41 in comparison with the comparison mAb to toxin B of CDB-1. The EC5o values and the maximum percent suppression are as shown in Table B of Example 7C. Figures 31G and H show the ELISA method and the results of evaluating the ability of the mAb to toxin A to block the internalization of toxin A into cells and evaluating the activities of these mAbs in preventing the internalization of toxin A against controls with goat polyclonal antibody to toxin A; and controls without antibodies.

On figa and 32B depict the amino acid sequences of the VH regions of humanized PA-39 (hPA-39), SEQ ID NO: 1 and SEQ ID NO: 2. Amino acid residues are shown using a single letter code. Numbers above sequences indicate positions according to Kabat et al. The provisions of the CDR are underlined.

On figa and 33B depict the amino acid sequence of the VL regions of humanized PA-39 (hPA-39), SEQ ID NO: 3 and SEQ ID NO: 4. Amino acid residues are shown using a single letter code. Numbers above sequences indicate positions according to Kabat et al. The provisions of the CDR are underlined.

On figa and 34B depict the amino acid sequence of the VH regions of humanized PA-50 (hPA-50), SEQ ID NO: 5 and SEQ ID NO: 6. Amino acid residues are shown using a single letter code. Numbers above sequences indicate positions according to Kabat et al. The provisions of the CDR are underlined.

On Fig depicts the amino acid sequence of the VL region of the humanized PA-50, SEQ ID NO: 7. Amino acid residues are shown using a single letter code. Numbers above sequences indicate positions according to Kabat et al. The provisions of the CDR are underlined.

On figa and 36B depict the amino acid sequences of the VH regions of humanized PA-41 (hPA-41), SEQ ID NO: 8 and SEQ ID NO: 9. Amino acid residues are shown using a single letter code. Numbers above sequences indicate positions according to Kabat et al. The provisions of the CDR are underlined.

On Fig depicts the amino acid sequence of the VL region of the humanized PA-41, SEQ ID NO: 10. Amino acid residues are shown using a single letter code. Numbers above sequences indicate positions according to Kabat et al. The provisions of the CDR are underlined.

On figa and 38B shows the nucleic acid sequence and the encoded amino acid sequence of a humanized monoclonal antibody to toxin A. C. difficile. On figa depicts the amino acid sequence of the light chain gumanitarnogo monoclonal antibodies to toxin A, described in SEQ ID NO: 16, which is encoded by the nucleic acid sequence described in SEQ ID NO: 17. On figv depicts the amino acid sequence of the heavy chain gumanitarnogo monoclonal antibodies set forth in SEQ ID NO: 14, which is encoded by the nucleic acid sequence set forth in SEQ ID NO: 15.

On figa and 39B shows the nucleic acid sequence and the encoded amino acid sequence of a humanized monoclonal antibody to toxin A. C. difficile. On figa depicts the amino acid sequence of the light chain gumanitarnogo monoclonal antibodies to toxin A, described in SEQ ID NO: 20, which is encoded by the nucleic acid sequence described in SEQ ID NO: 21. On Fig shows the amino acid sequence of the heavy chain gumanitarnogo monoclonal antibodies to toxin A, described in SEQ ID NO: 18, which is encoded by the nucleic acid sequence described in SEQ ID NO: 19.

40A and 40B show the nucleic acid sequence and the encoded amino acid sequence of a humanized C. difficile Toxin B monoclonal antibody. On figa depicts the amino acid sequence of the light chain gumanitarnogo monoclonal antibodies to toxin B, set forth in SEQ ID NO: 24, which is encoded by the nucleic acid sequence set forth in SEQ ID NO: 25. 40B depicts the amino acid sequence of the heavy chain of a humanized monoclonal anti-toxin B antibody set forth in SEQ ID NO: 22, which is encoded by the nucleic acid sequence set forth in SEQ ID NO: 23.

On figa-41C shows the neutralizing activity of in vitro Fab fragments of murine mAbs against toxin A and toxin B. C. difficile in comparison with the effectiveness of an equivalent whole antibody. (A): neutralizing activity of Fab mouse PA-39 and PA-39 mAbs on CHO-K1 cells; Toxin A (Techlab, 60 ng / ml); (B): neutralizing Fab activity of murine PAb-41 and PA-41 mAbs on CHO-K1 cells; Toxin B (Techlab, 2 pg / ml); (C): neutralizing activity of Fab mouse PA-50 and PA-50 mAbs on T-84 cells; Toxin A (Techlab, 60 ng / ml).

42A and 42B show antibody concentration profiles obtained from the pharmacokinetic (PK) study described in Example 13 herein. 42A shows PK results (serum antibody concentration in mg / ml) for 29 days from animals receiving a single dose of purified, humanized, mAb to RA-50 toxin A at a concentration of 1 mg / kg (▲) or 5 mg / kg (■) per day 0. FIG. 42B shows PK results (serum antibody concentration in mg / ml) for 29 days from animals receiving a single dose of purified, humanized, PA-41 toxin B mAb at a concentration of 1 mg / kg (▲) or 5 mg / kg (■) per day 0.

DETAILED DESCRIPTION OF THE INVENTION

The present invention encompasses antibodies and antigen-binding fragments thereof that provide therapeutic agents and non-antibiotic drugs that block the pathogenic effects of C. difficile infection and preferably provide time for treating the colon and / or normalizing the microflora of the gastrointestinal tract, for example , colon, intestines, intestinal tract, etc., until it is restored. The monoclonal antibodies described herein, their antigen binding fragments, such as, for example, their humanized or hybrid forms thereof, provide non-antibiotic therapies and drugs, both for treating an active disease and for preventing re-disease, in order to to allow a patient suffering from C. difficile infection and CDAD to recover from his illness without relapse into a further illness or a more serious illness or symptoms. In an embodiment, the antibodies of the present invention have therapeutic activity against an active disease caused or associated with infection of C. difficile subjects. In an embodiment, the antibodies of the present invention eliminate an active disease caused or associated with infected C. difficile subjects. In an embodiment, the antibodies of the present invention have a therapeutic effect in reducing the duration and / or severity of an active disease caused or associated with an infected C. difficile in subjects. In an embodiment, the antibodies of the present invention or parts, or fragments thereof, can be provided in combination with antibiotic therapeutic agents.

Monoclonal antibodies (mAb) to C. difficile toxins A and B were prepared as described herein. Toxin mAbs are highly active both in vitro and in preclinical models in animals with C. difficile infection in vivo. More specifically, the mAbs of the present invention effectively and permanently protect hamsters from mortality in a relevant or accurate model for hamsters with C. difficile infection.

Antibodies provide approaches to the treatment of CDAD without antibiotics and may provide an opportunity to stop taking antibiotics and block the pathogenic effects of C. difficile toxins, thus providing time for healing the intestines and restoring normal intestinal microflora. The mAbs described herein can provide therapeutic utility due to their ability to neutralize C. difficile toxins and can be used in passive or active treatment strategies for patients with multiple repeated manifestations of C. difficile. In particular, mAbs can be used to prevent re-infection, to treat severe active forms of the disease, and to treat patients with multiple relapses of diseases associated with C. difficile. The mAbs described herein can provide effective means of neutralizing C. difficile toxins A and B in order to prevent, block or suppress the re-emergence of infection, and / or severe and active forms of the disease and multiple relapses.

As used herein, “toxin A” and “toxin B” refer to the cytotoxic enterotoxins produced by the C. difficile microorganism. Toxins A and B are the main determinants of C. difficile virulence, and toxin-negative strains are non-pathogenic. Toxins A and B are transcribed from the pathogenicity locus, which includes the tocin genes tcdA (toxin A) and tcdB (toxin B), and three regulatory genes, one of which (tcdC) encodes a putative negative toxin transcriptional regulator. It turns out that the TcdC protein inhibits toxin transcription during the early phase of the exponential growth of the bacterial life cycle. An autocatalytic cleavage site between leucine ₅₄₃ and glycine ₅₄₄ for toxin B has been described. Cleavage occurs as a result of activation of the aspartyl protease domain by the cytosolic inositol phosphate of the host, and the active glycosyl transferase domain is released.

Antibodies and antigen-binding fragments thereof that specifically bind to C. difficile toxin A and / or C. toxin B are provided herein, compositions containing one or more of these antibodies or antigen-binding fragments thereof, vectors containing nucleic acid sequences that encode antibodies or their antigen binding fragments, hybridoma cell lines that produce antibodies, and methods for using antibodies or their antigen binding fragments to treat or prevent C. difficile sludge infection and diseases associated with C. difficile.

It should be understood that when referring to the expression “antibodies” or “immunoglobulins” in the description of the present invention and its various aspects and embodiments, this expression is intended to encompass antigen-binding fragments of such antibodies or immunoglobulins in order to avoid excessive repetition of the associated the phrase “antigen binding fragments” each time the expression “antibodies” or “immunoglobulins” is mentioned. Thus, the present invention encompasses not only antibodies directed against C. difficile Toxin A and Toxin B, i.e., Toxin A and Toxin B antigens, but also fragments of such antibodies that bind C. difficile Toxin A and Toxin B antigens described later in this document. In embodiments, such antigen binding fragments are capable of neutralizing the toxicity of toxin A and / or toxin B in the same way as in an intact antibody.

Antibodies that are encompassed by the present invention include isolated antibodies that specifically bind C. difficile toxin A and which competitively suppress or cross-hair compete for specific binding to C. difficile toxin A of an isolated monoclonal antibody produced by a hybridoma cell line deposited under accession number ATCC PTA -9692, PTA-9694 or PTA-9888. These antibodies also include isolated antibodies that specifically bind to C. difficile toxin A and that specifically bind to an epitope on C. difficile toxin A, determined by binding to the isolated monoclonal antibody produced by a hybridoma cell line deposited under ATCC accession number PTA-9692 , PTA-9694 or PTA-9888. In some embodiments, epitopes are located in the C-terminal receptor-binding domain of tcdA. In some of these embodiments, the antibodies competitively suppress or the crosshairs compete for binding of RA-50 to tcdA. In other embodiments, the epitope is located in the translational domain of tcdA. In some of these embodiments, the antibodies competitively suppress or the crosshairs compete for the binding of PA-39 to tcdA. Such isolated antibodies may include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, human antibodies, humanized antibodies, and antigen binding fragments or parts thereof.

Experiments using the enzymatic (enterokinase) proteolysis of toxin A to assess the specificity of the monoclonal antibodies of the present invention for C. difficile toxin A were performed as shown in Example 6. The epitope on toxin A, which is recognized and bound by PA-39 mAb, is within the region which is different from the receptor binding domain of the toxin A, that is, outside the receptor binding domain of the toxin A, and also different from the epitope bound by the human antibody to toxin A, which is reported to bind the C-terminal p tseptorsvyazyvayuschy domain of toxin A (7). As described in Examples 6 and 7 herein and shown in FIGS. 22 and 31, Biacore assays confirm a single toxin A binding site for PA-39. Detection of enzyme-cleaved toxin A via Western blotting demonstrates that RA-39 binds to a region on toxin A that is separated from the regions bound by RA-50 and CDA-1 mAb. The in vitro activity of PA-39 in a toxin efficacy study shows shifts in both the EU ₅₀ and the maximum percentage of suppression as more toxin A is added to the culture, indicating a mixed competitive mechanism for suppressing RA-39. Detection of toxin A by ELISA after protection with a 100-fold excess of RA-39 confirmed that the suppression of the toxin by RA-39 occurs by preventing the internalization of the toxin and the adverse effects of the toxin on the cell, for example, cytotoxicity.

In addition, as described in Examples 6 and 7 herein and shown in FIGS. 22 and 31, a Biacore assay confirms at least two toxin A binding sites for PA-50. Detection of enzyme-cleaved toxin A via Western blotting demonstrates that RA-50 binds to a region on toxin A similar to that associated with the mAb CDA-1 reference drug. The in vitro activity of RA-50 in a toxin efficacy study shows a shift in the EC ₅₀ as more toxin A is added to the culture, indicating a competitive suppression mechanism for RA-50. Detection of toxin A by ELISA after protection with a 100-fold excess of RA-50 confirmed that the suppression of toxin by RA-50 occurs by preventing internalization of the toxin and subsequent cytotoxicity.

Antibodies of the present invention also include isolated antibodies that specifically bind C. difficile Toxin B and which competitively suppress or cross-hair compete for specific binding to C. difficile Toxin B of an isolated monoclonal antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9693 or PTA-9692. Antibodies also include isolated antibodies that specifically bind to C. difficile Toxin B and that specifically bind to an C. difficile Toxin B epitope, as determined by binding to an isolated monoclonal antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9693 or PTA -9692. In some embodiments, the epitope is located in the N-terminal enzyme domain of tcdB. In some of these embodiments, the antibodies competitively suppress or cross-compete for binding of RA-41 to tcdB. In other embodiments, the epitope is located in a translocation domain, for example, amino acids 850-1330 tcdB. In some of these embodiments, the antibodies competitively inhibit or cross-compete for the binding of PA-39 to tcdB.

Experiments using enzymatic (caspase 1) toxin B proteolysis to assess the specificity of the C. difficile monoclonal antibodies to toxin B of the present invention were performed as described in Example 6. mAb PA-41 (PTA-9693) was shown to recognize fragments by approximately 103 and 63 kDa, which originate from the N-terminal enzymatic domain of toxin B. Analysis of the N-terminal sequence of most cleaved fragments confirmed this analysis. mAb RA-41 has been shown to bind a unique epitope within the N-terminal enzyme domain of toxin B, different from the C-terminal receptor-binding domain of toxin B, bound by the human antibody to toxin B (7). As described in Examples 6 and 7 herein and shown in FIGS. 19 and 31E and F, Biacore assays confirm a single toxin B binding site for PA-41. Detection of enzymatically cleaved toxin B via Western blotting indicates that RA-41 binds to a distinct region on toxin B that is different from that associated with the reference preparation mAb CDB-1. The in vitro activity of PA-41 in a toxin efficacy study shows shifts in both EC5o and the maximum percentage of suppression as more toxin B is added to the culture, indicating a mixed competitive mechanism for suppressing RA-41.

Antibodies provided herein include monoclonal antibodies produced by hybridomas that have been deposited and which have been given the following patent designation for deposition: PTA-9692 (for PA-39), PTA-9693 (for PA-41), PTA-9694 (for RA-50) and PTA-9888 (for RA-38). These hybridomas were deposited in accordance with and in order to comply with the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Techniques by the American Type Culture Collection (“ATCC”), P.O. Box 1549, Manassas, VA 20108 United States as International Depositary Office, January 6, 2009 (for PTA-9692, PTA-9693, PTA-9694) and March 24, 2009 (for PTA-9888), and they were given the above patent designations for escrow. As used herein, both deposited hybridomas and monoclonal antibodies produced by hybridomas can be referred to with the same designation for ATCC deposition or with numbers found among the designation for ATCC deposition. For example, PTA-9888 or 9888 can be used to refer to a deposited hybridoma or to a monoclonal antibody produced by a hybridoma. Accordingly, the names of the monoclonal antibodies described herein can be used interchangeably with the names of the hybridomas that produce them. One of ordinary skill in the art will understand when the name is intended to refer to an antibody or to a hybridoma that produces the antibody. Antigen binding sites provided herein include antigen binding sites of the aforementioned deposited antibodies.

The antibodies of the present invention exhibit a number of useful properties. For example, antibodies to toxin A neutralize or suppress the toxicity of toxin A both in vitro and in vivo. In vitro neutralization studies using IMR-90 cells humanized with RA-39 and humanized with RA-41 demonstrated neutralization efficiencies (for example, EC ₅₀ values) of 46 pM against toxin A on cells and 5 pM against toxin B on these cells , respectively. Compared with the values described in the literature for neutralizing human monoclonal antibodies to toxin A and toxin B (WO / 2006/121422; US2005 / 0287150; Babcock et al., Infect. Immun., 2006), (7), EC ₅₀ value the neutralization of 46 pM for hPA-39 was found to be higher than that described for the human mAb to toxin A, and the EC ₅₀ for the neutralization value of 5 pM hPA-41 was found to be higher than that described for the human mAb to toxin B. Accordingly, in the studies described herein, humanized monoclonal antibodies of the present invention to toxin C. difficile inu A and C. difficile toxin B, in particular the humanized forms of these monoclonal antibodies, show improved neutralization characteristics against toxins compared to other reported anti-toxin antibodies.

In one embodiment, the anti-Toxin A antibody of the present invention neutralizes or suppresses in vivo toxicity of C. difficile Toxin A at an effective dose. In another embodiment, antibodies to toxin B neutralize or suppress in vivo toxicity of toxin B. In an embodiment, an effective dose of one or more antibodies to toxin A of the present invention is provided to a subject infected with C. difficile. In an embodiment, an effective dose of one or more antibodies to toxin A of the present invention is provided in combination with an effective dose of one or more antibodies to toxin B of the present invention for a subject infected with C. difficile. In an embodiment, the anti-toxin A antibody of the present invention in combination 1: 1 with the anti-toxin B antibody of the present invention is provided as an effective dose for a subject infected with C. difficile. In an embodiment, an effective dose of an anti-toxin A antibody and anti-toxin B antibody of the present invention may be, for example, ½: 1, 1: 1, 2: 1, 3: 1, 4: 1, etc., a combination of antibodies provided for a subject infected with C. difficile. In an embodiment, the antibodies are humanized. In an embodiment, these antibodies are included in the composition. To illustrate, an effective dose of antibodies to toxin A and / or toxin B can range from 0.1 mg to 1000 milligrams (mg). Antibodies to toxin A and antibodies to toxin B or antigen binding sites 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 the amount of 1 mg / kg-50 mg / kg; in the amount of 2 mg / kg-40 mg / kg; in the amount of 2 mg / kg-50 mg / kg; in an amount of 5 mg / kg -50 mg / kg; in the amount of 5 mg / kg-25 mg / kg; in the amount of 10 mg / kg-40 mg / kg; in the amount of 10 mg / kg-50 mg / kg; in the amount of 10 mg / kg-25 mg / kg; or in an amount of 15 mg / kg-50 mg / kg. In an embodiment, the above amounts may include various ratios of antibodies to toxin A and antibodies to toxin B provided in combination.

As used herein, the term “neutralize” refers to reducing, suppressing, blocking, lowering the intensity or elimination of the adverse effect (s) of the toxin (s) that the antibody (s) specifically bind. The neutralization of the adverse effect (s) of the toxin (s) includes 1) delaying, reducing, suppressing or preventing the onset or progression of C. difficile infection or diarrhea or a disease associated with C. difficile, 2) increasing the survival of the subject compared to the average survival of the subjects that have not been treated with antibody (s) and that have a C. difficile infection or a disease associated with C. difficile, 3) eliminate one or more symptoms or adverse effects, or reduce the severity of one or not sharp symptoms or adverse effects associated with C. difficile infection or diarrhea, or a disease associated with C. difficile, 4) allowing the normal microflora of the gastrointestinal tract to repopulate subjects who are or have been infected with C. difficile, 5) prevention the re-manifestation of C. difficile infection or a disease associated with C. difficile in subjects who have suffered from a C. difficile infection or a disease associated with C. difficile, 6) a treatment efficacy met by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% of subjects who have C infection .difficile or disease associated with C. difficile and / or 7) prevention of death due to CDAD or other adverse events associated with C. difficile infection.

Antibodies to toxin A and toxin B. C. difficile of the present invention can be used to treat a large number of species of subjects, including humans (humans) and other non-humans (mammals) animals. Subjects to be treated in accordance with the present invention include humans, primates, other than humans, dogs, cats, mice, rats, hamsters, guinea pigs, cows, goats, sheep, pigs, horses and others. Human subjects may also be referred to herein as patients or individuals. In particular, the subjects include human patients having C. difficile infection or a disease associated with C. difficile. Such human patients include those who are elderly or immunocompromised.

In accordance with the present invention, antibodies to C. difficile toxin A and Toxin B can eliminate C. difficile disease and increase the survival of the subject. In one embodiment, one or more antibodies to toxin A and / or one or more antibodies to toxin B when administered to a subject, improves (improves) the survival of the subject compared to the average survival of subjects who have not been treated with antibody (antibodies) and who have infection C. difficile or a disease associated with C. difficile.

In some embodiments, the dose or amount of one or more antibodies to toxin A and toxin B can be, for example, in the range of 0.2 μg-250 μg, or 2 μg-50 μg, or 5 μg-50 μg, for example, based on in vivo studies in mice. In some embodiments, the dose or amount of one or more of its antibodies to toxin A and toxin B, and in particular, the combination of an antibody to toxin A and an antibody to toxin B can be, for example, in the range of 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, for example, based on studies on hamsters in vivo.

As another example, the antibodies of the present invention can perform a cure or survival rate of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99%. As another example, these antibodies can fulfill a cure rate or survival rate of 100%. In one embodiment, one or more antibodies to toxin A when administered to a subject, along with one or more antibodies to toxin B, perform a treatment effectiveness or survival rate of 50%, 55%, 60%, 65%, 70%, 75%, 80% , 85%, 90%, 95%, 97%, 99% or 100%. As used herein, “cure rate” refers to the percentage of subjects who, by the definition of a clinician, no longer have an infection or disease, from a population of subjects with an infection or disease who have been given one or more antibodies of the present invention or one or more of them compositions .. "Survival rate", as used herein, refers to the percentage of subjects that survive for the required period of time, from the population of subjects who were injected with one or not how many antibodies of the present invention or one or more of their compositions. Examples of such necessary time periods are provided elsewhere herein.

Antibodies to toxin A and toxin B provided by the present invention may provide an opportunity to restore normal intestinal flora in a subject infected with C. difficile. Thus, such antibodies can eliminate the disease in patients who are being treated. Antibodies to toxin A and toxin B of the present invention may also demonstrate favorable in vivo pharmacokinetics. Antibodies to toxin A and toxin B of the present invention can also provide prolonged or long-lasting therapy for a subject who has been infected with C. difficile. As used herein, the term “long lasting” refers to therapy that results in the absence of C. difficile infection or a disease associated with C. difficile one month or more after treatment is withdrawn. Preferably, the treatment results in the absence of C. difficile infection or a disease associated with C. difficile for two or more months. In some embodiments, therapy using the mAbs of the present invention results in the treatment or reduction of the activity of an active C. difficile infection and a decrease or decrease in the resistance of the infection. In other embodiments, the therapy provided by the present invention results in the absence of C. difficile infection or a disease associated with C. difficile in subjects for 1, 2, 3, 4, 5, or 6 months. In other embodiments, the therapy provided by the present invention results in the absence of a C. difficile infection or a disease associated with a C. difficile in a subject for more than 6 months. Antibodies to toxin A and toxin B of the present invention can prevent the re-manifestation of C. difficile infection and / or a disease associated with C. difficile.

The nature of the re-manifestation of CDAD is exacerbated by the advent of hypervirulent BI / NAP strains 1/027, which have been found to be resistant to two of the latest antibiotics of last resort, levofloxacin and moxifloxacin. Such strains caused outbreaks with increasing frequency throughout the United States, Canada and Western Europe. Hypervirulent strains include a group of closely related isolates characterized as North American Pulsed Field Type 1 (NAP1), type “BI” based on restriction mapping, and PCR ribotype 027, commonly known as BI / NAP1 / 027 (5). The hypervirulence of strains BI / NAP1 / 027 was explained at least in part by an increase in the production of toxins A and B, two virulent factors of CDAD (6). Isolates BI / NAP1 / 027 produce 16-23 times higher levels of toxins A and B than other strains (6). The apparent fitness of these strains poses a threat of spread around the world, jeopardizing the possibility of antibiotic treatment of other diseases, as well as an increase in the frequency of repeated manifestations and the severity of CDAD. Although there are antibiotics under development for the treatment of CDAD, such as nitazoxanide, rifaximin, ramoplanin, fidaxomycin, C. difficile clinical isolates that are resistant to rifaximin have been reported. In the recently completed phase 3 trial (91), fidaxomycin significantly reduced the overall frequency of re-onset of CDAD compared to vancomycin, but not in relation to strains BI / NAP1 / 027. Outbreaks of hypervirulent BI / NAP1 / 027 strains have led to an increase in hospitalization time, treatment failure, relapse rates and mortality rates (3). The new mAbs developed and described herein provide new therapies to combat the increasing incidence and severity of CDAD.

In an embodiment, the mAbs of the present invention are used in the treatment of infection caused by various C. difficile strains. In an embodiment, C. difficile strains are highly infectious and their toxins are neutralized by the mAbs of the present invention. In an embodiment, the toxins of the hypervirulent C. difficile strains, including BI / NAP1 / 027, are neutralized by the mAbs of the present invention. In an embodiment, the mAbs of the present invention provide a therapeutic effect in neutralizing toxins from a wide range of toxicogenic clinical isolates, including strains from outpatient isolates. Preferably, the mAbs neutralize the toxins of hypervirulent isolates, such as BI / NAP 1/027, and at least 90% or more of other clinically significant C. difficile cultures. In particular, and as illustrated in Example 8 herein, the mAbs of the present invention have been shown to significantly neutralize the toxicity / activity of nineteen different C. difficile clinical isolates, including BI / NAP 1/027 and other hypervirulent C. strains. difficile, e.g. CCL676, HMS553, Pitt45, CD196, montreal 5 and montreal 7.1. In accordance with the present invention, antibodies are provided that neutralize toxin A and toxin B of hypervirulent C. difficile strains, for example, without limitation, as determined by an EC ₅₀ value in the range of 7.7 ^-12 M to 4.8 ^-8 M for a mAb to toxin A and an EC ₅₀ value ranging from 1.1 ^-11 M to 6.5 ^-10 M for a mAb to toxin B. In addition, the mAbs of the present invention are provided for use in neutralizing hypervirulent C. difficile strains, including isolates of hospital and community origin, as a treatment for C. difficile infections and related diseases.

In other embodiments, and in a non-limiting example, the mAbs of the present invention show an EC ₅₀ neutralization value within 93 pM-30 nM or EC 5 about 46 pM to neutralize toxin A and an EC ₅₀ value ranging from 4 pM -9.5 pM, or EC ₅₀ 5 PM to neutralize toxin B, depending on the in vitro cell assay used. As described herein in Example 3, in an assay comprising CHO-K1 cells in which 8 μg / ml of toxin A was used, the mAb to toxin A of the present invention showed an EC _{50 of} 93 pM. In an assay involving CHO-K1 cells in which 8 pg / ml of toxin B was used, the mAb to toxin B of the present invention showed an EC ₅₀ value of 9.2 pM. In an assay involving T-84 cells in which 240 ng / ml of toxin A was used, the mAb to toxin A of the present invention showed an EC _{50 of} 146 pM and 175 pM. In a Caco-2 cell based assay, the mAb to toxin A of the present invention neutralized the toxicity of toxin A at EC levels of ₅₀ 196 pM and 485 pM. In red blood cells, a hemagglutination assay using 8 μg / ml of toxin A, mAb to toxin A of the present invention had an EC neutralization of _{50 of} 1.8 nM and 30 nM to prevent RBC hemagglutination.

The antibodies of the present invention may possess any of, combinations, or all of the above features.

As described in the examples herein, the mAbs of the present invention demonstrate superior in vitro and in vivo neutralization of toxins in the most affordable preclinical CDAD models. In addition, the mAbs of the invention demonstrate a uniquely extensive and effective neutralization of toxins from numerous strains of BI / NAP1 / 027. Moreover, such mAbs have demonstrated complete and long-lasting protection against mortality in high-precision hamster CDAD models. These results confirm the ability of the mAbs of the present invention to sufficiently and efficiently block the pathogenic effects of C. difficile toxins in such a way that they allow colon healing, restore normal intestinal microflora and eliminate CDAD disease and / or C. difficile infection.

In an embodiment, anti-toxin A antibodies include those that competitively suppress or cross-hair compete for specific binding to C. difficile toxin A of an isolated monoclonal antibody produced by a hybridoma cell line, deposited under accession number ATCC PTA-9692, PTA-9694 or PTA- 9888. Preferred antibodies to toxin B include those that competitively inhibit or cross-compete for specific binding to C. difficile toxin B of an isolated monoclonal antibody produced by a hybridoma cell line deposited under ATCC accession number PTA-9693 or PTA-9692. In some embodiments, antibodies include those that competitively inhibit or cross-compete for specific binding to C. difficile toxin A of an isolated monoclonal antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9692, PTA-9694 or PTA-9888, and competitively inhibit or cross-compete for specific binding to C. difficile Toxin B of an isolated monoclonal antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9693 and and PTA-9692. All embodiments further include humanized forms of the antibodies described above under Access Access No. ATCC PTA-9692, PTA-9694, PTA-9888 or PTA-9693.

Various assays known to those skilled in the art can be used to determine competitive inhibition or cross-competition of binding. For example, cross-competition assays can be used to determine if an antibody inhibits competitive binding to toxin A and / or toxin B of other antibodies. Such methods can be cellular methods using flow cytometry or solid-phase binding analysis. Other assays that evaluate the ability of antibodies to cross-compete for the binding of toxin A and / or toxin B in the solid phase or in the liquid phase can also be used. Examples of antibodies or antigen binding fragments thereof that are encompassed by the present invention include those that competitively inhibit specific binding by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90 %, 95% or 99%. The suppression can be evaluated at various molar ratios or mass ratios, for example, experiments with competitive binding can be performed with 2-fold, 3-fold, 4-fold, 5-fold, 7-fold, 10-fold or large molar excess of the first antibody in compared to the second antibody.

Other antibodies that are encompassed by the present invention include those that specifically bind to an epitope on C. difficile toxin A, defined by the binding of an isolated monoclonal antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9692, PTA-9694 or PTA-9888 . However, other antibodies or antigen binding fragments that are encompassed by the present invention include those that specifically bind to an epitope on C. difficile Toxin B, determined by the binding of an isolated monoclonal antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9693 or PTA -9692. However, other antibodies or antigen binding fragments that are encompassed by the present invention include those that specifically bind to an epitope on C. difficile toxin A, defined by the binding of an isolated monoclonal antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9692, PTA -9694, or PTA-9888, and specifically bound to an epitope on C. difficile Toxin B, determined by binding of an isolated monoclonal antibody produced by a hybridoma cell line deposited under access ATCC PTA-9693 or PTA-9692.

To determine the epitope, standard epitope mapping techniques known in the art can be used. For example, toxin fragments (peptides) (preferably synthetic peptides) that bind the antibody can be used to determine whether the antibody of interest or its antigen-binding fragment binds the same epitope. For linear epitopes, overlapping peptides of a certain length (for example, 8 or more amino acids) are synthesized. The peptides are preferably shifted by 1 amino acid, so that a series of peptides covering every 8 amino acid fragments from the toxin sequence is obtained. Fewer peptides can be obtained using larger biases, for example, by 2 or 3 amino acids. In addition, longer peptides can be synthesized (for example, 9-, 10- or 11-dimensional). The binding of peptides to antibodies can be determined using standard techniques, including surface plasma resonance (e.g., Biacore) and ELISA assays. Large fragments of peptides can be used to study conformational epitopes that the antibodies provided herein can bind in some embodiments. Other methods that use mass spectrometry to determine conformational epitopes have been described and can be used (see, for example, Baerga-Ortiz et al., Protein Science 11: 1300-1308, 2002 and references cited in this document) . However, other methods for determining epitopes are provided in reference lab standards, such as in Section 6.8 ("Phage Display Selection and Analysis of B-cell Epitopes") and in Section 9.8 ("Identification of Antigenic Determinants Using Synthetic Peptide Combinatorial Libraries ") Current Protocols in Immunology, Coligan et al., Eds., John Wiley & Sons. Epitopes can be confirmed by introducing one or more point mutations or deletions into a known epitope, and then testing the binding to one or more antibodies to determine which mutations reduce the binding of antibodies.

Antibodies or antigen binding fragments provided by the present invention may specifically bind toxin A and / or toxin B with subnanomolar affinity. Antibodies or antigen binding fragments can have a binding affinity of about 1 × 10 ^-9 M or less, about 1 × 10 ^-10 M or less, or about 1 × 10 ^-11 M or less. In a separate embodiment, the binding affinity is less than about 5 × 10 ⁻¹⁰ M.

Antibodies or antigen binding sites may have an association rate constant (Kon) for toxin A or toxin B of at least 10 ² M ^-1 s ^-1 ; at least 10 ³ M ^-1 s ^-1 ; at least 10 ⁴ M ^-1 s ^-1 ; at least 10 ⁵ M ^-1 s ^-1 ; at least 10 ⁶ M ^-1 s ^-1 ; or at least 10 ⁷ M ⁻¹ s ⁻¹ , measured by surface plasma resonance. Antibodies or antigen binding fragments may have a dissociation rate constant (Koff) for toxin A or toxin B not greater than 10 ⁻³ s ⁻¹ ; not more than 10 ⁻⁴ s ⁻¹ ; not more than 10 ⁻⁵ s ⁻¹ ; or no more than 10 ⁶ s ^-1 measured by surface plasmon resonance. Antibodies or antigen binding fragments may have a dissociation constant (K _D ) for a 7 A toxin or Toxin B no greater than 10 ⁻⁷ M; no more than 10 ^-8 M; no more than 10 ^-9 M; no more than 10 ^-10 M; no more than 10 ^-11 M; no more than 10 ^-12 M; or no more than 10 ^-13 M.

As used herein, the expressions “antibody” or “immunoglobulin” include glycoproteins containing at least two heavy (H) polypeptide chains and two light (L) polypeptide chains connected by disulfide bonds. Each heavy chain contains a variable region of the heavy chain (abbreviated herein as HCVR or V _H ) and a constant region of the heavy chain (C _H ). The constant region of the heavy chain consists of three domains CH1, CH2 and CH3. Each light chain consists of a variable region of the light chain (abbreviated herein as LCVR or V _L ) and a constant region of the light chain (C _L ). The constant region of the light chain consists of one domain, CL. The V _H and V _L regions can be further subdivided into hypervariability regions called hypervariable regions (CDRs), alternating with regions that are more conservative, called framework regions (FR). Each V _H and V _L consists of three CDRs and four FRs located from the amino end to the carboxy end in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In total, the variable regions of the heavy and light chain polypeptides contain or form a binding domain that interacts with / binds the antigen. Antibody constant regions can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system.

In addition, the present invention provides other forms of antibodies, such as single chain antibodies, recombinantly produced antibodies, bispecific, heterospecific or multimeric antibodies, diabodies, etc., as described later in this document.

The expression “antigen binding fragment” of an antibody, as used herein, refers to one or more portions of an antibody that retains the ability to specifically bind to an antigen (eg, toxin A, toxin B, toxin A and toxin B, etc.) or epitope areas of antigen. It has been shown that the antigen-binding function of an antibody can be carried out by full-length antibody fragments. In an embodiment, fragments of monoclonal antibodies function in a manner similar to intact analogous monoclonal antibodies. In an embodiment, fragments of monoclonal antibodies of the crosshairs react with intact analogous monoclonal antibodies. In an embodiment, fragments of monoclonal antibodies can be used interchangeably with intact analogous monoclonal antibodies. Examples of binding fragments encompassed by 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 1 domains; (ii) a F (ab ′) ₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) a Fd fragment consisting of the V _H and CH1 domains; (iv) an 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 hypervariable region (CDR). Moreover, although the two domains of the Fv fragment, V _L and V _H , are encoded by separate genes, they can be combined using recombinant methods using a synthetic linker that allows them to form a single protein chain in which the V _L and V _H regions are arranged in pairs for the formation of monovalent molecules (known as single chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85 : 5879-5883). It is contemplated that such single chain antibodies are encompassed by the expression “antigen binding fragment” of an antibody. These antibody fragments are obtained using conventional techniques, such as proteolytic fragmentation techniques, as described in J. Coding, Monoclonal Antibodies: Principles and Practice, pp 98-118 (NY Academic Press 1983), which is incorporated herein by reference and using other techniques known to those skilled in the art. Fragments are screened for activity or practicality in the same manner as intact antibodies.

In an embodiment, Fab mAb fragments of the present invention were created and tested for neutralization activity in cell assays as described in Example 10 herein. Thus, antibody fragments, such as Fab Fab fragments of the present invention, can also be used to bind and neutralize C. difficile Toxin A and / or Toxin B.

An “isolated antibody”, as used herein, is intended to mean an antibody that essentially does not contain other antibodies having different antigenic specificities (eg, an isolated antibody that specifically binds to toxin A essentially does not contain 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, but generally has cross-reactivity to other related antigens, for example, from other C. difficile strains. In addition, an isolated antibody that specifically binds to an epitope, isoform or variant of toxin A can also specifically bind toxin B, and an isolated antibody that specifically binds to an epitope, isoform or variant of toxin B can also specifically bind toxin A. In some embodiments, however, the isolated antibody or its antigen binding fragment that specifically binds to an epitope, isoform or variant of toxin A also does not specifically bind toxin B. In other still other variants, schestvleniya isolated antibody or antigen binding fragment thereof which specifically binds to an epitope, isoform or variant of toxin B is also not specifically binds toxin A. Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals. Antibodies that essentially do not contain other antibodies having different antigenic specificities, or other materials and / or chemicals, and / or proteins, can be isolated and / or purified antibodies. Antibodies can be purified by methods that are usually carried out by specialists in this field, for example, affinity chromatography, chromatography on protein A and the like. As used herein, “specific binding” refers to the binding of antibodies to a predetermined or related antigen. Typically, an antibody binds to an affinity that is at least two times greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than a predetermined antigen or closely related antigen. In an embodiment, an antibody of the present invention can bind a linear epitope of a target antigen, for example, toxin A and / or toxin B. In an embodiment, an antibody of the present invention can bind a conformational epitope of a target antigen, for example, toxin A and / or toxin B.

The isolated antibodies of the present invention encompass various isotypes of the heavy and light chains of antibodies (immunoglobulins), such as classes of heavy chains or isotypes of IgGI, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, IgE and their subtypes, for example, IgG2a, IgG2b; and isotypes of light chains k and X and their subtypes. In one embodiment, the isolated antibodies are isotype IgG2a or IgG1k. As used herein, an “isotype” refers to a class of antibodies (eg, IgM or IgGI, or λ1), which are encoded by the constant and light chain constant region genes. Antibodies or their antigen binding regions may be full length or may include only an antigen binding fragment, such as a constant and / or variable domain of antibodies IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD or IgE, or may consist of a fragment Fab, F (ab ') ₂ fragment and Fv fragment.

The antibodies of the present invention can be polyclonal, monoclonal, or a mixture of polyclonal and monoclonal antibodies. Antibodies can be obtained by various methods well known in the art. Techniques for inducing polyclonal antibodies are well known. By way of non-limiting example, polyclonal antibodies are induced by administering a protein of toxin A and / or toxin B subcutaneously to New Zealand white rabbits from which blood is first taken to produce pre-immune serum. Toxin A and / or toxin B can be injected in a total volume of 100 μl per plot in six different patches, typically with one or more adjuvants. Then, rabbits are bled two weeks after the first injection and the booster is periodically immunized with the same antigen three times every six weeks. A serum sample is collected 10 days after each booster immunization. Polyclonal antibodies are obtained from serum, preferably by affinity chromatography using toxin A and / or toxin B to capture the antibody. This and other polyclonal antibody induction techniques 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 incorporated herein by links.

The production of monoclonal antibodies can be carried out using methods that are also well known in the art. The expression "monoclonal antibody", as used herein, refers to the production of antibody molecules of the same molecular composition. A monoclonal antibody exhibits the only binding specificity and affinity for a single epitope of a given antigen or immunogen. The production process of monoclonal antibodies involves the production of immune somatic cells with the potential for the production of antibodies, in particular B lymphocytes, which were previously immunized with an antigen of interest either in vivo or in vitro, or both, which are suitable for fusion with B- cells of the myeloma line. Monoclonal antibodies can be obtained using immune cells and myeloma cells from various species, such as mouse and human cells and cell lines, or, for example, in mouse strains that have been genetically modified to support the human immune system, as described below.

Although monoclonal antibodies directed against toxin A and toxin B were typically obtained by immunizing animals with toxins (inactive forms of toxin A and toxin B) and / or inactive fragments of these toxins, the mAbs of the present invention were created by developing and using a new immunization strategy. In accordance with the present invention, the mAbs described herein and deposited were obtained by immunizing animals with toxoids followed by booster immunization of animals with an active (non-toxoid) form of toxin A and / or toxin B (see Example 1 herein). Booster immunization with the active form of toxin A and toxin B facilitated the identification of those immunized animals that produced unique protective antibodies by virtue of the new immunization schedule. Not wishing to be bound by theory, the booster immunization regimen with active toxin A and / or toxin B was more highly immunogenic in recipient animals. Those animals that tolerated increasing doses of the booster immunization of active toxin A and toxin B, which are typically detailed for animals not previously used in the experiments, produced highly effective neutralizing antibodies that protected these animals and contributed to their survival, despite the fact that they received an active toxin. The production of hybridomas from animals that showed an effective immune response against toxin A or toxin B gave highly effective monoclonal antibodies to toxin A and toxin B, which provide a high level of protection both in vitro and in vivo. In preparing antibodies, including polyclonal and monoclonal antibodies, adjuvants may be used. Non-limiting examples of adjuvants that are suitable for use include Freund's incomplete adjuvant, aluminum phosphate, aluminum hydroxide, Ribi (e.g. monophosphoryl lipid A, trehalose dimicolate, mycobacterium cell wall skeleton and Tween® 80, with 2% squalene), saponins, Quil A or alum. The response of cytotoxic T lymphocytes (CTLs) can be primed by conjugation of toxins (or fragments, inactive derivatives or their analogues) with lipids, such as, for example, tripalmitoyl-8-glycerylcysteinylserylserine.

In other embodiments, additional immunization methods can be used to create monoclonal antibodies directed against toxin A and / or toxin B. For example, in vivo immunization of animals (e.g., mice) can be carried out with the desired type and amount of protein or polypeptide, e.g., toxoid or toxin. Such immunizations, if necessary, are repeated at intervals of several weeks to obtain a sufficient antibody titer. After immunization, animals can be used as a source of antibody-producing lymphocytes. After the last booster immunization with antigens, animals are sacrificed and spleen cells are removed. Murine lymphocytes give a higher percentage of stable fusions to the murine myeloma lines described herein. Among them, the BALB / c mouse line is suitable. However, other mouse lines, rabbits, hamsters, sheep, goats, and frogs can also be used as recipients for the production of antibody-producing cells. Cm.; Coding (in Monoclonal Antibodies: Principles and Practice, 2d ed., Pp. 60-61, Orlando, Fla., Academic Press, 1986). In particular, lines of mice that have human immunoglobulin genes integrated in the genome (and which cannot produce murine immunoglobulins) can be used. Examples include HumAb mouse lines obtained by Medarex (now Bristol Myers Squibb) / GenPharm International, and XenoMouse lines obtained by Abgenix. Such mice produce fully human immunoglobulin molecules in response to immunization.

Those antibody-producing cells that are in the stage of dividing plasmoblast preferably fuse. Somatic cells can be obtained, for example, from the lymph nodes, spleens and peripheral blood of antigen-primed animals, and the preferred lymphatic cells depend to a large extent on their empirical applicability in a particular fusion system. Antibody-secreting lymphocytes are then fused to (mouse) myeloma B cells or transformed cells that are capable of replication indefinitely in cell culture, thereby creating an immortalized immunoglobulin-secreting cell line. The resulting fusion cells or hybridomas are cultured, and the resulting colonies are screened for the production of the necessary monoclonal antibodies. Colonies producing such antibodies are cloned, subcloned and grown both in vivo (as ascites) and in vitro to produce large quantities of the antibody. Descriptions of the hybridization methodology and technology can 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 incorporated herein by reference.

Alternatively, human somatic cells capable of producing an antibody, in particular B lymphocytes, are suitable for fusion with myeloma cell lines. While B lymphocytes from biopsied spleens, tonsils or lymph nodes of an individual can be used, more readily available peripheral blood B lymphocytes (PBLs) are preferred. In addition, human B cells can 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 techniques are preferred, in principle, other methods for producing monoclonal antibodies, such as viral or oncogenic transformation of B lymphocytes, can be used.

Myeloma cell lines suitable for use in hybridoma fusion techniques are preferably non-antibody producing, have high fusion efficiency and enzyme deficiencies that render them incapable of growth in certain selective media that support the growth of the required hybridomas. Examples of such myeloma cell lines that can be used to produce fused cell lines include P3-X63 / Ag8, X63-Ag8.653, NSl / l.Ag 4.1, Sp2 / 0-Agl4, FO, NSO / U, MPC- 11, MRSP-X45-GTG 1.7, S194 / 5XX0 Bul obtained from mice; R210.RCY3, Y3-Ag 1.2.3, IR983F and 4 B210 obtained from rats; and U-266, GM1500-GRG2, LICR-LON-HMy2, UC729-6, obtained from humans (Goding, in Monoclonal Antibodies: Principles and Practice, 2d ed., pp. 65-66, Orlando, Fla., Academic Press 1986, Campbell, Monoclonal Antibody Technology, Laboratory Techniques in Biochemistry and Molecular Biology Vol. 13, Burden and Von Knippenberg, eds. Pp. 75-83, Amsterdam, Elseview, 1984).

Merging with mammalian myeloma cells or other fusion partners that can replicate indefinitely in cell culture is accomplished by standard and well-known methods, for example, using polyethylene glycol ("PEG") or other fusion agents (see Milstein and Kohler, Eur. J. Immunol. 6: 511 (1976), which is incorporated by reference).

In other embodiments, the antibodies may be recombinant antibodies. The expression "recombinant antibodies", as used herein, is intended to include antibodies that are obtained, expressed, created or isolated by recombinant methods, such as antibodies isolated from animals (e.g. mice), which are transgenic for other types of immunoglobulins, antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial antibody library, or antibodies, obtained those expressed, created, or isolated by any other means that include splicing immunoglobulin gene sequences with other DNA sequences.

In still other embodiments, the antibodies may be chimeric or humanized antibodies. As used herein, the expression “chimeric antibody” refers to an antibody that combines the variable or hypervariable regions of a mouse immunoglobulin (Ig) with a constant region of human Ig, or constant and variable frame regions. In some embodiments, the chimeric antibody comprises a variable region of any of the antibodies deposited herein, and a human constant region. In some embodiments, the human constant region is a constant region of human IgG, such as a constant region of human IgGI. Chimeric antibodies can be obtained by the method described in the examples below, or by any method known to those skilled in the art. As used herein, the term “humanized antibody” refers to an antibody that retains essentially only antigen binding CDRs from the original antibody, for example, a murine monoclonal antibody in association with human framework regions (see, for example, Waldmaim, 1991, Science 252: 1657). Such chimeric or humanized antibodies that retain the binding specificity of a murine antibody but have constant / framework regions of human Ig are expected to have reduced immunogenicity when administered in vivo. Thus, chimeric and humanized antibodies preferably retain the toxin-neutralizing activity of the provided monoclonal antibodies and are suitable for repeated dosing (for example, in humans). One of skill in the art can use known methods (e.g., in vitro cell assays) to compare the activity of humanized antibodies with the deposited monoclonal antibodies described herein and to determine whether humanized antibodies are treated and / or to prevent recurrence of established C. difficile infection . One of skill in the art can also use the methods described herein, including the hamster C. difficile infection model described below.

Humanized mAb sequences can be constructed using the following illustrative, non-limiting method. First, framework amino acid residues that are important for the structure of CDRs are identified. In parallel, human V _H and V _L sequences having high homology with respect to mouse V _H and V _L , respectively, are selected from sequences of known human immunoglobulins (germ line). CDR sequences from mouse mAbs, together with frame amino acid residues that are important for maintaining the structure of CDRs, are transplanted into selected human frame sequences. In addition, human framework amino acid residues that have been found to be atypical in the corresponding subgroup V region are replaced with typical residues to reduce the potential immunogenicity of the resulting humanized mAb. These humanized regions of V _H and V _L are cloned into expression vectors, for example, 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 harvested after transfection, for example, three days later, and purified using protein A chromatography. Other expression vectors and host cells can be used to recombinantly produce the antibodies described, as will be appreciated by those skilled in the art .

Other methods for humanizing antibodies or antigen binding fragments are well known in the art and include methods that are described, for example, in US Pat. Nos. 5,550,089; 5,693,761; 5,693,762; and 6,180,370. The humanization techniques that are described in these patents are incorporated herein by reference in their entirety. Antibodies or antigen binding fragments, humanized in accordance with the methods that are described in these patents, are also given in this document.

In an embodiment, the humanized mAb (hmAb) to C. difficile toxin A of the present invention encompasses an immunoglobulin protein or fragment thereof, which consists of (i) two heavy (H) chain polypeptides, where each H chain contains a VH region comprising an amino acid sequence , which is set forth in SEQ ID NO: 1, and the human CH region, for example, the IgG1 C region, and (ii) two light polypeptides (L) chains, where each L chain contains a VL region including an amino acid sequence that set forth in SEQ ID NO: 3, and the CL region of a person, for example, C- Blast κ-chain. In an embodiment, the humanized C. difficile toxin A mAb of the present invention encompasses an immunoglobulin protein or fragment thereof, which consists of (i) two heavy (H) chain polypeptides, where each H chain contains a VH region comprising the amino acid sequence set forth in SEQ ID NO: 2 and the human CH region, for example, the IgG1 C region, and (ii) two light polypeptides (L) chains, where each L chain contains a VL region including the amino acid sequence set forth in SEQ ID NO: 3 and the human CL region, for example, C-region κ chains. In an embodiment, the humanized C. difficile toxin A mAb of the present invention encompasses an immunoglobulin protein consisting of (i) two heavy (H) chain polypeptides, where each H chain contains a VH region comprising the amino acid sequence set forth in SEQ ID NO: 1 and human CH region, for example, the IgG1 C region, and (ii) two light polypeptides (L) chains, where each L chain contains a VL region including the amino acid sequence set forth in SEQ ID NO: 4 and the human CL region, for example, the C region of the κ chain. In an embodiment, the humanized C. difficile toxin A mAb of the present invention encompasses an immunoglobulin protein that consists of (i) two heavy (H) chain polypeptides, where each H chain contains a VH region comprising the amino acid sequence set forth in SEQ ID NO: 2 and human CH region, for example, the IgG1 C region, and (ii) two light polypeptides (L) chains, where each L chain contains a VL region including the amino acid sequence set forth in SEQ ID NO: 4 , and the human CL region, for example, the C region of the κ chain. Such humanized C. difficile toxin A mAbs encompass the hPA-39 mAbs of the present invention.

In an embodiment, the humanized C. difficile toxin A mAb of the present invention encompasses an immunoglobulin protein that consists of (i) two heavy (H) chain polypeptides, where each H chain contains a VH region comprising the amino acid sequence set forth in SEQ ID NO: 5 and the human CH region, for example, the IgG1 C region, and (ii) two light polypeptides (L) chains, where each L chain contains a VL region including the amino acid sequence set forth in SEQ ID NO: 7 and the human CL region, for example, the C region of the κ chain. In an embodiment, the humanized C. difficile toxin A mAb of the present invention encompasses an immunoglobulin protein that consists of (i) two heavy (H) chain polypeptides, where each H chain contains a VH region comprising an amino acid sequence as set forth in SEQ ID NO: 6 and the human CH region, for example, the IgG1 C region, and (ii) two light polypeptides (L) chains, where each L chain contains a VL region including the amino acid sequence set forth in SEQ ID NO: 7 and the human CL region, for example, the C region of the κ chain. Such humanized C. difficile toxin A mAbs encompass the hPA-50 mAbs of the present invention.

In an embodiment, a humanized C. difficile toxin B mAb of the present invention encompasses an immunoglobulin protein that consists of (i) two heavy (H) chain polypeptides, where each H chain contains a VH region comprising the amino acid sequence set forth in SEQ ID NO: 8 and human CH region, for example, the IgG1 C region, and (ii) two light polypeptides (L) chains, where each L chain contains a VL region including the amino acid sequence set forth in SEQ ID NO: 10 and the human CL region, for example, the C region of the κ chain. In an embodiment, a humanized C. difficile toxin B mAb of the present invention encompasses an immunoglobulin protein that consists of (i) two heavy (H) chain polypeptides, where each H chain contains a VH region comprising the amino acid sequence set forth in SEQ ID NO: 9 and human CH region, for example, the IgG1 C region, and (ii) two light (L) chain polypeptides, where each L chain contains a VL region including the amino acid sequence set forth in SEQ ID NO: 10 and the human CL region, for example, the C region of the κ chain. Such humanized C. difficile toxin A mAbs encompass the bPA-41 mAbs of the present invention.

The C regions of the L chain and the H chains of the above-described humanized antibodies of the present invention may include the C region (C _L ) of the human L chain and the C region (C _H ) of the human IgGI H chain having sequences contained in Genbank under access no. NW_001838785 and in Genbank under access no. MW_001838121, respectively. In other embodiments, humanized antibodies include a human H chain C region selected from IgG2a, IgG2b, IgG3 or IgG4 isotypes.

In an illustrative embodiment, the present invention encompasses a monoclonal antibody or fragment thereof generated against C. difficile toxin A, wherein the antibody consists of two heavy chain polypeptides, each heavy chain containing a human VH region and a CH region, and two light chain polypeptides wherein each light chain contains a VL region and a human CL region. The nucleic acid sequence (or cDNA) encoding the subsequent 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 subsequent amino acid sequence of the light chain polypeptide of SEQ ID NO: 16 is set forth in SEQ ID NO: 17 (Fig. 38A).

In an illustrative embodiment, the present invention encompasses a monoclonal antibody or fragment thereof generated against C. difficile toxin A, wherein the antibody consists of two heavy chain polypeptides, each heavy chain containing a human VH region and a CH region, and two light chain polypeptides wherein each light chain contains a VL region and a human CL region. The nucleic acid sequence (or cDNA) encoding the subsequent 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 subsequent amino acid sequence of the light chain polypeptide of SEQ ID NO: 20 is set forth in SEQ ID NO: 21 (Fig. 39A).

In an illustrative embodiment, the present invention encompasses a monoclonal antibody or fragment thereof directed to C. difficile Toxin B, wherein the antibody consists of two heavy chain polypeptides, each heavy chain containing a human VH region and a CH region, and two light chain polypeptides wherein each light chain contains a VL region and a human CL region. The nucleic acid sequence (or cDNA) encoding the subsequent 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 subsequent amino acid sequence of the light chain polypeptide of SEQ ID NO: 24 is set forth in SEQ ID NO: 25 (Fig. 40A).

The present invention also encompasses portions or fragments of the above-described humanized antibodies to Toxin A and Toxin B of C. difficile. Such portions or fragments include the hypervariable regions (CDRs) of the V regions of the polypeptides of both H chains and L chains, as those skilled in the art can determine in the usual way; P (ab) fragments, F (ab ') fragments, P (ab') ₂ fragments, Fc fragments, Fd fragments, etc. In an embodiment, portions or fragments of humanized antibodies containing V regions or their functional parts will optimally bind to the corresponding toxin and neutralize the toxin activity. In an embodiment, such functional parts or fragments of humanized antibodies optimally neutralize the toxin activity at a level similar to that of a complete humanized antibody, if not better.

In accordance with the present invention, molecularly cloned, humanized mAbs directed against C. difficile toxins A or B are provided. Such humanized mAbs were isolated and characterized as described in Example 9, Sections D and E below herein. In an embodiment, the constant region (CL) of the light chain of each of the humanized antibodies is in the kappa (k) class. In an embodiment, the constant region (CH) of the heavy chain of each of the humanized antibodies is of the IgG1 isotype. In other embodiments, the implementation of the CH region of humanized antibodies refers to the isotype IgG2a, IgG2b, IgG3, IgG4, IgA, IgE, IgA or IgM. It was found that humanized mAbs containing unique variable (V) regions bind and neutralize the activity of either toxin A or toxin B of C. difficile. The VL and VH regions of humanized mAbs can form part of a complete immunoglobulin (Ig) molecule or antibody, or they can be used as parts or fragments of an antibody, in particular parts or fragments having binding and / or neutralizing activity. Non-limiting examples of antibody fragments include Fab, F (ab) 2, and F (ab ′) or F (ab ′) 2 fragments. Embodiments of the present invention are directed to humanized C. difficile toxin A mAbs, or fragments thereof having anti-C. difficile toxin A activity, wherein the L chain V region is selected from one or more of SEQ ID NO: 3, SEQ ID NO : 4 and SEQ ID NO: 7. In an embodiment, the present invention is directed to CDRs, namely CDR1, CDR2, and / or CDR3, in the VH and VL regions of the described antibodies. Embodiments of the present invention are directed to humanized C. difficile toxin B toxin B, or fragments thereof having anti-C. difficile toxin B activity, wherein the L chain V region is set forth in SEQ ID NO: 10. Embodiments of the present invention are directed to humanized C. difficile toxin A mAbs, or fragments thereof having anti-C. difficile toxin A activity, wherein the H chain V region is selected from one or more of SEQ ID NO: 1, SEQ ID NO : 2, SEQ ID NO: 5 and SEQ ID NO: 6. Embodiments of the present invention are directed to humanized C. difficile toxin B toxin B or fragments thereof having anti-C. difficile toxin B activity, wherein the H chain V region is selected from one or more of SEQ ID NO: 8 or SEQ ID NO :9. In an embodiment, the invention is directed to CDRs, namely CDR1, CDR2, and / or CDR3, in the VH and VL regions of the described antibodies.

In another embodiment, the present invention encompasses nucleic acids that encode antigen binding parts, CDRs, or variable (V) regions of antibodies to C. difficile toxin A and / or Toxin B of the present invention. In various embodiments, portions of the CDR or V region are derived from PA-38, PA-39, PA-41, or RA-50, or their humanized variants described herein. In further embodiments, the present invention encompasses amino acid sequences of antigen binding parts, CDRs, or V regions that are encoded by the corresponding nucleic acids.

In accordance with another embodiment, 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. Non-limiting examples of bispecific and heterospecific antibodies and procedures for preparing such antibodies can be found in a number of publications illustrating similar examples, for example, UA20090060910, WO 2009/058383, WO 2009/030734, WO 2007/093630, USP 6071517, WO 2008/024188, UA 20070071675, USP 7442778, USP 7235641, USP 5932448 and USP 5292668. The term “bispecific antibody” is understood to include any agent, for example, a protein, peptide, or a protein or peptide complex that has two different binding specificities, and which binds to or interacts with ( a) C. difficile toxin A and (b) C. difficile toxin B . In one embodiment, the bispecific antibody comprises PA-39 or PA-50, or an antigen binding fragment thereof, and PA-41, or an antigen binding fragment thereof. In an embodiment, the bispecific antibody comprises a chimeric or humanized form of RA-39 or RA-50, or an antigen binding fragment thereof, and RA-41, or its antigen binding fragment. Accordingly, a bispecific antibody comprising PA-39 and PA-41, or their chimeric or humanized forms, or an antigen-binding fragment thereof, will bind both toxin A and C. difficile B toxin. Similarly, a bispecific antibody comprising PA-50 and PA-41, or their chimeric or humanized forms, or an antigen-binding fragment thereof, will bind both toxin A and C. difficile Toxin B. The term “multispecific antibody” is understood to include any agent, for example, a protein, peptide, or protein or peptide complex that has more than two different binding specificities and that binds to or interacts with (a) C. difficile toxin A, (b) toxin In C. difficile and (c) at least one other component. Accordingly, the present invention includes, but is not limited to, bispecific, trispecific, tetraspecific and other multispecific antibodies. In one embodiment, the antibodies or antigen binding fragments of the bispecific or multispecific antibodies are humanized.

The term “bispecific antibodies” further includes diabodies. Diabodies provide therapeutic antibodies that have dual specificity and are capable of targeting many different epitopes in a single molecule. Diabodies are bivalent, bispecific antibodies in which the V _H and V _L domains are expressed on the same polypeptide chain, but using a linker that is too short to allow pairing of two domains on the same chain, thereby forcing the domains to mate with complementary domains another chain and creating two antigen-binding sites (see, for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poijak, RJ, et al. (1994) Structure 2: 1121-1123). Two antigen-binding regions of a bispecific antibody are either chemically linked or expressed by a cell created by genetic engineering to produce a bispecific antibody. (see, in general, Fanger et al., 1995 Drug News & Perspec. 8 (3): 133-137). In an embodiment, an effective amount of a bispecific antibody can be administered to a subject with C. difficile infection and / or a disease associated with C. difficile, and the bispecific antibody neutralizes the toxicity of toxin A and toxin B in the subject.

In certain embodiments, the antibodies may be human antibodies. The term "human antibody", as used herein, is understood to include antibodies having variable and constant Ig regions derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced randomly or by site-specific in vitro mutagenesis or in vivo somatic mutation). However, the term “human antibody” as used herein is not understood to include antibodies in which CDR sequences derived from the germ line of another mammalian species, such as mice, 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 obtained using transgenic mice genetically modified and bred for expression of the components of the human immune system, and not the mouse system.

Fully human monoclonal antibodies can also be obtained by immunizing mice transgenic to large portions of the human immunoglobulin heavy and light chain loci. See, for example, US patents 5591669, 5598369, 5545806, 5545807, 6150584 and references cited therein, the contents of which are incorporated herein by reference. These animals were genetically modified in such a way that there is a functional deletion in the production of endogenous (eg, murine) antibodies. Animals are further modified to contain all or part of the locus of the human germline immunoglobulin gene, so immunization of these animals results in the production of fully human antibodies to the antigen of interest. After immunization with these mice (e.g., XenoMouse (Abgenix), HumAb (Medarex / GenPharm) mice), monoclonal antibodies are prepared according to standard hybridoma technology. These monoclonal antibodies have the amino acid sequences of a human immunoglobulin and therefore will not provoke a human humoral immune response to a murine antibody (NAMA) when administered to humans.

Those skilled in the art will understand that nucleic acids and polynucleotides that encode the described antibodies or antigen binding fragments thereof are also provided herein. It will also be understood that nucleic acids and polynucleotides are provided herein that contain a sequence encoding antibodies or antigen binding fragments thereof. Therefore, the present invention provides vectors and plasmids designed to contain and / or express antibody-encoding nucleic acids and polynucleotides. As used herein, a “coding region” refers to a region of a nucleotide sequence that encodes a polypeptide sequence;

the coding region may include a region encoding a portion of a protein that is later cleaved, such as, for example, a signal peptide. In some cases, the nucleotide and amino acid sequences may include sequences that encode signal peptides or which are signal peptides. The present invention includes each of these sequences with or without part of a sequence that encodes a signal peptide or is a signal peptide.

Antibodies provided herein can be cloned using the following method, as well as other methods known to those of ordinary skill in the art. As a non-limiting example, total RNA is obtained from hybridoma cells and cDNA is transcribed back using an oligo-dT primer. RNase H can be used to remove RNA to produce single stranded cDNA. To remove free nucleotides, purification using a centrifuge column can be used. Then, using a terminal transferase, you can add 3 ’poly-dG-tail. PCR amplification can be carried out using an oligo-dC primer with the addition of a degenerate primer to the constant region. About 40 cycles can be performed to reliably amplify the heavy chain. Then, direct sequencing of PCR products can be performed.

In certain embodiments, the antibody or antigen binding fragment thereof is encoded by a nucleic acid molecule that is highly homologous to the aforementioned nucleic acid molecules. A homologous nucleic acid molecule may include a nucleotide sequence that is at least 90% identical to the nucleotide sequence described herein. A homologous nucleic acid molecule may include a nucleotide sequence that is at least 95% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the nucleotide sequence described herein. Homology can be calculated using various, commonly available software tools well known to those of ordinary skill in the art. Illustrative tools include the BLAST system, available on the website of the National Center for Biotechnology Information (NCBI) at the National Institute of Health.

One way to identify highly homologous nucleotide sequences is through nucleic acid hybridization. Also provided herein are antibodies having the binding properties of toxin A and / or toxin B and other functional properties described herein, which are encoded by nucleic acid molecules that hybridize under high stringency conditions with nucleic acid molecules encoding the antibodies of the present invention. Identification of related sequences can also be carried out using polymerase chain reaction (PCR) and other amplification techniques suitable for cloning related nucleic acid sequences. For such techniques, PCR primers are typically selected to amplify portions of nucleic acid sequences of interest such as CDRs.

The term "high stringency conditions" as used herein refers to parameters that are familiar with the art. Parameters for nucleic acid hybridization can be found in references that compile such methods, for example, 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, FM Ausubel, et al., Eds., John Wiley & Sons, Inc., New York. One non-limiting example of high stringency conditions is hybridization at 65 ° C. in hybridization buffer (3.5X SSC, 0.02% ficol, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5 mM NaH ₂ PO ₄ ( pH 7), 0.5% SDS, 2 mM EDTA). SSC is 0.15 M sodium chloride / 0.015 M sodium citrate, pH 7; SDS means sodium dodecyl sulfate; and EDTA means ethylenediaminetetraacetic acid. After hybridization, the membrane onto which the nucleic acid is transferred is washed, for example, in 2X SSC at room temperature, and then in 0.1-0.5X SSC / 0.1X SDS at temperatures up to 68 ° C.

Also provided herein are vectors (e.g., expression vectors) or plasmids containing the described nucleic acid molecules, in addition to other nucleic acid sequences, for example, a replication origin, promoter, enhancer, terminator sequences necessary for expression of a protein, polypeptide or peptide. These vectors can be used to transform or transfect host cells to produce antibodies or antigen binding fragments thereof with binding specificity and / or binding characteristics of the antibodies or antigen binding fragments described herein. In one embodiment, the vectors may comprise an isolated nucleic acid molecule encoding a heavy chain or a portion of these antibodies and antigen binding fragments. In another embodiment, the vectors may contain nucleic acid sequences encoding a light chain or part thereof. In a further embodiment, the vectors of the present invention may comprise a sequence for a heavy chain or part thereof and a sequence for a light chain or part thereof. In a further embodiment, plasmids are provided that produce the antibodies or antigen binding fragments described herein.

Modified versions of the antibodies of the present invention are also shown. Modifications to the antibody or antigen binding fragment thereof are typically performed on a nucleic acid that encodes the antibody or antigen binding fragment thereof, and may include deletions, point mutations, truncations, amino acid substitutions, and additions of amino acids or non-amino acid fragments. Alternatively, modifications can be made directly to the polypeptide, such as, for example, by cleavage, addition of a linker molecule, addition of a detectable fragment, such as biotin, addition of a fatty acid and the like. Modifications also encompass hybrid proteins containing all or part of the amino acid sequence of an antibody or antigen binding fragment. The modifications further encompass the addition or combination of the antibody with another agent, such as a cytotoxic agent, drug or therapeutic agent.

Modified polypeptides include polypeptides that are specifically modified to alter a trait of a polypeptide that is not related to its physiological activity. For example, cysteine residues can be replaced or removed to prevent unwanted disulfide bonds. Similarly, some amino acids can be altered to enhance expression of the polypeptide by eliminating proteolysis by proteases in the expression system (for example, dibasic amino acid residues in yeast expression systems in which KEX2 protease activity is present). In addition, one or more amino acids, in particular in the constant region of Ig, can be changed to prevent proteolytic degradation of antibodies by enzymes after a specific route of administration, for example, oral administration, as described, for example, in WO 2006/071877, published July 6, 2006 of the year.

Modifications are conveniently obtained by modifying a nucleic acid molecule that encodes a polypeptide. Mutations of a nucleic acid that encodes a polypeptide preferably preserve the amino acid reading frame of the coding sequence, and preferably do not create regions in the nucleic acid that can hybridize to form secondary structures, such as hairpins or loops, that may be harmful to the expression of the modified polypeptide.

Modifications can be made in any antibodies or their antigen binding fragments, by selecting an amino acid substitution or by random mutagenesis of a selected site in a nucleic acid that encodes a polypeptide. Modified polypeptides can then be expressed and tested for one or more types of activity to determine which mutation yields a modified polypeptide with the desired properties. Additional mutations can be introduced into modified polypeptides (or unmodified polypeptides) that are silent with respect to the amino acid sequence of the polypeptide, but which provide preferred codons for translation in a particular host. Preferred codons for translation of a nucleic acid, for example in E. coli, are well known to those of ordinary skill in the art. However, other mutations can be introduced into the non-coding sequences of the clone sequence or cDNA clone to enhance expression of the polypeptide. The activity of the modified polypeptides can be tested by cloning a gene encoding the modified polypeptide into an expression vector, introducing the vector into a suitable host cell, expressing the modified polypeptide and testing the functional ability of the polypeptides described herein. The above procedures are well known to those of ordinary skill in the art.

The specialist will also be clear that conservative amino acid substitutions can be made in the polypeptides to obtain functionally equivalent polypeptides. As used herein, a “conservative amino acid substitution” refers to an amino acid substitution that does not change the relative characteristics of the charge or size of the protein in which the amino acid substitution is made. Modified polypeptides can be obtained in accordance with methods for changing the polypeptide sequence, which are known to the ordinary person skilled in the art, such as those that can be found in the directories in which such methods are collected, for example, 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. Conservative amino acid substitutions include substitutions made among amino acids within the following illustrative 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.

Conservative amino acid substitutions in polypeptides are typically carried out by modifying a nucleic acid encoding a polypeptide. Such replacements can be made using a number of methods known to the ordinary person skilled in the art. For example, amino acid substitutions can be made by PCR-directed mutation, site-directed mutagenesis, or by chemical synthesis of a gene encoding a polypeptide. If amino acid substitutions are carried out in a small fragment of the polypeptide, substitutions can be made by direct synthesis of the peptide. The activity of functionally equivalent fragments of polypeptides can be tested by cloning a gene encoding an altered polypeptide into a bacterial expression vector or expression vector in a mammal, introducing the vector into a suitable host cell, expressing the altered polypeptide, and testing the functionality of the polypeptides disclosed herein.

An anti-toxin antibody or antigen-binding portion thereof of the present invention can be modified or linked to another functional molecule, for example, another peptide or protein. In addition, the antibody or part of the antibody can be functionally linked, for example, by chemical binding, genetic fusion, non-covalent association, etc., with one or more other molecular structures, 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, the core region of streptavidin or the polyhistidine tail.

A derivative protein or antibody can be obtained by cross-linking or binding of two or more proteins or antibodies of the same or different types. Suitable crosslinking or binding agents include those that are heterobifunctional, with two different reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimidine ester), or homobifunctional (e.g., disuccinimidyl suberate), and are commercially available ( Pierce Chemical Company, Rockford, IL). The toxin antibody or antigen binding fragment of the present invention can be conjugated to another molecular structure, such as a label. Detectable agents or labels by 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 structures and radioactive materials. Examples of 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 (AR), horseradish peroxidase, beta-galactosidase, acetylcholinesterase, glucose oxidase, etc. Such enzymatically derivatized proteins or antibodies become detectable by adding a specific substrate or enzyme to produce a detectable reaction product. Proteins derivatized with a prosthetic group, such as biotin, can be detected by indirectly measuring the binding of avidin or streptavidin.

Labeled proteins and antibodies can be used as diagnostic and / or experimental agents or reagents to isolate a known or predefined antigen using standard techniques such as affinity chromatography or immunoprecipitation, or to detect a known or predefined antigen in order to determine protein levels in tissues as part of a clinical testing procedure, for example, to monitor the effectiveness of a given treatment regimen. In an embodiment, the antigen to be determined may be a toxin in a cell lysate or in a sample from a patient.

In a particular embodiment, the antibodies or antigen binding fragments of the present invention are used in combination, for example, in the form of a pharmaceutical composition containing two or more different antibodies or antigen binding fragments thereof (for example, one or more directed to toxin A and one or more directed to toxin B, two or more directed to toxin A, or two or more directed to toxin B, etc.). Combinations of antibodies or antigen-binding fragments thereof can be combined in a single therapy (i.e., administered simultaneously) to achieve the desired therapeutic effect. In another embodiment, antibodies or antigen binding fragments thereof can be administered separately (i.e., at different points in time). It follows that antibodies or their antigen binding fragments can be stored together or separately. Antibodies or antigen binding fragments thereof can be stored in an aqueous medium or in lyophilized form, which can be resuspended before use.

In another embodiment, compositions are provided comprising one or more isolated antibodies or an antigen binding fragment thereof. Also provided are compositions comprising a combination of one or more of the aforementioned antibodies or antigen binding fragments thereof. Compositions are also provided, 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, medium or diluent. A physiologically or pharmaceutically acceptable carrier, excipient, medium or diluent may be mixed with an isolated antibody or antigen binding fragment thereof. In an embodiment, the compositions comprise a combination of multiple (e.g., two or more) isolated antibodies or antigen binding fragments thereof. In an embodiment, one or more antibodies or their antigen binding fragments of the composition specifically bind C. difficile toxin A and neutralize its toxic effects, while one or more antibodies or their antigen binding fragments specifically bind C. difficile toxin B and neutralize its toxic effects, In one embodiment, as one or more antibodies or antigen-binding fragments thereof that specifically bind C. difficile toxin A and neutralize its toxic effects, or one or several only those that specifically bind C. difficile Toxin B and neutralize its toxic effects are humanized.

In a particular embodiment, the composition comprises a combination of an anti-toxin A antibody or antigen binding fragment thereof described herein and one anti-toxin B antibody or antigen binding fragment thereof described herein. In such a composition, an anti-toxin A antibody and an anti-toxin B antibody may be present in equal amounts or in ratios, for example, 1: 1. In another embodiment, in such a composition, an anti-toxin A antibody and an anti-toxin B antibody may be present in various amounts and ratios, such as, for example, 1/2: 1.2: 1.3: 1; 4: 1, etc. In an embodiment, the antibodies of the composition are humanized. In one embodiment, the composition comprises a combination of a PTA-9888 mAb, its antigen binding fragment, or its humanized form, and mAb 9693, its antigen binding fragment, or its humanized form. In an embodiment, the composition comprises a combination of a PTA-9694 mAb, its antigen binding fragment or its humanized form and mAb 9693, its antigen binding fragment or its humanized form. In an embodiment, the composition comprises a combination of any of the following: mAb PTA-9692, its antigen binding fragment or its humanized form and mAb 9693, its antigen binding fragment or its humanized form; or mAb PTA-9888, its antigen binding fragment or its humanized form and mAb 9692, its antigen binding fragment or its humanized form; or mAb PTA-9694, its antigen binding fragment or its humanized form, and mAb 9692, its antigen binding fragment or its humanized form.

Pharmaceutical compositions may also be administered in combination therapy, i.e. in combination with other therapeutic agents. For example, combination therapy may include a composition comprising one or more antibodies or antigen binding fragments thereof, provided herein with at least one other conventional therapy. Such additional therapeutic agents include therapeutic agents that are antibiotics, and therapeutic agents that are not antibiotics. Additional therapeutic agents include the C. difficile toxoid vaccine, ampicillin / amoxicillin, vancomycin, metronidazole, fidaxomycin, linezolid, nitazoxanide, rifaximin, ramoplanin, diphimycin (also called PAR-101 or ORT-80), clindamycin, such as cephalospins second and third generation cephalosporins), fluoroquinolones (such as gatifloxacin or moxifloxacin), macrolides (e.g. erythromycin, clarithromycin, azithromycin), penicillins, aminoglycosides, trimethoprim-sulfamethoxazole, chloramphenicol, tetracycline, foam and meropenem. Additional therapeutic agents also include antibiotics, antibacterial agents, bactericidal agents or bacteriostatic agents. In an embodiment, the additional therapeutic agent may be a small molecule or a low molecular weight chemical compound that targets C. difficile and / or its toxins. In an embodiment, the additional therapeutic agent is ORT-80. Non-antibiotic therapies include tolever, a high molecular weight anionic polymer that binds toxins A and B through non-specific charge-based mechanisms.

Alternatively, it is contemplated that the antibodies or antigen binding fragments of the present invention can be used in combination with other antibodies or antigen binding fragments thereof. Other additional therapeutic agents include normal combined immunoglobulin, intravenous immunoglobulin or polyclonal immunoglobulins to toxin A and toxin B in serum. Other antibodies include human mAbs directed to C. difficile toxin A or toxin B, which are described and reported in the published literature (e.g., WO / 2006/121422; US2005 / 0287150).

Also provided herein is a method that includes the use of antibodies or antigen binding fragments of the present invention for the treatment or prophylaxis, i.e. for the treatment, elimination, attenuation, elimination, prevention or retardation of C. difficile infection or disease associated with C. difficile, pathology, or their development or progression. CDAD is usually triggered by a violation of the intestinal flora through the use of antibiotics such as clindamycin, cephalosporins and fluoroquinolones. This disorder of the intestinal microenvironment, along with exposure to C. difficile spores, leads to the colonization of affected individuals. Approximately one third of all colonized patients develop CDAD, which can lead to severe diarrhea, colon perforation, colectomy, and death. Therefore, methods are provided in which one or more antibodies of the present invention or a composition described herein are administered to a patient for treating C. difficile infection or CDAD.

As used herein, “treat” refers to any beneficial effect on a patient with C. difficile infection or a disease associated with C. difficile, reported by administering antibodies or a composition or combination of compositions provided herein. For example, and without limitation, such beneficial actions may be the elimination of one or more symptoms or adverse effects, or the reduction or amelioration of the severity of one or more symptoms or side effects that result from an infection or disease; slowing, stopping or stopping the progression of an infection or disease; re-colonization, resurgence or secondary population of normal and natural microflora of the gastrointestinal tract, colon, intestines, etc., or cure for infection or disease (i.e., the doctor must 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, cramps, kidney failure, intestinal perforation, toxic megacolon, which can lead to rupture of the colon and death. The compositions may be used to reduce, reduce, mitigate or eliminate any or all of the symptoms or adverse effects described herein.

As used herein, “C. difficile infection” refers to an infection caused by 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 (eg, an increase in the total amount of C. difficile by against one or more other bacteria, etc.), which leads or can lead to an adverse effect (s) and / or an increase in the level of toxins A and / or B in the intestine or other organs and tissues that make up the gastrointestinal intestinal tract. Typically, the cause of CDAD is the acquisition and propagation of C. difficile in the intestine. In vivo, toxins A and B exhibit various pathological profiles with strong synergies that cause the disease. In rabbits and mice, for example, toxin A is enterotoxin, which causes diarrhea, while toxin B does not cause a fluid response in these species. However, toxin B is more strongly cytotoxic in vitro. Toxin-A-negative, toxin-B-positive (A- / B +) strains of C. difficile are increasingly reported. A- / B + strains do not produce toxin A due to deletions of the repeating domains of the tcdA gene, but still can cause clinical disease. In contrast, to date, there are no reports of toxin-A-positive, toxin-B-negative (A + / B-) strains in humans.

C. difficile infection usually manifests as mild to moderate diarrhea, sometimes with abdominal cramps. Sometimes pseudomembranes are observed, which are grown yellowish-white plaques on the intestinal mucosa. In rare cases, patients with C. difficile infection may have acute abdominal and transient, life-threatening colitis, which occurs as a result of a disturbance in the normal bacterial flora of the colon, C. difficile colonization, and the release of toxins that cause inflammation and damage to the mucous membrane. Antibiotic treatment is a key factor changing intestinal flora. While the normal intestinal flora resists colonization and increased growth of C. difficile, the use of an antibiotic that suppresses the normal flora allows C. difficile bacteria to multiply. C. difficile is present in 2–3% of healthy adults and up to 70% of healthy children. In one of its aspects, the mAbs of the present invention are used to treat subjects who do not exhibit symptoms but who are susceptible or at risk of acquiring C. difficile infection and become affected by diseases associated with it. Such subjects can be hospitalized or left out of the hospital setting.

A major risk factor for a disease associated with C. difficile is prior exposure to antibiotics. The most common antibiotics implied in colitis caused by C. difficile include cephalosporins (especially second and third generation), ampicillin / amoxicillin and clindamycin. Rarely implied antibiotics are macrolides (e.g., erythromycin, clarithromycin, azithromycin) and other penicillins. Compounds or other agents that are sometimes described in the literature as causing disease include aminoglycosides, fluoroquinolones, trimethoprim-sulfamethoxazole, metronidazole, chloramphenicol, tetracycline, imipenem and meropenem. Even a short-term exposure to any single antibiotic can cause C. difficile colitis, especially if the normal intestinal flora is adversely affected or destroyed. A long course of antibiotics or the use of two or more antibiotics increases the risk of illness. It has been shown that antibiotics traditionally used to treat C. difficile colitis cause the disease. Other risk factors associated with C. difficile infection include old age (> 65 years); weakened immune system; recent hospitalization (especially sharing a hospital room with an infected patient, intensive care, staying in a ward, and staying in a hospital for a long time); living in a nursing home, hospice or other long-term care facility; abdominal surgery; chronic colon diseases (for example, inflammatory bowel disease (IBD) or colorectal cancer); prescription or over-the-counter antacids that can lower gastric acidity and allow easier passage of C. difficile into the intestinal tract, and previous infection with C. difficile. Other factors associated with C. difficile disease include antitumor agents, primarily methotrexate, hemolytic uremic syndrome, malignant neoplasms, intestinal ischemia, renal failure, necrotic enterocolitis, Hirschsprung's disease, IBD, and non-surgical procedures of the gastrointestinal tract, including nasogastric tubes. Subjects to which the compositions provided herein may be administered include any of the described subjects at risk of infection with C. difficile.

Although most patients with C. difficile colitis recover without special treatment, the symptoms can be long-term and debilitating. Associated with C. difficile diarrhea can be a serious condition with a mortality rate of up to 25% in elderly patients who are painful. Surveys that focus on more seriously ill patients indicate mortality rates of 10-30%. C. difficile infection is more common in older people, and older age may contribute to susceptibility to colonization and disease. Although infants and young children often carry C. difficile and its toxins, manifest infection is rare. Cross-infection with C. difficile is common in neonatal wards, but it appears that newborns do not develop diarrhea associated with C. difficile.

A number of methods using the humanized antibodies of the present invention and / or the compositions provided herein are described herein. For example, a method is provided for treating a subject who has a C. difficile infection or disease, exhibits any of the symptoms or adverse effects described herein, or has any of the diseases described herein. In one embodiment, the method reduces, reduces, or attenuates the severity of a disease associated with a C. difficile infection or C. difficile-associated disease in a subject. As another example, a method of treating a subject that suffers from C. difficile-associated diarrhea is provided.

Also provided is a method for neutralizing toxin A and / or C. difficile toxin B in a subject. As an example, a method of neutralizing the combined systemic toxin A and toxin B C. difficile is given. In one embodiment, the combined systemic toxin A and C. difficile toxin B are neutralized by administering either a humanized anti-toxin A antibody or antigen binding fragment thereof, or a humanized anti-toxin B antibody or antigen binding fragment thereof, or a composition containing these antibodies. In another aspect, the combined systemic toxin A and C. difficile toxin B are neutralized by administering an antibody or an antigen binding fragment thereof that specifically binds both toxin A and toxin B, or a composition containing antibodies (e.g., in a humanized form). In some embodiments, humanized antibodies or compositions are administered in combination with another therapeutic agent that targets C. difficile.

As another embodiment, a method is also provided for restoring the normal flora of the gastrointestinal tract in a subject infected with C. difficile, thereby effectively treating an infection caused by C. difficile and / or its toxins. As yet another embodiment, a method of reducing a subject's susceptibility to C. difficile infection or a disease associated with C. difficile is also provided. As another embodiment, a method of preventing C. difficile infection or a disease associated with C. difficile in a subject is provided.

In the above methods, one or more antibodies or compositions provided herein are administered to a subject (e.g., a composition comprising a monoclonal antibody or its antigen binding fragment directed against C. difficile toxin A and a composition containing a monoclonal antibody or its antigen binding fragment directed against Toxin B. C. difficile). Compositions can be administered to a subject at one time or at different points in time. The compositions may be administered to a subject as a mixture in a composition containing a pharmaceutically acceptable carrier, medium or excipient, and, optionally, another antibiotic, non-antibiotic agent, drug or therapeutic effective agent against C. difficile and / or its enterotoxins.

The humanized monoclonal antibody to toxin A or its antigen binding fragment and / or the humanized monoclonal antibody to toxin B or its antigen binding fragment, or a pharmaceutically acceptable composition containing the humanized antibodies or their antigen binding fragments, individually or together, can be used in any of the methods described the present invention.

As used herein, a “pharmaceutically acceptable carrier” or “physiologically acceptable carrier” includes all kinds of salts, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are physiologically compatible. Preferably, the carrier is suitable for oral, intravenous, intraperitoneal, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, that is, the antibody, may be coated with a material to protect the compound from the effects of acids and other natural conditions that can inactivate the compound.

When administered, the pharmaceutical preparations of the present invention are used in pharmaceutically acceptable amounts and in pharmaceutically acceptable compositions. The term “pharmaceutically acceptable” means a non-toxic, physiologically acceptable material that does not affect the effectiveness of the biological activity of the active ingredients. Such formulations may typically contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents, such as additional immunostimulatory agents, including adjuvants, chemokines, and cytokines. When used in medicine, the salts must be pharmaceutically acceptable, but pharmaceutically unacceptable salts can usually be used to obtain pharmaceutically acceptable salts and are not excluded from the scope of the present invention.

The salt retains the desired biological activity of the parent compound and does not cause any undesirable toxicological effects (see, for example, 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 salts derived from non-toxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, iodic, phosphoric and the like, as well as from non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl substituted alkanoic acids hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Base addition salts include salts derived from alkaline earth metals such as sodium, potassium, magnesium, calcium and the like, as well as non-toxic organic amines such as N, N'-dibenzylethylenediamine, N-methylglucamine, chlorprocaine, choline , diethanolamine, ethylenediamine, ethylene diamine acetate (EDTA), with or without a counterion such as sodium or calcium, procaine, and the like.

Any of the compositions of the present invention, if desired, can be combined with a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” as used herein means one or more compatible solid or liquid excipients, diluents, or encapsulating substances that are suitable for administration to humans. The term “carrier” means an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate use. The components of the pharmaceutical compositions are also miscible with the molecules of the compositions and with each other so that there is no interaction that would significantly impair the required pharmaceutical efficacy.

The pharmaceutical compositions may contain suitable buffering agents, including: acetic acid in salt, citric acid in salt, boric acid in salt, and phosphoric acid in salt.

The pharmaceutical compositions may also contain, if necessary, suitable preservatives, such as: benzalkonium chloride; chlorobutanol and parabens.

Pharmaceutical compositions may traditionally be presented in unit dosage form and may be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of bringing the active agent into association with a carrier, which is one or more accessory ingredients. In general, compositions are prepared by uniformly and thoroughly bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, molding the product.

Antibodies to toxin A and toxin B of the present invention or parts thereof can be given in accordance with the regimens that can be adjusted to obtain the optimal desired response, such as a therapeutic or prophylactic response, in a single subject. For example, you can enter a single bolus dose, you can enter several separate doses over time, or the dose can be proportionally reduced or increased, as may be required by a particular therapeutic situation. Parenteral compositions can be packaged or prepared in unit dosage form for ease of administration and uniformity of dosage. A unit dosage form refers to physically discrete units provided as single doses for subjects to be treated, where each unit contains a predetermined amount of the active compound calculated to produce the desired therapeutic effect, in combination with the required pharmaceutical carrier, medium, excipient or diluent. The requirements for the standard dosage forms of the present invention are dictated and directly depend on (a) the unique characteristics of the active compound and the specific therapeutic effect that must be achieved, and (b) the limitations inherent in the technical field related to the preparation of compositions of such active compounds for treating sensitivity in individuals .

Compositions suitable for parenteral administration usually include a sterile aqueous or non-aqueous preparation, which is preferably isotonic with the blood of the recipient. This preparation can be formulated in accordance with known methods using suitable dispersing or moisturizing agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspensions in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Suitable media and solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally used as a solvent or suspension medium. For this purpose, you can use any soft fatty oil, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can be used in the preparation of injection dosage forms. Carrier formulations suitable for oral, subcutaneous, intraperitoneal, intravenous, intramuscular, etc. introduction can be found in Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.

Active components can be formulated with carriers that will protect the components from rapid release, such as, for example, a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, for example, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid. Many methods for preparing such formulations are patented or generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

The antibody and compositions of the present invention as therapeutic agents can be administered by any conventional route, including injection or gradual infusion over time. The route of administration may, by way of non-limiting examples, be oral, intravenous, subcutaneous, intraperitoneal, intramuscular, intrathecal, intracavitary, retroorbital, vaginal, rectal, inhalation, aspiration, cutaneous, with a suppository or transdermal.

Antibodies and compositions of the present invention are administered in effective amounts or doses. An “effective amount” is that amount of an antibody or antigen binding fragment or composition (s) provided herein that, alone or with additional doses or other therapeutic agent (s), produces the desired response, for example, treats, weakens, eliminates, eliminates or prevents C. difficile infection, diarrhea, or the disease associated with C. difficile in a subject. This may include only slowing the progression of infection, diarrhea, or disease over an extended period, for example, longer than one week, two weeks, three weeks, a month, two months, three months, or more than three months. However, such effective amounts are optimally treated indefinitely or stop the progression of infection, diarrhea or disease. This can be tracked using standard methods. The desired response to treating a disease or condition may also be to slow the onset or even prevent the onset of an infection or disease.

Of course, effective amounts will depend on the particular infection or disease being treated, the severity of the infection or disease, the patient’s individual characteristics, including age, physical condition, size and weight, duration of treatment, nature of concomitant therapy (if any), specific routes of administration, and similar factors within the knowledge and competence of the practitioner. These factors are well known to those skilled in the art and can be studied by merely routine testing or experimentation. As a rule, it is preferable that the maximum dose of the individual components or their combination be used, namely the highest safe dose in accordance with the results of a medical evaluation. However, it will be understood by those skilled in the art that the patient may insist on a lower dose or maximum dose for medical reasons, psychological reasons, or virtually any other reason.

The pharmaceutical compositions used in the aforementioned methods are preferably sterile and contain an effective amount of one or more of the antibodies or antigen binding fragments described herein to obtain the desired response, per unit mass or volume, suitable for administration to a patient. For example, the response can be measured by determining the physiological effects of the composition, such as amelioration of disease symptoms. Other assays will be known to those skilled in the art and can be used to measure the level of response.

Doses or amounts of compositions administered to a subject can be selected in accordance with various indicators, in particular in accordance with the method of administration used and the condition of the subject. Other factors include the desired treatment period. In case the response in the subject is insufficient, when using the initial doses, higher doses (or effectively higher doses along a different, more localized delivery route) can be used to the extent that patient tolerance allows.

In general, doses or amounts may range from about 1 μg / kg to about 100,000 μg / kg. Non-limiting examples of dose ranges comprise a therapeutically or prophylactically effective amount of an antibody, portions of an antibody or composition of the present invention include from 0.1 mg / kg to 100 mg / kg; from 0.1 mg / kg to 60 mg / kg; from 0.5 mg / kg to 75 mg / kg; from 0.5 mg / kg to 25 mg / kg; from 0.75 mg / kg to 40 mg / kg; from 1 mg / kg to 50 mg / kg; or from 1 mg / kg to 5 mg / kg. It should be borne in mind that for any specific person, patient or subject, specific dosages and regimens should be adjusted over time in accordance with the individual need and professional judgment of a qualified practitioner who administers or coordinates the administration of antibodies and / or compositions. Such dose ranges are exemplary only and are not intended to limit the scope or application of this invention. Based on the composition, the dose can be delivered continuously, for example, by continuous pumping or after certain periods of time. The desired time intervals of multiple doses of a particular composition can be determined without undue experimentation by those skilled in the art. Other protocols for administering compositions in which the dose amount, schedule of administration, place of administration, route of administration, and the like will be known to those skilled in the art. differ from those mentioned earlier.

The introduction of the composition to mammals other than humans, for example, for test purposes or veterinary medicinal purposes, is carried out essentially under the same conditions as described above.

Also provided herein are kits containing antibodies of the present invention or compositions containing antibodies of the present invention and instructions for use. The kits may further comprise at least one additional reagent, such as an additional therapeutic agent, or one or more additional antibodies or antigen binding fragments, as described herein (for example, an antibody or antigen binding fragment thereof to toxin A if the first antibody or antigen binding thereof the fragment in the kit is an antibody or antigen-binding fragment thereof to toxin B, and vice versa).

The components of the kits may be packaged, either in an aqueous product or in a lyophilized form. If antibodies or antigen-binding fragments thereof are used in conjugate kits (for example, a bispecific antibody conjugate), then the components of such conjugates can be supplied either in fully conjugated form, in the form of intermediates, or in separate particles to be conjugated by the kit user in accordance with the instructions for use.

The kit may include a carrier that is divided into compartments to provide, in strict isolation, one or more container devices or a series of container devices, such as test tubes, ampoules, vessels, bottles, syringes, etc. The first container device or series of container devices may contain one or more antibodies or antigen binding fragments thereof. The second container device or series of container devices may contain one or more antibodies or antigen binding fragments thereof, in which the antibodies or antigen binding fragments thereof are different from those in the first container devices or some other additional therapeutic agent. The kits provided herein may further include a third container that contains a molecule for binding antibodies or antigen binding fragments contained in the first and second containers.

As used herein with respect to polypeptides, proteins, or fragments thereof, “isolated” means separated from its natural environment and present in an amount sufficient to allow its determination or use. Isolated, if you mean a protein or polypeptide, means, for example: (i) obtained selectively by expression cloning or (ii) purified, as by chromatography or electrophoresis. Isolated proteins or polypeptides may, but may not, be substantially pure. The term “substantially pure” means that the proteins or polypeptides are substantially free of other substances with which they can be found in nature, or in t vivo systems, to some extent practical and suitable for their intended use. Almost pure polypeptides can be obtained by methods well known in this field. Since the isolated proteins can be mixed with a pharmaceutically acceptable carrier in a pharmaceutical preparation, the protein can comprise only a small percentage by weight of the preparation. Protein, however, is secreted in that it was separated from substances with which it can be naturally associated in living systems, i.e. isolated from other natural proteins.

Methods for evaluating a candidate agent for effectiveness in treating C. difficile infection or a disease associated with C. difficile are also provided. Such methods may include the steps of treating a subject with an agent that increases the risk of C. difficile infection or a C. difficile associated disease in a subject, inoculating a C. difficile subject, treating the subject with a candidate agent, and evaluating the effectiveness of the treatment with the candidate agent. As used herein, “an agent that increases the risk of C. difficile infection or C. difficile associated disease” is any agent that is believed to contribute to the onset or progression of C. difficile infection or C. difficile associated disease. Such an agent may be an antibiotic or nonantibiotic agent. For example, the agent may be any of the antibiotics described herein. Illustratively, such an antibiotic may be clindamycin, metronidazole, vancomycin, fidaxomycin, nitazoxanide, rifaximin, ramoplanin, or combinations thereof.

In these methods, a candidate agent may be administered to a subject before or after inoculation of C. difficile. A candidate agent may be any agent that is believed to have the potential to treat or prevent C. difficile infection or a disease associated with C. difficile. Candidate agents, which may be antibiotics or nonantibiotics, include antibodies or antigen binding fragments thereof that specifically bind C. difficile toxin A and / or toxin B.

These methods include any of the in vitro and in vivo methods described in the examples below.

The ultimate goal of treating CDAD is to stop all antibiotics and restore normal intestinal microflora. In accordance with the present invention, the toxin A and B mAb described herein can provide non-antibiotic therapies aimed at blocking the pathogenic effects of C. difficile toxins, allow antibiotics to stop, and thereby provide time for healing the colon and restoring normal intestinal microflora . The monoclonal antibodies of the present invention demonstrated complete and long-term (> 37 days) protection in a rigid hamster CDAD model. Based on their exceptional characteristics and properties, C. difficile toxin A and B mAbs of the present invention provide new treatment options and medications for patients who have been poorly assisted by existing therapies, including those individuals who suffer from the most severe cases of the disease.

The present invention further includes a vaccine or immunogen containing parts, fragments, or peptides of C. difficile Toxin A and / or Toxin B containing epitope regions recognizable and / or linked by one or more of the RA-39 monoclonal antibody (ATCC Accession No. PTA-9692 ), a humanized form of RA-39, a monoclonal antibody RA-50 (ATCC accession No. РТА-9694), a humanized form of RA-50, a monoclonal antibody RA-41 (ATCC accession number РТА-9693), a humanized form of RA-41, antibody, which competes for the binding of toxin A to monoclonal ant hetel RA-39 or its humanized form, an antibody that competes for the binding of toxin A with the monoclonal antibody RA-50 or its humanized form or an antibody that competes for the binding of toxin B with the monoclonal antibody RA-41 or its humanized form. In an embodiment, the vaccine or immunogen includes portions, fragments or peptides of C. difficile toxin A and toxin B containing epitope regions recognized and / or linked by one or more of the RA-39 monoclonal antibody (ATCC Accession No. PTA-9692), humanized form PA-39 or an antibody that competes for the binding of toxin A and toxin B with the RA-39 monoclonal antibody or its humanized form. In an embodiment, the epitope-containing portions, fragments or peptides of the toxin A and / or toxin B of a given vaccine or immunogen are prepared from a protein of the toxin A or toxin B by proteolytic cleavage. In an embodiment, fragments, parts or peptides of the toxin A of a given vaccine or immunogen are prepared by proteolytic cleavage by enterokinase. In an embodiment, toxin fragments, parts or peptides of a given vaccine or immunogen are obtained by proteolytic cleavage of caspase (caspase 1). In an embodiment, epitope-containing parts or fragments of a vaccine or immunogen chemically or recombinantly synthesize peptides of a toxin A and toxin B protein. In an embodiment, fragments, parts or peptides of a vaccine or immunogen containing one or more epitope regions of toxin A and / or toxin B recognized and bound by an antibody are derived from one or more of the amino terminus of toxin A; amino end of toxin B; the carboxy terminus of toxin A; carboxy terminus of toxin B; toxin A receptor binding domain; areas outside the receptor binding domain of toxin A; N-terminal enzymatic region of toxin B; toxin A glycosyltransferase domain; glycosyltransferase domain of toxin B; the proteolytic domain of toxin A; the proteolytic domain of toxin B; hydrophobic, pore-forming domain of toxin A; or hydrophobic, pore-forming domain of toxin B.

In some embodiments, fragments containing one or more epitope regions recognized and bound by antibodies are derived from the amino terminus of toxin A or toxin B. In some embodiments, fragments containing one or more epitope regions recognized and bound by antibodies are derived from C -end of toxin A or toxin B. In some embodiments, fragments containing one or more epitope regions recognized and bound by antibodies are derived from a glycosyl transferase domain then xin A or toxin B. In some embodiments, fragments containing one or more epitope regions recognized and bound by antibodies are derived from the proteolytic domain of toxin A or toxin B. In some embodiments, fragments containing one or more epitope regions are recognized and bound antibodies are obtained from the hydrophobic, pore-forming domain of toxin A or toxin B. In some embodiments, fragments containing one or more epitope regions recognized and called antibodies are obtained from the receptor-binding domain of toxin A. In some embodiments, fragments containing one or more epitope regions recognized and bound by antibodies are obtained from the receptor-binding domain of toxin B. In some embodiments, fragments containing one or more epitope regions are recognized and bound antibodies are obtained from a region outside the receptor binding domain of toxin A. In some embodiments, fragments containing one or more epitope regions recognized and bound by antibodies are obtained from the N-terminal enzymatic region of toxin B. In an embodiment, epitope-containing fragments or portions of toxin A and / or toxin B are <300 kDa. Epitope-containing fragments or portions of toxin A and / or toxin B have a size of ~ 158-160 kDa, ~ 100-105 kDa, for example 103 kDa, ~ 90-95 kDa, for example, 91 kDa, and / or ~ 63-68 kDa, for example, 63 kDa or 68 kDa. In other embodiments, epitope-containing fragments or portions of toxin A have a size of ~ 158-160 kDa; ~ 90-95 kDa, for example, 91 kDa, and / or ~ 63-68 kDa, for example, 68 kDa. In other embodiments, epitope-containing fragments or portions of toxin B have a size of ~ 100-105 kDa, for example, 103 kDa, and / or ~ 63-68 kDa, for example, 63 kDa.

Such portions, fragments, or peptides of toxins when administered in the form of a vaccine or immunogen to a subject infected with C. difficile or afflicted with a disease associated with C. difficile can elicit a humoral response in the subject, i.e. antibodies having specificities for toxin A and / or toxin B thus allowing the subject to provide an immune response to toxins and neutralize, block, reduce, reduce the intensity, cure or treat C. difficile-associated disease, infection or CDAD in the subject. Accordingly, in another embodiment, there is provided a method of neutralizing, blocking, reducing, decreasing the intensity, curing or treating a 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 vaccine or immunogen. In an embodiment, the subject exhibits a humoral response to C. difficile toxin A and / or Toxin B, thereby neutralizing, blocking, decreasing, decreasing intensity, curing or treating the C. difficile associated disease, infection or CDAD in the subject. In another embodiment, the subject exhibits a cellular immune response to Toxin A and / or C. difficile Toxin B. In another embodiment, the subject exhibits both a humoral and a cellular immune response to toxin A and / or C. difficile toxin B.

In another embodiment, the present invention includes a method of neutralizing, suppressing, or blocking the activity of toxin A and / or toxin B in or in relation to 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, where the antibody or its antigennegative fragment neutralizes, inhibits or blocks the activity of toxin A and / or toxin B in or in relation to the cell using a competitive or mixed competitive anizma action. In an embodiment, the antibody is one or more of a monoclonal antibody, a humanized antibody, or a chimeric antibody. In an embodiment, the cell is in the subject, and the antibody or antigen binding fragment thereof is administered to the subject in an effective amount. In an embodiment, the cell is located within the gastrointestinal tract of a subject, for example, an intestinal epithelial cell. In an embodiment, the toxin is toxin A. In an embodiment, the toxin is toxin B. In an embodiment, the toxin is toxin A, and the mechanism of action of the antibody is the mechanism of action of competitive suppression. In an embodiment, the toxin is toxin A, and the antibody or antigen binding fragment thereof is PA-50 (accession number ATCC PTA-9694), its humanized form, or an antibody or fragment thereof that competes with RA-50 to neutralize the activity of toxin A. In an embodiment, the toxin is toxin A, and the mechanism of action of the antibody is the mechanism of action of mixed competitive inhibition. In an embodiment, the toxin is toxin A, and the antibody or antigen binding fragment thereof is PA-39 (accession number ATCC PTA-9692), its humanized form, or an antibody or fragment thereof that competes with RA-39 to neutralize the activity of toxin A. In an embodiment, the toxin is toxin B, and the mechanism of action of the antibody is the mechanism of action of mixed competitive inhibition. In an embodiment, the toxin is toxin B, and the antibody or antigen binding fragment thereof is PA-41 (accession number ATCC PTA-9693), its humanized form, or an antibody or fragment thereof that competes with RA-41 to neutralize the activity of toxin B.

As used herein, the term “competitive inhibitor” of a toxin refers to a toxin-neutralizing inhibitor, for example, an antibody, agent or small molecule or chemical structure, that exhibits a right-handed shift of the EC ₅₀ on the neutralization curve without changing the maximum percentage of neutralization as the concentration toxin increases in culture. Thus, a competitive inhibitor is generally able to overcome the cytotoxic effect of the toxin by adding more inhibitor. The expression “non-competitive inhibitor” of a toxin refers to a toxin neutralizing inhibitor that exhibits a decrease in the maximum neutralization percentage without changing the concentration, giving a half-maximum response value (EC ₅₀ ) as the concentration of the toxin increases in culture. Thus, a non-competitive inhibitor, as a rule, is not able to completely overcome the cytotoxic effect of the toxin by adding more inhibitor. The term “mixed competitive inhibitor” of a toxin refers to a toxin neutralizing inhibitor that exhibits some degree of both competitive and non-competitive inhibition as the concentration of the toxin increases in culture. For example, a mixed competitive toxin inhibitor can bind the toxin and detects its effect by blocking the binding of the toxin to the cell, as well as blocking the other cytotoxic effects of the toxin; thus discovering a mixed competitive mechanism of action.

Examples

Example 1

Obtaining neutralizing monoclonal antibodies against toxin A and / or toxin B. C. difficile

A. Preparation of the immunogen

Neutralizing monoclonal antibodies directed against C. difficile toxin A and / or Toxin B were obtained by immunizing mice with C. difficile toxin A toxin A (an inactive form of the toxin) and active forms of Toxin A and / or Toxin B. Mouse mAb (PA-38: mAb to toxin A, ATCC No. PTA-9888, PA-39: mAb to toxins A and B, ATCC No. PTA-9692, PA-41: mAb to toxin B ATCC No. PTA-9693 and PA-50: mAb to toxin A, ATCC No. PTA-9694) was obtained by immunizing animals with toxoid A and subsequent immunizations with the active form of toxin A and / or toxin B. Toxin A toxin, Toxin A, Toxin B (List Biological Laboratories Inc., Campbell, CA) and Toxin A (TechLab Inc, Blacksburg, VA) were stored at 4 ° C until use. Toxins and toxoid were obtained from strain VPI 10463, a widely used reference strain of C. difficile. Quil A adjuvant (Accurate Chemical, Westbury, NY) was added to the desired volume of toxoid or toxin and mixed. The mixture was prepared 60 minutes before immunization and stored on ice until immunization. For the final booster dose before fusion, the required toxin was diluted in PBS and stored on ice until used in immunization.

B. Immunization and fusion

Thirty female BALB / c mice (Charles River Labs, Wilmigton, MA) received two immunizing doses (for PA-50) or three immunizing doses (for PA-38, PA-39 and PA-41) of toxin A toxin (10 μg) subcutaneously at intervals of three weeks before receiving booster immunizations with increasing doses of active toxin A or active toxin B, also at intervals of three weeks. For RA-38, one mouse received booster immunization every three weeks with a total of three booster doses with toxin A (List Biological Laboratories Inc.), with each booster dose increasing in dose from 500 ng to 2 μg, and the final booster dose of toxin A (8 mcg) three days before splenectomy. For PA-39 and RA-41, two mice received three or five booster immunizations, respectively, every three weeks with toxin B, with each booster dose increasing in a dose from 2 μg to 12.5 μg, and the final booster dose of toxin In (20 mcg) three days before splenectomy. For RA-50, one mouse received booster immunization every three weeks with a total of four booster doses with toxin A (TechLab Inc), with each booster dose increasing in dose from 20 ng to 2.5 μg, and the final booster dose (10 mcg) three days before splenectomy.

Immunizing and booster doses of toxoid and toxin, respectively, were administered in combination with an adjuvant, for example Quil A (10 μg). Booster immunization with the active form of toxin A or toxin B was used to identify animals that may have developed protective antibodies. Sera from immunized animals were serially diluted and tested to neutralize the cytotoxic effect of toxin A on CHO-K1 cells, as described below. Animals with the highest titer of neutralizing antibodies were selected for fusion and booster immunization with toxin without adjuvant.

After booster immunization, the animals were sacrificed, and isolated splenocytes were fused to the Sp2 / 0 cell line using standard methods. Hybridomas were suspended in selective 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 RA-41 and RA-50), which contained 100 μM hypoxanthine, 1 μg / ml azaserin and 16 μM thymidine for selective pressure. Hybridomas were plated in 96-well flat-bottomed tissue culture plates (BD Biosciences, San Jose, CA). The plates were incubated at 37 ° C for 3 days, followed by the addition of NT growth medium (selective medium without azaserin). Incubation continued for 4–7 days prior to screening the hybridoma supernatants for neutralizing activity.

In an initial screening for RA-38, 608 hybridoma supernatants were tested for their ability to neutralize the cytotoxic effect of toxin A (List Laboratories) on CHO-K1 cells (ATCC No. CCL-61, Manassas, VA). In the primary screening for PA-39 and PA-41, 2416 hybridoma supernatants were tested for their ability to neutralize the cytotoxic effect of Toxin B (List Laboratories) on CHO-K1 cells. In the initial screening for RA-50, 1,440 hybridoma supernatants were tested for the ability to neutralize the cytotoxic effect of toxin A (Techlab Inc.) on T-84 cells. In a second assay, toxin-mediated rabbit erythrocyte agglutination inhibition was investigated. Based on the screening procedure, four mAbs were identified, which were designated RA-38 (to toxin A), PA-39 (to toxin A / B), RA-50 (to toxin A) and PA-41 (to toxin B) and which was effective suppressed or neutralized C. difficile toxins in screening assays. Hybridoma cell lines that produced mAb data were cloned twice using limiting dilution to obtain clonal cell lines. Using the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche Applied Science, Indianapolis, IN), it was determined that the mAbs PA-38, PA-39, PA-41 and PA-50 are isotypes IgG2a, K, IgG1.K, IgG1.K and IgG1.K, respectively. Hybridoma cell lines producing mAbs are denoted by the same name as the mAbs that they produce.

C. Screening: neutralizing the cytotoxic effect of toxin A or B on cells

Hybridoma supernatants were screened for their ability to neutralize the cytotoxic effect of toxin A or toxin B on cells. Developed a high-performance method for processing thousands of hybridoma supernatants simultaneously. For analysis of cytotoxicity, either CHO-K1 cells (for RA-38, PA-39, and PA-41) or T-84 cells (for RA-50) were used. Cells were added to assay plates (96-well plates, white opaque wall, clear flat bottom; Perkin Elmer, Waltham, MA) using a Biomek FX robotic system (Beckman Coulter, Brea, CA). Assay plates were incubated for 4 hours at 37 ° C to allow cells to adhere to the wells. For analysis on T-84, toxin A was diluted to 240 ng / ml. For analysis on CHO-K1, toxin A was diluted to 2 μg / 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 box (BSC). Hybridoma supernatants were manually collected and added to wells of reagent dilution plates using the Biomek FX system. A mixture of supernatant and toxin was incubated at 37 ° C for 1 hour and introduced into analysis plates containing cells using the Biomek FX system. After incubation at 37 ° C for 72 hours, 20 μl / well of CellTiter-Blue (Promega, Madison, WI) was added to each well. The plates were incubated for an additional 4 hours, and then read on a SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, Calif.) Using an excitation wavelength of 560 nm and an emission wavelength of 590 nm. Cell survival was compared in untreated and toxin-treated cultures. The percentage of cell survival was plotted against concentration.

D. Preparation of the murine mAbs of the present invention In vivo and in vitro production methods were used to obtain the isolated and / or purified mAbs of the present invention. For in vivo production of murine mAb, ascites fluid was obtained by injecting the corresponding hybridoma cell line into the abdominal cavity of the attached BALB / c mice. mAbs were purified to> 95% homogeneity by precipitation with ammonium sulfate, followed by chromatography with protein A. The purified antibodies were resuspended in phosphate-buffered saline (PBS).

For in vitro production on a small scale (<100 mg), murine mAb antibodies were purified from cultured hybridoma supernatants. Hybridomas were cultured in Hybridoma-SFM (Invitrogen) and 10% FBS. Cell lines were reseeded and propagated in T-150 mattresses three times a week to ensure that the cell concentration does not exceed 2 × 10 ⁶ cells / ml. Supernatants containing PA-39 (IgG1, κ), PA-41 (IgG1, κ) and PA-50 (IgG1, κ) were purified from impurities by centrifugation at 2000 rpm for 10 minutes and filtration. The material purified from impurities was diluted to a final concentration with a movable buffer (60 mM glycine / 3 M NaCl, pH 8.5) and loaded onto a protein A column equilibrated with a movable buffer. After washing the column, the PAb-39 or PA-41 mAb was eluted with 0.1 sodium acetate, pH 5.5, and neutralized to pH 7.0. Supernatants containing PA-38 (IgG2a, κ) were purified from impurities by centrifugation at 2000 rpm for 10 minutes and filtration. Purified from impurities, the material was brought 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, the PA-38 mAb was eluted with 0.1 M sodium acetate, pH 3.0, and then the eluted antibody was neutralized to pH 7.0.

For in vitro production in large quantities of mAbs (> 100 mg), hybridomas were inoculated into a WAVE bioreactor (GE Healthcare, Piscataway, NJ) at an initial density of 2 × 10 ⁵ cells / ml with Hybridoma-SFM with 5% Ultra Low IgG FBS. Cell counting was performed and cell viability was monitored daily. At about day 6 or 7, when antibody production slowed down, cultivation was stopped. The culture was purified and then concentrated 10-20 times using tangential flow filtration. The antibody was loaded onto 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 combined antibody was neutralized to pH 7.4 with 1 M Tris, concentrated to 10 mg / ml, and diafiltered in PBS. The purified mAbs were sterilized by filtration and stored at -80 ° C.

Example 2

The specificity and affinity of the mAb for toxin A and / or toxin B C. difficile of the present invention toxin A and / or toxin B

A. ELISA for determining the specificity of mAb to toxin A and / or toxin B

ELISA plates (BD Biosciences) covered 50 ng / well of toxin A (List Laboratories) or 25 ng / well of toxin B (List Laboratories) overnight at 4 ° C. After washing the plates in PBS- (PBS without calcium or magnesium, 0.05% Tween 20), the wells were blocked with 200 μl blocking buffer (PBS without calcium or magnesium, 0.1% Tween 20, 2.5% skim milk) in for one hour at 37 ° C. The washing step was repeated, hybridoma supernatants or purified mAbs were added for one hour at 37 ° C. The plate was washed and incubated for one hour at 37 ° C with horseradish peroxidase conjugated (HPR) goat anti-mouse IgG-Fc antibody (Jackson Immunoresearch, West Grove, PA). The plate was processed with ABTS peroxidase substrate system (KPL, Gaithersburg, MD), ABTS peroxidase stop solution (KPL) and read on a SpectraMax plate reader (Molecular Devices) at 405 nm.

Titration data are shown in FIGS. 1A-C. FIG. 1A shows that RA-38 binds to toxin A, but not to toxin B. FIG. 1B shows that RA-39 can bind to both toxin A and toxin B. FIG. 1C shows that RA-41 binds to toxin B, but not to toxin A. B. Reactivity of mAb to toxin A and B in Biacore A Biacore 3000 device (GE Healthcare) was used to determine the specificity of the binding of the mAb of the present invention to toxin A and / or B. mAb immobilized at approximately 10,000 resonance units (RU) in CM5 sensor chips (GE Healthcare) in accordance with the manufacturer's instructions amino accession. A control surface with isotypically matching antibodies of inappropriate specificity (Southern Biotech) was used as a control. Binding experiments were carried out at 25 ° C in HEPES-based HEPES buffer (GE Healthcare). Purified toxin A or toxin B (30 nM; List Biological Laboratories) was passed over the control and test flow cells at a speed of 5 μl / min. If required, additional mAbs (100 nM) were passed over the flow cell at 5 μl / min to check for multivalent or competitive binding.

As shown in FIGS. 2A-D, the mAb PA-38 (FIG. 2A) and the mAb PA-50 (FIG. 2C) specifically bind to toxin A; mAb PA-41 (Figure 2D) binds specifically to toxin B and mAb PA-39 (Figure 2B) binds predominantly to toxin A, but also shows binding to toxin B. The results from these data are consistent with ELISA data (Figure 1A) -C) and demonstrate the binding specificity of the mAbs of the present invention to toxin A and / or toxin B.

C. Binding Affinity

The Biacore assay was also used to determine the avidity of binding of the mAbs of the present invention to its corresponding toxin. mAbs were captured using a CM5 sensor chip prepared using a Biacore mouse antibody capture kit. Then the toxin was passed through the flow cells at various concentrations (0.4-100 nM, double increase). All concentrations of toxins were tested in duplicate, and the surface of the chip was regenerated after each experiment using the conditions indicated in the kits. Changes in RU were recorded and analyzed using the 1: 1 binding model (Langmuir) in the Bia Evaluation Software, which calculates kd mAb for toxin. Association and dissociation data and approximation are illustrated in FIGS. 3A-E.

Biacore analysis determined that the kd mAb for toxin A was 1.0 nM for RA-38, 0.16 nM for RA-39, and 0.16 nM for RA-50. The kd mAb for toxin B was determined to be 2.4 nM for PA-39 and 0.59 nM for PA-41. These results showed that the mAbs of the present invention bind toxin A and / or toxin B with nanomolar and subnanomolar affinity.

Example 3

In vitro cell-based neutralization assays

Cell-based cytotoxicity analyzes using CHO-K1 cells or T-84 cells were used to evaluate the neutralizing activities of the described mAbs for toxin A and toxin B.

A. Neutralization of the cytotoxic effect of toxin A on CHO-K1 cells

CHO-K1 cells were seeded (2000 cells in 50 μl / well) onto assay plates (96-well plates, white opaque wall, clear flat bottom (Perkin Elmer)). Cells were allowed to attach for 4 hours before treatment. Equal volumes (35 μl) of 2 μg / ml of Toxin A (List Biological Laboratories) and serially diluted mAbs were mixed on reagent dilution plates (96-well round-bottom plates (Falcon)) for 1 hour at 37 ° C and then 50 μl of the mixture was added to each well of the plates. After incubation for 72 hours, 20 μg / ml CellTiter-Blue (Promega) was added to each well. The plates were incubated for an additional 4 hours, then read on a SpectraMax M5 plate reader (Molecular Devices) using an excitation wavelength of 560 nm and an emission wavelength of 590 nm. Cell survival was compared in untreated and toxin-treated cultures. The percentage of cell survival was plotted against mAb concentration. Inhibition data were approximated with a sigmoidal dose-response curve with non-linear regression using GraphPad Prism software, and the mAb concentration required for 50% cytotoxicity neutralization (EC ₅₀ ) was calculated. As shown in figure 4, the mAb PA-39 completely neutralized the activity of toxin A on CHO-K1 cells with EC ₅₀ - 93 PM. B. Neutralization of the cytotoxic effect of Toxin B on CHO-K1 cells A cytotoxicity assay on CHO-K1 was used to evaluate the neutralizing activity of specific mAb to toxin B. As in the case of evaluating the mAb to toxin A, toxin B (8 pg / ml, TechLab) was incubated for 1 hour at 37 ° C with serially diluted mAb before application to CHO-K1 (2000 cells / well) in a 96-well plate. After 72 hours, 20 μl / well of CellTiter-Blue (Promega) was added to each well. The 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. Cell viability was determined using CellTiter-Blue; cell survival was compared between treated and untreated cultures. Inhibition data were approximated with a sigmoidal dose-response curve with non-linear regression using GraphPad Prism software, and the mAb concentration required for 50% cytotoxicity neutralization (EC ₅₀ ) was calculated. As shown in FIG. 5, RA-41 showed a high degree of activity (EC ₅₀ - 9.2 pM) in neutralizing the cytotoxicity of toxin B in CHO-K1 cells.

Although PA-39 mAbs showed binding to toxin B based on ELISA and Biacore assays, this mAb did not have in vitro activity against toxin B in assays based on CHO-K1 cells and other cells. Antibodies that bind both toxin A and toxin B but have no functional activity in neutralizing toxin A or toxin B in w vitro cell-based assays have already been reported (46, 92 and 93). The present invention includes a novel mAb with the dual ability to bind both toxin A and toxin B, as well as neutralize the cytotoxicity of C. difficile toxin, for example, toxin A.

C. Neutralization of the cytotoxic effect of toxin A on T-84 cells

A cytotoxicity assay on T-84 was used to evaluate the neutralizing activity of the described mAbs for toxin A. T-84 cells were seeded (15,000 cells in 50 μl / well) in assay plates (96-well plates, white opaque wall, transparent flat bottom (Perkin Elmer)). Cells were allowed to attach for 4 hours before treatment. Equal volumes (35 μl) of 240 ng / ml of toxin A (TechLab) and serially diluted mAb were mixed in reagent dilution plates (96-well round-bottom plates (Falcon)) for 1 hour at 37 ° C. and then in each well of the plates 50 μl of the mixture were added to the assay. After incubation for 72 hours, 20 μl / well of CellTiter-Blue (Promega) was added to each well. The plates were incubated for an additional 4 hours, then read on a SpectraMax M5 plate reader (Molecular Devices) using an excitation wavelength of 560 nm and an emission wavelength of 590 nm. Cell survival was compared in untreated and toxin-treated cultures. Inhibition data were approximated with a sigmoidal dose-response curve with non-linear regression using GraphPad Prism software, and the mAb concentration required for 50% cytotoxicity neutralization (EC ₅₀ ) was calculated. As shown in Fig.6, the mAb RA-38 and RA-50 completely neutralized the activity of toxin A on T-84 cells with EC ₅₀ - 175 PM and 146 PM, respectively. In a T-84 cell assay, RA-39 mAb showed minimal activity against toxin A, and RA-41 was not active.

D. Hemagglutination of rabbit red blood cells (RBC)

The ability of the mAbs of the present invention to block the binding of toxin A to cell receptors was assessed by hemagglutination assay. For this analysis, equal volumes (30 μl / well) of toxin A (8 μg / ml; TechLab) and serially diluted mAb 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 three times with PBS and resuspended in PBS. 60 μl of a 1% RBC suspension was added to the wells of 96-well plates containing a mixture of toxin A-mAb, the plates were incubated at 4 ° C for 4 hours. Free toxin A causes RBC hemagglutination. Accordingly, the addition of a mAb to toxin A, which binds to toxin A, is expected to prevent hemagglutination. The degree of hemagglutination was determined using an ImageQuant 400 device; complete hemagglutination gave a stronger signal than RBC in suspension. EC5o values were calculated from the suppression data using an approximation of the sigmoidal dose-response curve with non-linear regression from GraphPad Prism. As shown in Figure 7, mAb PA-38 (filled squares) and mAb PA-50 (closed triangles) completely neutralized toxin A activity at a RBC EC ₅₀ - 30 nM and 1.8 nM, respectively. It turned out that RA-38 and PA-50 neutralize toxin A, blocking the binding of toxin A to its receptor. Found that the mAb PA-39 and PA-41 were inactive in this analysis.

E. Monolayer analysis on Saso-2

Caco-2 cells were plated (25,000 cells in 75 μl / well) in the upper chamber of 96-well Multiscreen Caco-2 plates (Millipore Billerica, MA) with 250 μl of medium added to the lower chamber. Cells were allowed to grow for 10 days with regular medium changes every 3-4 days. After incubation for 10-14 days, the formation of a dense monolayer was confirmed by measuring transepithelial electric resistance (TEER) using an epithelial voltmeter (model: EVOMX, World Precision Instruments, Sarasota, FL). After the monolayer integrity was determined and determined, equal volumes (60 μl) of toxin A (at 50 ng / ml) and serially diluted mAb were mixed for 1 hour at 37 ° C, and then introduced into the upper chamber of the assay plate. The plates were incubated for 18-24 hours, and then the TEER value was measured using a multimeter. Monolayer integrity was compared in untreated and toxin-treated wells. As shown in FIG. 8, the suppression data were approximated with a sigmoidal dose-response curve with non-linear regression using GraphPad Prism software to determine the mAb concentration required for 50% toxin suppression (EC ₅₀ ). PAb-38 and PA-50 mAbs neutralized the disruption of Caco-2 monolayers by toxin A with EC ₅₀ -485 pM and 196 pM, respectively. Other mAbs were found to be inactive in this assay.

Without wanting to be bound by theory, in vitro cell-based results show that PA-38 and PA-50 appear to be one class of mAb to toxin A, while PA-39 is another class of mAb to toxin A. mAb PA-38 and PA-50 appear to bind toxin A epitope, which is important for receptor binding; The PA-39 mAb appears to bind the toxin in a manner that more directly blocks the cytotoxic effects of toxin A in vitro.

Example 4

In vivo Evaluation of the Effectiveness of a mAb for Toxin A and Toxin B C. difficile of the Present Invention in Mice

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. In vivo, the neutralizing activities of PAb-38, PA-39, PA-41, or PA-50 mAbs, administered individually or in combination, were tested for the effects of combined systemic toxins A and B. C. difficile (Techlab) on test animals.

Swiss Webster females, 4-6 mice / group (age: ~ 6-8 weeks at baseline; Charles River Laboratories) were used in the experiments. Mice were acclimatized in the laboratory for at least 4 days, and their health was checked before use. Animal experiments were carried out in accordance with the IACUC approved protocol.

Initial experiments were performed to determine the toxicity of toxins A and B in mice. Animals were injected with 0, 20, 100, 500, 2500 ng / toxin / animal intraperitoneally (i.p.) and the dose that was lethal for animals was chosen for use in subsequent antibody neutralization experiments. Control mice injected with PBS were unaffected. A dose of Toxin A (TechLab) of 100 ng was chosen for neutralization experiments, as it was found to be the lowest dose level, which was lethal for 100% of mice within 24 hours after injection. Similarly, a dose of Toxin B (TechLab) of 100 ng was chosen for neutralization experiments, as it was the lowest dose level that was lethal for 100% of mice within 24 hours after injection.

To assess the neutralizing activity of the mAb to the toxin, mice (5 per group) were intraperitoneally injected with one injection of each mAb at different dose levels on day 0, followed by an intraperitoneal injection of 100 ng / 200 μl of toxin A or toxin B on day 1. Animals were observed daily for 3 days and then weekly up to 21 days after the toxin injection. Animal survival was the main expected outcome of the study.

For neutralization experiments, all doses of the antibody were in PBS without calcium or magnesium (PBS-, Invitrogen, Carlsbad, CA). On day 0, mice were intraperitoneally injected with a single injection of PAb-38, PA-39, PA-41, or PA-50 mAb at different dose levels (200 μl / dose / animal), followed by injection of the toxin (intraperitoneally to a place different from the injection site antibodies) per day 1. The health status of animals was monitored daily for the first 3-4 days and then twice a week for 21 days after administration of the toxin. Observations of animals in the cages (for example, bent posture, matted coat, sedentary) were recorded in the same way as survival.

Differing dose levels of RA-38 (0.2 μg - 250 μg / animal) and RA-50 (0.2 μg - 100 μg / animal) were evaluated. In this model, it was found that RA-38 and RA-50 neutralize the dose of toxin A 100 ng and provide 100% survival at such low dose levels as 2 μg mAb / animal, as shown in figa and B. In contrast to them , the human monoclonal toxin A reference antibody (WO / 2006/121422 and US2005 / 0287150), referred to herein as CDA-1 comparison mAbs, at 5 μg / animal, did not protect animals from toxin-related death, unlike mAbs according to the present invention (figs). The PA-41 doses were evaluated in the range of 0.5 μg to 250 μg, and it was found that a single dose of 5 μg mAb / animal completely neutralizes the toxicity of the dose of 100 ng toxin B, as shown in FIG. It was not observed that the mAb RA-39 (100 μg / animal) provided a slowdown in the toxin-related death of mice from either toxin A or toxin B.

After the neutralizing activity of individual antibodies to toxin A (PA-38, PA-50) or toxin B (PA-41) was clearly shown t vivo, an experiment was performed to test the combination of mAb (PA-38 + PA-41) with dose levels of 5 and 50 μg of each mAb relative to the combined lethal dose of toxins (100 ng of toxin A and 100 ng of toxin B) in the same mouse model of v vivo. In addition, individual monoclonal antibodies were included as controls. As shown in FIG. 11, the combination of PAb-38 and PA-41 mAbs showed protection against a combination of toxins at both 50 μg / animal (4 out of 5 survived) and 5 μg / animal (1 out of 5 survived) compared to the activity of each mAb separately (all animals died within 24 hours after administration of the toxin).

Example 5

Evaluation of the mAb for Toxin A and Toxin B of C. difficile of the present invention in a model of C. difficile associated diarrhea (CDAD) in Syrian Golden Hamsters

The hamster CDAD model reproduces key aspects of human CDAD disease. As a result of antibiotic treatment, the normal colon flora is destroyed and the hamsters become easily susceptible to C. difficile infection. Infection leads to severe diarrhea, pseudomembranous colitis and death. The hamster CDAD model was used to evaluate the potential efficacy of the mAbs of the present invention to prevent disease and death in animals associated with infection with live C. difficile bacteria. These experiments were performed in accordance with IACUC approved protocols.

A. Pharmacokinetic analysis

Before conducting an efficacy study on a hamster model using hamsters infected with C. difficile live microorganisms, pharmacokinetic studies were performed on normal uninfected hamsters. Syrian Golden Hamsters (Harlan) were intraperitoneally injected with 0.2 mg / animal or 1 mg / animal of purified mAb RA-38 or mAb RA-41. Blood samples were collected using retro-orbital techniques or with a puncture of the heart (terminal) blood sampling after 0.125, 0.25, 1, 2, 4, 7, 10, 14 and 21 days. Blood samples were centrifuged at 8000 rpm for 10 minutes to obtain serum.

Serum mAb concentration was determined by 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. The plates were washed three times with PBS / 0.05% Tween-20® (PBS-T) and blocked with 200 μl blocking buffer (PBS without calcium or magnesium, 0.1% Tween 20®, 2.5% skim milk) for one hours at room temperature. A standard antibody sample (purified mAb PA-38 or mAb PA-41) was diluted in 1% pooled serum from unexposed hamsters to obtain a standard curve with a range of 0.3-1000 ng / ml. Diluted test and standard samples were incubated for one hour at room temperature.

The plates were washed (as described above) and incubated for one hour at room temperature with goat HRP-conjugated antibodies specific for mouse Fey IgG (Jackson Immunoresearch). The plates were treated 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. The concentration of mAb in each hamster at different points in time was calculated using standard curves. Approximately 10% of the samples did not have an antibody titer, probably due to missed injection or lack of absorption, these samples were not included in the calculation of PK parameters. A non-compartmental pharmacokinetic analysis was performed using WinNonLin, version 4.0 (Pharsight Corp, Mountain View, CA), and the data are shown in Table 1 and FIGS. 12A and B. As indicated, C _max and area under the curve (AUC) were dose dependent . Each of the antibodies showed a half-life of more than 6 days, which ensured the conservation of antibodies in the efficacy studies described below.

Table 1 Parameters of PK mAb in hamsters mAb AUC _INF-obs (day * mcg / ml) Tmax (day) C _max (μg / ml) Half-life _λ (day) Rsq RA-38 2 mg / kg 303.8 2.00 25,2 7.4 0,999 RA-38 10 mg / kg 1522.6 0.25 128.0 10.6 0.988 RA-41 2 mg / kg 202.2 0.25 20.5 6.2 1,000 RA-41 10 mg / kg 696.7 1.00 78.1 6.8 1,000

B. Evaluation of the mAb for Toxin A and Toxin B of C. difficile of the present invention, in combination, in a model associated with C. difficile diarrhea (CDAD) in Syrian Golden Hamsters

An efficacy experiment was performed to evaluate the ability of murine mAbs to toxin A and toxin B of the present invention to influence the survival of infected animals in an in vivo model of hamster-associated C. difficile diarrhea. Male Syrian golden hamsters (~ 90 g) (Crl: LVG (SYR)) (Charles River Laboratories, Inc., Kingston, NY) were pretreated with a single subcutaneous dose of clindamycin (Sigma, St. Louis, in PBS at 5 mg / ml ) at 50 mg / kg to disrupt the normal flora of the colon. The next day, hamsters in the respective test groups received an oral dose (1 × 10 CFU in 0.5 ml) of a suspension of C. difficile (strain ATCC 43596). Strain 43596 was previously used in hamster models to evaluate neutralizing antibodies. Animals were weighed weekly and monitored for health and survival.

Test antibodies included combinations of murine mAbs of the present invention, i.e. a combination of mAb RA-38 and RA-41; or a combination of mAb RA-39 and RA-41. Goat polyclonal antibodies to Toxin A and Toxin B. C. difficile (Techlab) were included as a positive control. mAb and control reagents were administered as described in table 2.

table 2 Treated groups in a hamster efficacy study Gr. Treatment Dose (mg / kg) Way timetable Number of hamsters one Uninfected NA * NA NA four 2 Uninfected + clindamycin NA NA NA four 3 Infected control NA NA NA 8 four Vancomycin twenty PO BIDX 5 days 8 5 Polyclonal Ab Goats 1 ml / hamster IP Q2dX4 8 6 RA-38 + RA-41 50.50 IP Q2dX4 8 7 PA-39 + RA-41 50.40 IP Q2dX4 8 Not applicable

Hamsters from group 1 did not receive any treatment throughout the study. Hamsters from groups 2-7 were pretreated with a single subcutaneous dose of clindamycin phosphate at 50 mg / kg (day -1). The hamsters from groups 5-7 were administered Polyclonal goat antibodies (group 5) or combinations of mAb (group 6 and 7), as indicated in table 2, by intraperitoneal administration immediately after treatment with clindamycin. After 24 hours, each hamster from groups 3-7 was inoculated with 0.5 ml of a corresponding suspension of C. difficile ATCC 43596 (10 ⁶ -10 ⁷ CFU / ml) through a gastric tube (day 0). After the initial antibody treatment on day -1, the following three treatments of these groups were carried out every two days, once a day, on days 1, 3 and 5. Vancomycin (20 mg / kg BID) was administered to animals from group 4 through a gastric tube twice a day with an interval of about 6 hours on days 1-5. The introduction of vancomycin (animals from group 4) began approximately 20-24 hours after the animals were inoculated with C. difficile.

The survival results in the treated mAbs and control groups are illustrated in FIG. A generalization of mortality of hamsters in all groups is given in Table 3. All hamsters infected with C. difficile without any treatment (infected control, group 3) were found dead on day 2 or day 3 of the study. In the vancomycin-treated group (group 4), seven out of eight hamsters were found dead between days 15 and 19. As is usually observed in this model, most (88%) of the hamsters treated with vancomycin recovered and died from C. difficile infection through two weeks after discontinuation of therapy. In contrast, all hamsters treated with a combination of mAb PA-39 + PA-41 (group 7) and 7 out of 8 hamsters treated with a combination of mAb PA-38 + PA-41 (group 6) survived until the end of the study (37 days after infection ) In addition, all animals from the group treated with goat polyclonal antibodies (group 5) were alive until the end of the study. All surviving hamsters had normal gastrointestinal tract post mortem (see Fig. 15A, C and D).

Table 3 The mortality rate and day of death of hamsters in each group Gr. Treatment Number of animals % mortality Study day 2 3 5 7 fifteen eighteen 19 37 one Uninfected four 0 2 Uninfected + clindamycin four fifty one one 3 Infected control 8 one hundred 5 3 four Vancomycin 8 88 one 5 one 5 Polyclonal Ab Goats 8 0 6 RA-38 + RA-41 8 13 one 7 PA-39 + RA-41 8 0

These results show that the combination of mAb PA-39 and PA-41 and the combination of mAb RA-38 and RA-41 effectively and permanently protected hamsters from serious illness, both initially and from subsequent relapses of the disease. The duration of the effect of mAb treatment (37 days) significantly exceeded the interval (two weeks) for the occurrence of C. difficile infection after treatment with clindomycin in a hamster model.

The body weight of animals in groups treated with polyclonal antibodies and mAb, as well as in control groups is illustrated in Fig. 14. Hamsters in the uninfected control group (group 1) constantly gained weight, in the range from 13 to 29 g, throughout the study. All animals of the infected control died before the first, after inoculation, weight measurement. The average body weight of animals treated with vancomycin, goat polyclonal antibodies, the combination of mAb PA-38 + PA-41 and the combination of mAb PA-39 + PA-41 decreased significantly during the first week after infection. After that, the average body weight in the groups treated with mAb, as well as groups treated with polyclonal antibodies, was constantly increasing, and was similar to that in the uninfected control until the end of the study, indicating that there was no apparent toxicity.

In general, in this study on hamsters, it was shown that the mAb combinations of the present invention effectively and permanently protected hamsters from mortality in appropriate and rigorous hamster models of C. difficile infections. These results confirm the mechanism by which mAb combinations protected animals from C. difficile disease for a time long enough to allow the natural development and repopulation of normal intestinal flora in treated mAb animals compared to uninfected animals (Fig. 15A-D). Thus, the mAbs of the present invention provided therapeutic protection for infected animals and influenced the elimination of C. difficile-associated disease, restoration of gastrointestinal tract health and survival.

C. Evaluation of individual mAbs for toxin A and / or toxin B of C. difficile of the present invention in a model of C. difficile associated diarrhea (CDAD) in Syrian golden hamsters

An additional study on hamsters was performed to evaluate the efficacy of individual mouse mAbs of the present invention administered to an infected animal compared to mAbs administered in combination. The treatment groups of this study are presented in table 4.

Table 4 Study of the effectiveness of individual mAbs compared to a combination of hamsters Gr. Treatment Dose (mg / kg) Way Scheme Number of hamsters one Infected control NA ¹ NA NA 7 2 Vancomycin twenty RO BIDX 5 days 7 3 RA-39 + RA-41 50.50 IP Q2dX4 7 four RA-41 fifty IP Q2dX4 7 5 RA-38 fifty IP Q2dX4 7 6 RA-39 fifty IP Q2dX4 7 7 RA-50 fifty IP Q2dX4 7 ¹ Not applicable

In this study, 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 intraperitoneally injected with mAb immediately after treatment with clindamycin. After 24 hours, each hamster from groups 1-7 was inoculated with 0.5 ml of a suspension of C. difficile through a gastric tube (day 0), as described in section B above. Treatments with test mAbs were administered to animals in groups 3-7 in a single dose on days 1, 3 and 5. Vancomycin was administered to animals twice a day on days 1-5 (group 2). Hamsters were examined for viability twice a day. Body weight was noted once a week. An autopsy was performed on animals that were found dead or euthanized during the study. At the end of the study (40 days after inoculation), the final dissection of all other hamsters was performed.

The survival of the treated mAbs and control groups of animals is shown in FIG. 16A, and the day of hamster death in all groups is summarized in Table 5.

Table 5 Mortality and Day of Death Data Gr. Treatment Number of hamsters % mortality Study day 2 3 four 8 eleven 12 fourteen fifteen eighteen 19 40 one Infected control 7 one hundred 7 2 Vancomycin 7 one hundred one 2 one one 2 3 RA-39 + RA-41 7 fourteen one four RA-41 7 one hundred 6 one 5 RA-38 7 one hundred 5 2 6 RA-39 7 one hundred one one one 2 one one 7 RA-50 7 one hundred 3 four

In the infected control group (group 1), all seven hamsters were found dead on day 2. All of these hamsters had post-mortem examination with inflamed gastrointestinal tract. In the vancomycin-treated group (group 2), all seven hamsters died between study days 12 and 19. The timing of these deaths was similar to that previously observed on this model when treated with vancomycin. A post-mortem examination showed that all hamsters had inflamed gastrointestinal tract, indicating a C. difficile infection.

Treatment with the PAb-39 + PA-41 mAb combination (group 3) in this study was very effective in protecting infected hamsters. Six of the seven hamsters in group 3 survived until the end of the study. One hamster was found dead on day 12 of the study. A post-mortem examination showed that this hamster had an inflamed gastrointestinal tract, which is typical of C. difficile infection.

Among the treatments with antibodies alone (groups 4-7), only mAb RA-39 (group 6) showed some protective activity in the treated animals. In this group, hamsters were found dead for 2-12 days. In the groups treated with individual mAbs, RA-41 (group 4), RA-38 (group 5) or RA-50 (group 7), hamsters were found dead on days 2 and 3. At the final autopsy, all hamsters in these groups had inflamed gastrointestinal tract, indicating a C. difficile infection. In contrast, all treated hamsters that survived had normal gastrointestinal tract.

The results of this study show that treatment with a combination of mAb RA-39 + RA-41 successfully protected hamsters from developing the disease for more than one month after stopping treatment, and they are similar to those obtained in the above study from Example 5 B, in which eight out of eight hamsters treated with the combination of RA-39 + RA-41, survived with C. difficile infection. In this study, RA-39 mAbs, as a treatment with mAbs alone, showed some activity in protecting C. difficile infected hamsters from C. difficile disease.

D. Determination of antibody concentration in final blood samples and assessment of the presence of C. difficile in final hamster cecal samples

Blood was collected from animals that were found dying during the study. At the end of the study, blood was also collected from all animals that remained alive. Blood samples were processed to collect serum unless otherwise indicated below. Treated samples were frozen at <-70 ° C for possible further analysis.

After the in vivo efficacy studies on hamsters described above, the presence of mAbs in the final blood samples from the animals was investigated. To study the mAb combinations described in Example 5B, final blood samples were obtained on day 37 of the study from eight animals from group 7, which received a dose (Q2d × 4) of the combination mAb PA-39 (50 mg / kg) + mAb PA-41 ( 40 mg / kg). RA-39 at 3.3 ± 3.4 μg / ml was detected in serum collected from these final blood samples, and PA-41 was found at 2.4 ± 1.7 μg / ml. In the case of the study of individual mAbs described in Example 5C, final blood samples were obtained on day 40 of the study from six animals from group 3, who received a dose (Q2d × 4) of the combination mAb PA-39 (50 mg / kg) + mAb PA- 41 (50 mg / kg), and blood samples were processed to obtain plasma. In the plasma collected from these final blood samples, RA-39 was detected at 1.8 ± 1.4 μg / ml, and PA-41 was found at 3.4 ± 3.2 μg / ml. The detection limit of antibodies in these assays was 1.6 ng / ml. Thus, detectable mAb levels were measured in animals for several weeks. This supports a mode of action in which these mAbs provide a therapeutic effect throughout the treatment regimen and after administration of the last doses of mAbs.

At the final autopsy, each hamster from group 3 in Example 5C examined the cecum, and each turned out to be normal. No inflammation or redness was observed, and the contents of the cecum were relatively solid in consistency. The wall of each cecum was cut 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 10 μl inoculation loop was used to collect the stool sample from the test tube and inoculate the sample in a Petri dish with agar containing CCFA with horse blood medium (Remel, Lot 735065), which is selective for C. difficile.

Petri dishes were placed in an anaerobic box and incubated for 48 hours at 37 ° C. One Petri dish was streaked with C. difficile ATCC 43596 from the original culture and incubated with fecal line cultures to compare colonies. Colonies resembling C. difficile were found on Petri dishes from all six hamsters. The results of these experiments show that although the surviving animals treated with the mAbs of the present invention were still hosts of C. difficile, their normal flora repopulated to restore normal microbial balance in the intestine, which contributed to their overall survival.

E. Evaluation of Humanized C. difficile Toxin A and / or Toxin B mAb of the present invention and Comparison to a Toxin A and / or Toxin B mAb in a C. difficile Associated Diarrhea (CDAD) Model in Golden Syrian Hamsters

Additional studies on hamsters were performed to evaluate in vivo the effectiveness of the combination of humanized mAb to toxin A and toxin B of the present invention compared to a combination of human mAb comparison to toxin A CDA-1 and human mAb comparison to toxin B CDB-1 with appropriate combinations antibodies infected with C. difficile animals. The treatment groups of this study are presented in table 5A.

Table 5A Treatment groups in a comparative study of hamster efficacy Group Treatment Dose (mg / kg) Way Scheme Number of hamsters one Uninfected control NA ¹ NA NA four 2 Infected control NA NA NA 8 3 Vancomycin twenty BID BIDX 5 days 8 four hPA-41 + hPA-50 50.50 IP Q2dX4 10 5 hPA-41 + hPA-50 20,20 IP Q2dX4 10 6 CDA-1 + CDB-1 50.50 IP Q2dX4 10 7 CDA-1 + CDB-1 20,20 IP Q2dX4 10 ¹ Not applicable

Test antibodies included combinations of the humanized mAb of the present invention, i.e., a combination of the humanized PA-50 mAb to toxin A and the humanized PA-41 mAb to toxin B (hPA-41 + hPA-50), or a combination of a human comparison mAb to toxin A, referred to as a comparison mAb for CDA-1, and a human comparison mAb for toxin B, referred to as a CDB-1 comparison mAb (CDA-1 + CDB-1), in amounts shown in Table 5A. comparison mAbs were synthesized (DNA 2.0) based on published regions of the heavy and light chain 3D8 and 124 (WO2006 / 121422 and US2005 / 0287150) cloned in full-size IgGI expression vectors (pCON-gamma and pCON-kappa) expressed in CHO-KSV1 cells and purified using the methods described herein. Combinations of mAb and control were used as described in table 5A.

The processing methods were mainly as described in part B of this example above. Briefly, in Example 5E of this study, golden Syrian hamsters were used (Charles River Laboratories, Stone Ridge, NY, 50 days old). The hamsters in control group 1 were uninfected (and untreated). Animals in groups 2-7 were pretreated with a single subcutaneous dose of clindamycin phosphate at 50 mg / kg to disrupt the normal flora of the large intestine (day -1). Animals of groups 4-7 were intraperitoneally dosed immediately after treatment with clindamycin. After 24 hours, each animal from groups 2-7 was inoculated with 0.5 ml of a suspension of C. difficile (ATCC 43596, strain 545) through a gastric tube (day 0), (i.e., an oral dose). Additional administrations of test treatments were applied to animals in groups 4-7 in a single dose on day 1, 3 and 5. Vancomycin was administered to animals of group 3 twice a day, with a difference of about 6 hours, on day 1-5. The animals were weighed weekly and daily monitored for health and survival for 39 days. After completion, an autopsy was performed, and after anaerobic cultivation at 37 ° C for 48 hours, the titers of C. difficile microorganisms in the cecum were determined on selective medium. The detection limit was 20 CFU / g cecal contents. This study and the hamster studies described above were conducted at Ricerca Biosciences (Concord, OH) in accordance with the guidelines of the Institutional Animal Care and Use Committee.

Survival results for the treated mAbs and control groups are shown in FIG. 16B-1. Mortality studies are presented in table 5B below. A brief description of hamster survival is presented in table 5C below.

Table 5B Data on mortality and day of death of hamsters in each group Gr. Treatment Number of animals % mortality Study day 2 3 5 8 from 11 to 14 from 18 to 20 22 ...
thirty one Uninfected control four 0 2 Infected control 8 one hundred 7 one 3 Vancomycin 8 one hundred one 5 2 four hPA-50 + hPA-41 50, 50 mg / kg 10 10 one 5 hPA-50 + hPA-41 20, 20 mg / kg 10 0 6 CDA-l + CDB-1 comparisons 50, 50 mg / kg 10 one hundred one 8 one 7 CDA-1 + CDB-1 comparisons 20, 20 mg / kg 10 one hundred 2 8

As noted in table 5B, all 4 uninfected control hamsters (group 1) survived until the end of the study. All infected control animals (group 2) died on days 2 and 3. In the vancomycin-treated group (group 3), all the animals studied died between day 13 and 22. In group 4 with hPA-50 + hPA-41 (50, 50 mg / kg) nine out of ten animals survived until the end of the study. One hamster from this group was found dying on day 8, with a change in the color of the gastrointestinal tract to red, while the remaining nine surviving animals in this group had a normal gastrointestinal tract. All ten hamsters in group 5 survived until the end of the study and had normal gastrointestinal tract. In the comparison mAb groups, nine out of ten animals that were given a dose of 50 mg / kg (group 6) died between days 5 and 14; one animal died on day 28 of the study. All ten hamsters treated with a 20 mg / kg combination mAb comparison (group 7) died between study days 5 and 14.

Table 5C Average and overall survival of animals treated with the humanized mAbs of the present invention and comparison mAbs

Group / Processing Dose (mg / kg) Average survival (days) Day 40 Survival (%) Group 1 Uninfected NA * 40 one hundred Group 2 Infected NA * 2 0 Group 3 Vancomycin twenty twenty 0 Group 4 hPA-50 + hPA-41 50.50 NA 90 Group 5 hPA-50 + hPA-41 20,20 NA one hundred Group 6 CDA-l + CDB-1 50.50 fourteen 0 Group 7 CDA-l + CDB-1 20,20 eleven 0 ^* Not applicable

As noted in table 5C, all four animals in the uninfected control group 1 survived until the end of the study (40 days). All hamsters infected with C. difficile without any treatment (infected controls, group 2) had an average survival of 2 days; animals in this group did not survive until the end of the study. In the vancomycin-treated group (group 3), the average survival was 20 days, the animals did not survive until day 40. Both treatments with hPA-50 + hPA-41 mAb combinations were effective in protecting infected animals in this study (groups 4 and 5). All (100%) hamsters treated with a combination of humanized mAb PA-50 + PA-41 (20 mg / kg each, group 5) and 90% hamsters treated with a combination of humanized mAb PA-50 + PA-41 (50 mg / kg each ; group 4), survived until the end of the study (40 days after infection). At post-mortem, all surviving hamsters had mostly normal gastrointestinal tract. In contrast, the average survival of animals treated with a combination of mAb comparisons to toxin A and toxin B was similar for both doses of the mAb comparison; in two groups treated with mAb comparison combinations, all animals died. In particular, for animals treated with a combination of CDA-1 + CDB-1 (50 mg / kg each; group 6), the average survival was 14 days, and for animals treated with a combination of CDA-1 + CDB-1 (20 mg / kg each; group 7), mean survival was 11 days.

Additional assessments in the study included body weight measurements, macroscopic autopsy, and determination of the titer of C. difficile microorganisms in the cecum at the end of the study. The average body weight of animals treated with vancomycin or a combination of RA-50 / RA-41 decreased during the first week after infection, and then recovered (figv-2). On day 39, the average body weight of the animals treated with the RA-50 / RA-41 combination was similar to that in healthy uninfected animals that were kept in parallel (P> 0.05). The average body weight of animals treated with the combination of CDA1 / CDB1 reference antibodies steadily decreased during the study.

On day 39 of the gastrointestinal tract, 19 surviving animals treated with the RA-50 / RA-41 combination were found to be similar to those in uninfected animals; and C. difficile titers in the cecum were either undetectable (<1.3 log ₁₀ CFU, n = 11) or low (4.15 ± 0.76 log ₁₀ CFU, t = 8). In contrast, inflamed gastrointestinal tract was observed in some or all animals in other treated groups at the time of death. C. difficile was found in 4 of 4 untreated animals (mean CFU = 8.96 ± 0.59 log ₁₀ , P <0.0001 with respect to RA-50 / RA-41) and in 4 of 4 animals treated with vancomycin (mean CFU = 6.01 ± 0.93 log ₁₀ , P <0.017 with respect to RA-50 / RA-41), for which the cecum was analyzed. Most hamsters treated with a combination of CDA1 / CDB1 reference antibodies had practically no cecal contents, which precludes quantitative analysis of C. difficile titers.

For statistical analysis in this study and the aforementioned studies, neutralization data was suitable for a four-parameter logistic equation using GraphPad Prism (v. 4.0 GraphPad Software, San Diego, CA). To compare the average and overall survival data, we used bilateral t-criteria or logarithmic rank criteria, respectively.

The results of the study show that treatment of C. difficile infected animals with a combination of humanized PAb-50 and PA-41 mAbs at both dose levels contributes to effective and long-term protection of hamsters from serious diseases, both initially and in subsequent relapses of the disease. The duration of the treatment effect with a combination of humanized mAbs (40 days) significantly improved the long-term survival of animals compared to treatment with vancomycin or mAbs compared to toxin A and toxin B as a control.

According to in vivo animal studies, combination treatment with the combination of humanized PA-50 / PA-41 mAbs was highly effective against C. difficile infection in a well-organized model of Syrian Golden Hamsters for CDI and therapy. A short course of treatment with PA-50 / PA-41 resulted in 95% survival by day 39 after infection, compared with 0% survival of animals that were not treated, received standard antibiotic therapy or mAb comparisons. 39 days after infection, the animals treated with PA-50 / PA-41 had normal weight and there were no obvious gastrointestinal lesions. C. difficile cannot be isolated in most animals, reflecting> 7-log ₁₀ clearance relative to untreated animals. One possible explanation for these results is that mAb-mediated neutralization of toxins in the absence of antibiotics ensured the restoration of protective microflora in the digestive tract of animals.

It has been reported that either toxin A or toxin B alone can cause fatal disease in a hamster CDI model and, as a rule, mAb to both toxins is required for maximum treatment effectiveness. In the studies described above, murine mAb PA-50 and PA-41 did not show a beneficial effect on survival when used individually at doses of 50 mg / kg in a hamster model, which emphasizes the need for combined treatment.

In general, in this study on hamsters, similar to the results of the use of mouse mAb combinations described above, it was demonstrated that the combinations of humanized mAbs of the present invention contribute to the effective and long-term protection of hamsters from mortality in a strict model for hamsters of C. difficile infection. Without being limited by theory, these data confirm the mechanism of action by which combinations of humanized mAbs protect animals from C. difficile disease and / or ensure that animals respond to C. difficile infection for a time long enough to allow natural development and repopulation of normal intestinal flora in animals treated with humanized mAbs, thus providing therapeutic protection for infected animals, effectively eliminating the associated with C. difficile disease and restoring health and survival of the digestive tract.

Example 6

The binding of mAb to the areas of toxin A and toxin B C. difficile

The experiments were performed to determine the epitope regions of the toxin A and toxin B of C. difficile, to which the mAbs of the present invention bind. Both toxin A and toxin B produced by C. difficile are approximately 300 kDa and have significant sequence and structure homology. Both have a C-terminal receptor binding domain that contains clostridial repeating oligopeptides (CROPs), a central hydrophobic domain that is believed to cause pore formation and mediate the incorporation of toxin into the endosome membrane, and a proteolytic domain that cleaves the N-terminal enzymatic domain, such thus allowing glucosyltransferase to enter the cytosol. Nucleic acid sequences encoding C. difficile toxins, as well as other C. difficile proteins, have been published and are also available on the National Center for Biotechnological Information (NCBI) database (i.e., www.ncbi.nlm.nih.gov). For example, for C. difficile strain VPI 10463, DNA sequences encoding toxin A and toxin B can be found under accession number NCBI x92982; in addition, NCBI accession number NC_009089, region 795842-803975, provides the DNA sequence for toxin A from the complete genomic sequence of C. difficile 630, while accession number NCBI NC_009089, region 787393-794493, provides the DNA sequence encoding toxin B from the chromosome sequence of C. difficile 630.

A. Mapping of the Antibody-binding Domain of Toxin B C. difficile

Toxin In C. difficile, the full length consists of three main domains: the N-terminal enzymatic domain (63 kDa) exhibiting glucosyl transferase (GT) activity, and the C-terminal (59 kDa) binding cell receptor, which are located at both ends of the putative translational domain (148 kDa) (figa and C). Some fragments of toxin B were obtained by enzymatic digestion of toxin B full length using caspase 1 enzyme (FIG. 17C). Following the processing of toxin B in caspase 1 (enzyme / toxin ratio ~ 1 U / μg toxin) at 37 ° C for four hours, four main fragments were obtained, including two fragments that contain the C-terminus (193 and 167 kDa), and two fragments that contain the N-terminus (103 and 63 kDa), as detected by SDS-PAGE (pigv). Other smaller fragments, such as 26 and 14 kDa, apparently also received, but could not be detected in the analysis with 3-8% Tris-acetate gel.

SDS-PAGE and Western blotting analyzes were performed on toxin B that was not treated or was treated with caspase 1 (Fig. 18A-C). PAb 41 mAbs recognized both 103 kDa and 63 kDa fragments of caspase 1-treated toxin B (right row in FIG. 18B), thus indicating that RA-41 binds to the N-terminal enzyme domain of toxin B. Analysis N -terminal sequence confirmed that RA-41 binds to 63 kDa of the N-terminal enzymatic domain of toxin B. It is interesting to note that untreated toxin B with a 63 kDa band (left row, Fig. 18 B) was not recognized by RA-41, which suggests that in the bands two fragments with the same molecular weight (63 kDa) are apparently different proteins.

mAb PA-39 bound a 167 kDa fragment of toxin B treated with caspase 1 (Fig. 18C, right row), as well as 63 kDa protein in the preparation of untreated toxin B (Fig. 18C, left row), thus indicating that PA-39 binds the epitope in the translocation domain of toxin B. Thus, based on the results of SDS-PAGE / Western blotting of toxin B of C. difficile treated with caspase 1, it was observed that the PAb-41 mAb and PA-39 interacted differently with toxin B. While it was found that the mAb RA-41 binds the epitope in the N-terminal enzymatic domain of toxin B, found We lived that mAb PA-39 binds to an epitope of toxin translocation domain (amino acids 850-1330). These results were also confirmed by SDS-PAGE / Western blot analysis of toxin B fragments using enterokinase digestion.

Competitive binding of mAbs to toxin B with toxin B was also carried out using Biacore. As can be seen from FIGS. 19A-E, PAb-39 and PA-41 mAb bind different epitope regions of mouse toxin B. mAb PA-39 and PA-41 are found to bind to a single site or epitope on toxin B; these mAbs were not found to bind to the C-terminal binding cell receptor (SLE) domain. For mouse RA-41, the toxin B binding affinity was 0.59 mM. Additionally, the toxin B site bound to RA-41 did not block the binding of the mAb to toxin B of CDB-1 (WO / 2006/121422; US2005 / 0287150), (FIG. 19D). These results are consistent with those from Western blot analyzes. According to the observations of FIGS. 19C and E, the toxin B comparison mAb CDB-1 binds toxin B through epitopes different from those for PAb-39 and PA-41 mAbs.

B. Mapping of the Antibody Domain of Toxin A C. difficile

The full length C. difficile toxin A has a molecular weight of 310 kDa (Fig. 20A) and contains three main domains: an N-terminal functional domain with glucosyl transferase (GT) activity (~ 63 kDa) and a C-terminal SLE domain (~ 101 kDa), which are located at both ends of the hydrophobic domain (~ 144 kDa).

Some fragments of toxin A were obtained by enzymatic cleavage of a full-length toxin using the enterokinase enzyme (EC). Following treatment of toxin A with enterokinase (enzyme / toxin ratio: approximately 3 mU / μg toxin), nine main fragments were obtained at 25 ° C for 48 hours, 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. 20B and C).

SDS-PAGE and Western blot analyzes were performed on toxin A, which was treated or not treated with enterokinase (figa-C). Full length toxin A and fragments thereof with a molecular weight of ~ 223, ~ 158-160, ~ 91, and ~ 68 kDa were recognized by mAb PA-50 (Fig. 21B); Determination of the N-terminal sequence confirmed that the 68 kDa fragment contains a portion of the C-terminal receptor binding (CRB) domain. The PA-50 mAb binding pattern indicates that the mAb binds to toxin A fragments containing the C-terminus. Taken together, the results indicate that PAb-50 mAb binds to C-terminal receptor-binding epitopes on toxin A. mAb PA-39 binds fragments containing the C-terminus (-223 and -158-160 kDa), as well as -181 kDa fragments containing the N-terminus (Fig. 21C), thus indicating that the PA-39 mAb binds to the epitope outside the toxin A receptor binding domain. Multiple binding sites (at least two binding sites) were also identified in the interaction study PAb-50 mAb and Toxin A using Biacore analysis (FIG. 22A-1). In comparative assays using the Biacore assay, immobilized mouse PA-50, in particular, binds toxin A with an affinity of 0.16 nM. It was also found that after capturing the mouse PAb-50 mAb on a sensor chip, toxin A was able to bind an additional RA-50 and, subsequently, a comparison mAb to CDA-1 toxin A (WO / 2006/121422; US2005 / 0287150), (FIG. .22A-2). Additionally, the toxin A captured by the comparison mAb to the CDA-1 toxin A on a Biacore chip further linked the additional CDA-1 and PA-50 mAbs, indicating that the CDA-1 comparison mAbs bind to multiple repeats on the toxin A, which are different from the binding epitopes for mAb RA-50 on toxin A (pigv). Thus, as determined from these results, the mAb RA-50 epitope is present in multiple copies on toxin A and does not overlap with the epitope for CDA-1. In addition, the RA-39 mAb binds to the toxin A epitope (s) different from the toxin A epitope (s) that bind to the CDA-1 reference mAbs (Fig. 22C). mAb RA-39 and RA-50 were found to bind different epitopes on toxin A (Fig.22D). Western blot analysis showed that the PA-39 and the CDA-1 mAb comparisons have different binding patterns for the EK-treated toxin A, thus indicating different binding domains and different epitopes on toxin A (FIG. 22E). Western blot analysis showed that the RA-50 and the CDA-1 mAb comparisons bind to the same EK-treated toxin A domain (Figure 22F), but with different epitopes (Figure 22B).

As described in A and B of this example, the binding sites for murine mAbs of RA-50 and RA-41 were localized in certain areas of toxins by limited proteolysis of toxins followed by western blotting. The RA-50 mouse mAb recognized full-length toxin A and some enterokinase cleavage products, including a large 223 kDa fragment and carboxy-terminal fragments of 68, 91 and 160 kDa sizes (Fig. 22F). Determination of the N-terminal sequence confirmed that the 68 kDa fragment corresponds to the carboxy-terminal domain of toxin A. The same fragments were recognized by the CDA-1 mAbs. The murine mAb RA-41 bound the full length toxin B as well as the amino terminal fragments of 63 and 103 kDa obtained by caspase-1 cleavage (FIG. 18B), while the CDB-1 mAb comparison recognized a different set of caspase-1 cleavage products. Determination of the N-terminal sequence confirmed that the 63 kDa fragment corresponds to the amino-terminal domain of toxin B. Together, the data indicate that mPA-50 binds multiple sites within the receptor-binding domain of toxin A, and mPA-41 binds one site within the enzymatic domain of the toxin AT.

Example 7

mAb to Toxin A and Toxin B. C. difficile - studies of the mechanism of action

A. Cellular in vitro assays used to study the mechanism of action

In order to evaluate the mechanism of action of the mAb to the toxin, in vitro assays were performed using different concentrations of toxin A or toxin B. In these assays, either CHO-K1 or T-84 cells were used, as described in Example 3 above.

Briefly, a CHO-K1 assay was used to evaluate the neutralizing efficacy of a mAb for toxin A and toxin B (PA-39 and PA-41). CHO-K1 cells (2000 cells / well) were seeded in 96-well plates. Cells were allowed to adhere within 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 mAb, mixed in 96-well round-bottom plates for 1 hour at 37 ° C, and the mixture was then added to cell culture plates. After incubation for 72 hours, 20 μl / well of CellTiter-Blue was added; mixtures were incubated for an additional 4 hours; and measured the percentage of cellular survival compared to controls.

A cytotoxicity assay on T-84 was also used to evaluate the neutralizing efficacy of the mAb to toxin A. T-84 cells (human colon carcinoma cell line) were seeded (15,000 cells / well) in 96-well plates. Cells were allowed to adhere within 4 hours prior to treatment. Different concentrations (240, 120, 60, or 30 ng / ml) of C. difficile toxin (strain VPI 10463) were incubated with serially diluted mAb, mixed in 96-well round-bottom plates for 1 hour at 37 ° C, and the mixture was then added to cell culture plates. After incubation for 72 hours, 20 μl / well of CellTiter-Blue was added; mixtures were incubated for an additional 4 hours; and measured the percentage of cellular survival compared to controls.

B. ELISA showing that toxin A mAbs prevent the internalization of toxin A into cells

In an experiment designed to further evaluate the mechanism of action of mAbs against C. difficile toxin, each test antibody (PA-39, PA-50, CDA-1 toxin A comparisons and control goat polyclonal antibodies to toxin A) was mixed and incubated in for 1 hour at 100x its EC ₉₀ values with a CC ₉₀ concentration of toxin A for Vero cells to ensure complete neutralization at a high cytotoxic concentration of toxin A. The mixture was then incubated with Vero cells at 37 ° C for 15 minutes. Cells were then washed with PBS, fixed and permeabilized. Antibody (PA-38) to toxin A labeled with horseradish peroxidase (HRP), which does not compete for binding to test antibodies, was used to study for internalized toxin A and was detected using chemiluminescence (Fig. 31G). In this assay, only toxin A, which has bound and internalized into the cell, will be detected by the probe, thus producing a chemiluminescent signal based on the chemiluminescent HRP reaction. In chemiluminescent detection, an enzyme is used to catalyze a reaction (i.e., catalyzed oxidation of luminol by peroxide) between the HRP enzyme and its substrate in the presence of peroxide, resulting in the appearance 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 using a luminometer (Analyst GT).

C. Results of neutralizing activity and MOA studies

mAb to toxin A

In a cell cytotoxicity assay that was used to evaluate the neutralizing activity and mechanism of action of antibodies to toxin A, toxin A was added in increasing concentrations to the cells, as described above in Part A of this example. The results of these experiments, in which the neutralization of toxin A mAb to toxin A PA-39, PA-50 and CDA-1 toxin A were evaluated, are shown in FIG. 31B-D and below in Table A.

Table a The results of the experiment on the effectiveness of toxin A and the maximum percentage suppression mAb [Toxin A] (ng / ml) [EC ₅₀ ] *
(nM) Maximum percent suppression RA-39 60 0.340 69 thirty 0,080 86 fifteen 0.003 96 6 0.002 97 PA-50 240 0.91 105 120 0.54 one hundred 60 0.27 114 thirty 0.10 118 mAb comparison CDA-1 240 9.0 52 120 6.6 74 60 5.1 103 thirty 2.0 110 *: EC _{5o was} calculated as 50% of the maximum percent suppression in cases where the curves did not reach 100% control.

As can be seen from the data presented in table A, the activity of in vitro mAb PA-39 in the toxin efficacy assay shows shifts in both the EC ₅₀ and the maximum percentage of suppression as more toxin A is added to the culture, which indicates to a mixed competitive suppression mechanism for RA-39. ELISA detection of toxin A after protection with a 100-fold excess of RA-39 confirmed that the suppression of the toxin by RA-39 occurs by preventing the internalization of the toxin and the cytotoxic effect of the toxin. The in vitro activity of PAb 50 mAb in a toxin efficacy assay shows a shift in EC5o as more toxin A is added to the culture, indicating a competitive suppression mechanism for RA-50. ELISA detection of toxin A after protection with a 100-fold excess of RA-50 confirmed that the suppression of the toxin by RA-50 occurs by preventing the internalization of the toxin.

mAb to toxin B

In a cell cytotoxicity assay that was used to evaluate the neutralizing activity and mechanism of action of an anti-Toxin B antibody, toxin B was added in increasing concentrations to the cells, as described above in Part A of this example. The results of efficacy experiments in which the neutralization of toxin B mAb to toxin B RA-41 and CDB-1 toxin comparison mAb were evaluated are shown in FIGS. 31E and F and table B below. As can be seen from the data presented in the table, the in vitro activity of PA-41 in the toxin efficacy analysis shows shifts in both the EU ₅₀ and the maximum percentage of suppression as more toxin B is added to the culture, indicating mixed competitive suppression mechanism for RA-41.

Table B The results of the experiment on the effectiveness of toxin B and the maximum percentage suppression mAb [Toxin A] (ng / ml) [EC ₅₀ ] * (nM) Maximum percent suppression RA-41 twenty 0.82 49 10 0.33 80 5 0.13 96 2 0.05 96 mAb comparison CDA-1 twenty 4.1 23 10 3.0 51 5 1,1 75 2 0.2 95 *: EC _{5o was} calculated as 50% of the maximum percent suppression in cases where the curves did not reach 100% control.

Based on the above analyzes in this example, an inhibitor that has the ability to completely neutralize increasing concentrations of toxin A or toxin B simply by adding higher concentrations of the inhibitor will show a shift in the EC ₅₀ as the antibody binds and neutralizes higher concentrations of the toxin, and therefore, it will be considered a competitive inhibitor. An inhibitor that is not able to attenuate the toxic effects of increasing concentrations of toxin will exhibit a reduced maximum percent effect at higher inhibitor concentrations, but will not exhibit a shift in the EC ₅₀ and, therefore, will be considered a non-competitive inhibitor. Additionally, an inhibitor that exhibits both a shift in the EC ₅₀ and a reduced maximum percentage of the effect as a result of increasing concentrations of the inhibitor at higher concentrations of the toxin will be considered a mixed competitive inhibitor. To some extent, the assessment of the mechanism of action using cell cytotoxicity assays should be carried out taking into account the error associated with the reproducibility of the assay and the background cytotoxicity observed in control wells that do not contain an inhibitor that acts on the plateau of the maximum percent suppression. Therefore, a slight rightward shift in EC ₅₀ values can be observed as toxin concentrations increase due to these effects.

In accordance with the foregoing, regarding the neutralization of toxin A and MOA, the RA-39 mAb is considered to be a mixed competitive inhibitor due to a shift in the EC ₅₀ and a reduced plateau observed in relation to the RA-39 cytotoxicity curves (Fig. 31B), as well as a reduced maximum percent effect. calculated in Table A. These data are confirmed by ELISA results (Fig.31H), which show the degree of cytotoxic effect at high concentrations of RA-39, thus indicating that at least some of the MOA RA-39 occurs intracellularly it is. It was observed that the PAb 50 mAb is a competitive inhibitor, as evidenced by a right shift in the EC ₅₀ values observed in the cytotoxicity analysis curves (Fig. 31C) and the data presented in Table A showing the minimum change in the maximum percent inhibition. These data are confirmed by ELISA results (Fig.31H), which show a complete suppression of toxin A binding and internalization at high concentrations of PA-50.

According to the observations of FIG. 31D, the CDA-1 comparison mAb shows a minimal shift in the EC ₅₀ , but a significant decrease in the maximum percentage of the effect as the amount of toxin increases. If the results shown in Fig. 31D are considered with the data presented in Table A, the CDA-1 comparison mAb shows a non-competitive mechanism of action, since all its activity is observed outside the cell. The steric mismatch between toxin binding and cell internalization appears to be responsible for the output of the plots with the data in FIG. 31D, thereby confirming the non-competitive MOA for the CDA-1 mAb comparison.

In accordance with the foregoing, regarding neutralization and MOA of toxin B, it is believed that PAb-41 mAb exhibits a mixed competitive mechanism of action due to a rightward shift of EC ₅₀ values and a reduced maximum percent effect observed in FIG. 31E and in the data presented in Table B.

The results observed for the PAb-41 mAb contradict those observed for the CDB-1 mAb comparison, which exhibits a reduced maximum percentage of effect, but a lower degree of EC ₅₀ shift. In this case, the mechanism of action for the activity of the comparison mAb with respect to toxin B is less understood, especially considering the consideration of the error mentioned above.

Example 8

Testing the mAbs of the present invention with respect to a panel of C. difficile isolates or strains, including hypervirulent isolates or strains

To evaluate the C. difficile toxin A and B mAb ability of the present invention to neutralize toxins from a wide range of corresponding C. difficile isolates, the neutralizing activity of the mAbs was tested against a set of twenty toxicogenic clinical isolates or C. difficile strains, including the hypervirulent BI / NAP1 / 027 isolates . Since C. difficile exhibits significant interstrain heterogeneity in genes encoding toxins A and B, these studies were performed to study the degree of toxin neutralization by the described mAbs, and in particular, the PA-50 and PA-41 mAbs.

A panel of C. difficile toxicogenic clinical isolates (Table 6) was selected for geographic and genetic diversity based on the type of toxin, ribotype, and restriction endonuclease assays from the international collection of C. difficile isolates or strains supported by TechLab (Blacksburg, VA).

As classified in Table 6, C. difficile strains include 3 reference strains (VPI 10463 (ATCC 43255), 630 (ATCC BAA-1382) and 545 (ATCC 43596), six hospital-derived strains BI / NAP1 / 027 (CCL678, NMS553, Pitt45, CD196, 5, 7.1), two toxin A- / toxin B + (tcdA-tcdB +) strain (F1470, 8864), three outpatient isolates (MH5, CCL13820 and CCL 14402) and other common clinical isolates (Pitt2 , CCL14137, UVA17, UVA30 / TL42, Pittl02, Pitt7) Additionally, isolates 13 (CCL13820) and 19 (Pitt102), which are classified as “ambulatory isolate” and “common clinical isolate (other than ribotype 127)”, respectively, in the table 6 also are toxA- / toxB + strains. Cultural supernatants containing these C. difficile toxins were obtained in TechLab, sterilized by filtration and stored at 4 ° C. The presence of toxins in the culture supernatants was confirmed using a C. difficilelsamt OKKvoi toxin kit (TechLab) and cytotoxicity analysis .

The cytotoxic activity of each toxin-containing culture supernatant was evaluated on CHO-K1 cells (used to determine the toxin B activity of the culture supernatants) and T-84 cells (used to determine the toxin A activity of the culture supernatants) by treating the cells with titrated culture supernatants.

Table 6 Isolates / strains of C. difficile used to produce supernatants No. Strain Category Ribotype Type of toxin REA Type Year Location one VPI 10463 ATCC 43255 A + B + reference strains 003 0 Not known ~ 1975 USA 2 630 ATSS VAA-1382 Not known Not known Not known 1982 Switzerland 3 545 ATCC 43596 Not known Not known Not known Not known Leuven, Belgium four CCL 678 Ribotip 027 (hospital isolates) 027 III BI 2007 Virginia, USA 5 HMS553 2008 Pennsylvania, USA 6 Pitt45 2001-2002 Pennsylvania, USA 7 Cdl96 1988 Paris, France 8 5 2004 Quebec Canada 9 7.1 2004 Quebec Canada 10 F1470 tcdA strains ^- tcdB ⁺ 017 VIII CF1 1990 Leuven, Belgium eleven 8864 CCUG 20309 036 X CY1 1986 Birmingham england 12 Mh5 Outpatient Isolates 001 Not known Not known 2008 Virginia, USA 13 CCL 13820 017 VIII CF 2008 Virginia, USA fourteen CCL 14402 027 III BI 2008 Virginia, USA fifteen Pitt2 Common clinical isolates (other than ribotype 027) 001 Not known J 2001-2002 Pennsylvania, USA 16 CCL 14137 001 Not known J 2008 Virginia, USA 17 UVA17 002 He famous G 2006-2007 Virginia, USA eighteen UVA30 / TL42 014 Not known Y 2006-2007 Virginia, USA 19 Pitt102 017 VIII CF 2001-2002 Pennsylvania, USA twenty Pitt7 078 V Bk 2001-2002 Pennsylvania, USA

To test the neutralizing activities of the mAbs of the present invention, toxin containing culture supernatants were used at maximum dilution, resulting in> 95% loss of cell viability. Toxin supernatants were pre-mixed with various mAb concentrations for 1 hour and then added to the cells for incubation at 37 ° C for 72 hours. Cell viability was measured using Cell-Titer Blue (Promega). The percentage of survival in the treated wells was compared with untreated control wells and a graph was plotted to calculate the in vitro neutralizing activity of the mAb (EC ₅₀ ). In a first series of experiments, FIG. 23A shows the activity of RA-41 mAb in neutralizing toxin-containing supernatants using CHO-K1 cells. RA-41 effectively (EC ₅₀ range from 1.1 ^-11 M to 6.5 ^-10 M) neutralized the supernatants of all toxicogenic C. difficile strains, including all hypervirulent strains, with the exception of three toxin A- / toxin B + strains. It was reported that there are significant differences in the sequences of the enzymatic domains of toxin A- / toxin B + from standard C. difficile strains. Since RA-41 binds to the enzymatic domain of toxin B, the diversity of the sequences of this domain may explain the less effective neutralizing activity of RA-41 in relation to toxin B from two strains of toxin A- / toxin B +.

Experiments were also conducted to evaluate the activity of both hPA-41 and the human mAb comparing CDB-1 with respect to toxin B (WO / 2006/121422; US2005 / 0287150) to the hypervirulent C. difficile strains on the CHO-K1 cell line. In these studies, it was observed that hPA-41 showed significant neutralizing activity to supernatants from all 6 BI / NAP strains 1/027, while the CDB-1 comparison mAb showed minimal activity (Figure 23B). Also in these studies, it was observed that the neutralizing activity of the hPA-41 mAb was> 1000 times greater than that of the CDB-1 mAb for neutralizing the toxicity of the BI / NAP strains 1/027. The neutralizing activity of hPA-41 and mAb comparing CDB-1 against 2 reference strains (VPI 10463 and ATCC 43596) and 6 strains BI / 027/027 (CCL678, HMC553, Pitt 45, CD196, Montreal 5.1 and Montreal 7.1) is illustrated Fig. 23 B. In these studies, hPA-41 was found to be inactive with respect to the three isolates of ribotype 017 (Table 6), which are toxin A- / B +, although the mAbs of hPA-41 to toxin B showed a significantly greater neutralizing activity than mAb comparisons with respect to other strains in the panel.

The activity of RA-50 mAb in neutralizing toxin A-containing supernatants of C. difficile cultures using T-84 cells ranged from 2.6 ^-12 M to 7.7 ^-11 M, as shown in FIG. 24A. RA-50 completely neutralized supernatants from all available strains that produce toxin A, including hypervirulent strains. RA-50 did not neutralize four strains of toxin A- / toxin B + (F1470, 8864, CCL 13820, CCL 14402), since these strains did not produce any A. toxin. HPA-50 was also significantly more effective in neutralizing activity remaining strains in the panel. Other comparative studies found that the neutralizing activity of hPA-50 against all 6 C. difficile producing BI / NAP 1/027 strains producing toxin A was> 100 times greater than that of the CDA-1 mAb comparison (WO / 2006 (121422; US2005 / 0287150) (Fig. 24B). hPA-50 also showed greater neutralizing activity than the CDA-1 mAb comparisons against toxin A-producing reference strains VPI 10463 and 545 C. difficile. Similarly, RA-39 mAb neutralized toxin A in supernatants of C. difficile cultures, with EC ₅₀ values ranging from 7.7 ^-12 M to 4.8 ^-8 M, as shown in (Fig. 25A). As can be seen with respect to the mAb RA-50, the four strains of toxin A- / toxin B + were not neutralized by RA-39. Moreover, the results of comparative studies have shown that the neutralizing activity of the RA-39 mAb in relation to all 6 BI / NAP strains 1/027 was> 100 times greater than that of the human mAb comparison to CDA-1 toxin A; RA-39 also showed a significantly greater neutralizing activity with respect to the remaining strains in the panel (FIG. 25B). It was noted that one strain with a toxin, CCL 14402, did not reduce the viability of T-84 cells enough to enable accurate measurement of the neutralizing activity of mAb in this assay.

In these studies, on the CHO-K1 cell line, hPA-41 showed high levels of neutralizing activity against supernatants from all 6 BI / NAP strains 1/027, while the CDB-1 mAb comparison showed minimal activity. It was observed that the humanized RA-41 had a neutralizing activity> 1000 times greater than that of the CDB-1 mAb when neutralizing the hypervirulent BI / NAP 1/027 C. difficile strains. The neutralizing activities of hPA-41 and CDB-1 in these studies with respect to 2 reference strains (VPI 10463 and ATCC 43596) and 6 strains BI / 027/027 (CCL678, HMS553, Pin 45, CD196, Montreal 5.1 and Montreal 7.1) illustrated in figv. Similarly, hPA-41 showed a significantly higher neutralizing activity in these studies compared to the CDB-1 comparison mAb in relation to other strains in the panel, with the exception of three isolates of ribotype 017, which are A- / B +. Similar experiments were carried out to evaluate the neutralizing activity of hPA-39 and that of the human mAb comparing to CDA-1 toxin A on T-84 cells. The results showed that the neutralization of all 6 strains of BI / NAP 1/027 hPA-39 was> 100 times greater than that of the CDA-1 mAb comparison (FIG. 25B). Humanized RA-39 also showed a significantly higher neutralizing activity in the studies than the CDA-1 reference mAb, relative to the remaining strains in the panel. Thus, the mAbs hPA-41 and hPA-39 showed high levels of neutralizing activities for hypervirulent C. difficile strains with respect to all tested strains. This indicates that the epitopes recognized by these humanized mAbs are highly conserved for a combination of different strains.

Similar to table 6, table 7 presents the results of the above in vitro experiments to neutralize C. difficile toxin, showing a panel of toxicogenic C. difficile strains isolated from North America and Europe and the resulting EC ₅₀ values obtained using humanized toxin mAb and mAb CDA-1 and CDB-1 comparison drugs. The panel includes the ribotypes 001, 002, 003, 012, 014, 017, 027, and 078 approximately proportionally to the frequencies observed clinically (94, 95), with the exception of the ribotype 017 of the tcdA ^- tcdB ⁺ strains, which are presented in an excessive amount in the panel. Supernatants from tcdA ^- tcdB ⁺ strains were used as means for identifying cells that were not cytolizable by supernatants containing only toxin B, and thus would be suitable for studying cytotoxicity mediated by toxin A. VPI 10463 was included in the panel, and this allowed us to compare results obtained with purified and untreated toxins.

In these studies, humanized mAb PA-50 neutralized toxin A in a strain-independent manner. The EC ₅₀ median value was 32 pM (range: 20 to 127 pM, Table 7), and steep dose response curves were observed with Hill slopes, which typically were more than two (FIG. 25C). RA-50 was more active than CDA1 with respect to each test isolate. The greatest difference in efficacy was observed for hypervirulent strains 027, for which RA-50 was approximately 1000 times more effective than CDA1 (P = 0.0002), and for strain ribotype 078 included in the panel.

The humanized PAb-41 mAb effectively suppressed each of the tcdA ^- tcdB ⁺ strains with an EC ₅₀ median value of 23 pM (range: 7.7 to 129 pM, Table 7), and, in effect, complete neutralization was observed at higher concentrations (FIG. .250). PA-41 was generally more effective than CDB1 in relation to tcdA ^- tcdB ⁺ strains and was approximately 500 times more effective against hypervirulent strains 027 (P = 0.003). CDB1, but not PA-41, was effective in neutralizing toxin B from the tcdA strains ^- tcdB ^{+ of} ribotype 017. In the end, PA-41 and RA-50 showed similar activities with respect to crude and purified forms of the toxin from the reference strain VPI 10463 (Table 7 and FIGS. 25C and 25D).

Table 7 Neutralization of toxins from various strains of C. difficile in vitro EC ₅₀ , PM mAb to toxin A mAb to toxin B Ribotype Strain RA-50 CDA1 PA-41 Cdb1 001 CCL14137 39 611 9.7 129 001 MH5 127 384 eighteen 73 001 Pitt2 27 247 13 92 002 UVA17 26 825 129 671 003 VPI 10463 twenty 1271 7.7 136 012 545 38 4552 fifteen 153

012 630 54 1019 111 1782 014 UVA30 / TL42 51 625 21 324 017 CCL13820 N / a N / a > 10 ⁵ 72 017 F1470 N / a N / a > 10 ⁵ 46 017 Pitt102 N / a N / a > 10 ⁵ 8.1 027 CCL678 29th 58950 77 > 10 ⁵ 027 CCL14402 N / d N / d 19 4678 027 CD196 61 132600 16 9812 027 Hmc553 29th 109,000 24 14730 027 Montreal 5 29th 87090 36 25810 027 Montreal 7.1 31 109400 29th 16800 027 Pitt45 43 108100 19 26510 078 Pitt07 32 > 10 ⁵ 59 8415

As shown in this example, the humanized PA-50 and PA-41 mAbs showed a high level of neutralizing activity against C. difficile toxins A and B, respectively, from genetically different strains illustrative of the current CDI outbreaks. The degree of activity of these mAbs is significant in light of the genotypic and phenotypic variation within the toxins. In particular, RA-50 and RA-41 with picomolar activity neutralized toxins produced by the hypervirulent antibiotic-resistant strains 027, while the comparison mAbs were found to have nanomolar activity. This result may reflect the weakened binding of CDA-1 to toxin A from strains 027, as previously reported (90). Toxin B from strains 027 shows a noticeable discrepancy in sequences in relation to other strains of C. difficile. Sequence differences are concentrated within the carboxy-terminal receptor binding domain and are associated with enhanced in vitro cytotoxicity. However, this sequence discrepancy does not affect the neutralizing activity of RA-41, which binds the epitope within the amino-terminal domain of toxin B. RA-50 and RA-41 with picomolar efficiencies neutralized all six strains 027 in the panel, including the CD 196 strain, which precedes the recent an increase in the prevalence of 027. In general, the results indicate that the epitopes for RA-50 and RA-41 are widely conserved in the origin of 027.

CDI is typically caused by C. difficile strains that produce both toxins A and B. However, tcdA ^- tcdB ⁺ strains have also been associated with the disease. Clinically relevant strains of tcdA ^- tcdB ⁺ are predominantly ribotype 017. The strains of ribotype 017 were reported to exhibit reduced pathogenicity for hamsters and encoded an atypical tcdB whose amino-terminal region has 70-80% sequence identity with both tcdB from VPI 10463 and with lethal toxin (tcsL) from C.sordellii. Phenotically, the tcdB of ribotype 017 has the characteristics of a hybrid and exhibits receptor-binding properties and glycosylation specificity typical for tcdB and tcsL toxins, respectively. The atypical amino terminal region of tcdB 017 provides a likely explanation of why this toxin was not neutralized by RA-41 in this study. Although strains 017 may regionally predominate and cause localized outbreaks of CDI, in general, they have been determined to include <2% of the strains found in recent clinical trials of international phase 3 research therapies for treating CDI (94, 95).

Example 9

Obtaining chimeric mab

Chimeric monoclonal antibodies, which include the variable region of the original mouse mAb and the constant region of human IgG1, were obtained and characterized. Chimeric mAbs were obtained to confirm that cloned mouse variable regions with suitable toxin activity and binding specificity, and that constructing variable regions of a mouse with a human constant region slightly alter the binding and neutralizing properties of each cloned mAb. Chimeric mAbs typically exhibit the same binding activity as the original mouse mAbs.

All of the PAb-38, PA-39, PA-41, and PA-50 mAbs contain light Kappa chains. To obtain chimeric mAb, nucleic acid sequences encoding the variable regions of the heavy and light chains were placed inside suitable expression vectors, such as, but not limited to, pCON gamma 1 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 heavy chain of human IgG1.

To obtain a chimeric mAb, the variable region of the heavy chain of each mAb was cloned into the pCON gamma 1 plasmid. The full heavy chain gene was subcloned into a plasmid containing the light chain gene to form one 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 proposed protocol. The cell supernatant containing secreted chimeric mAb was harvested seven days after transfection and purified using Protein A chromatography. The efficacy and activity of the chimeric (chimeric) mAb were compared with those of mouse mAbs in cytotoxicity and hemagglutination assays.

In accordance with the above procedure, chimeric mAb (CPA-39 and CPA-41) were formed on the basis of the original mouse PA-39 and PA-41 mAbs. The concentrations of these chimeric mAb in crude cell supernatants were in the range of about 2-11 μg / ml. In particular, the crude supernatant from the transfected 293F cell culture producing CPA-39 contained 10.6 μg / ml of chimeric mAb, while the crude supernatant from several transfected 293F cell cultures producing CPA-41 contained 9.6-10 9 μg / ml chimeric mAb.

Chimeric mAbs were compared with their respective starting mouse mAbs with respect to their ability to neutralize C. difficile toxins in vitro by cytotoxicity assays (CPA-39: CHO-K1 cells, CPA-41: CHO-K1 and CPA-50: T-84 cells ), as described previously (example 3). All chimeric mAbs were found to be equally effective when compared to their original mouse mAbs, as shown in 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 with toxin neutralization efficiencies equivalent to those of the original mouse mAbs.

Example 10

Humanization of mouse mAbs and testing of humanized mAbs of the present invention for in vitro neutralization of toxins

Humanized mAbs were prepared 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), milotarg (73, 74), xolar (75-77), raptiva (78-80) , avastin (81, 82) and tisabri (83). Humanized monoclonal antibodies can be prepared that are effective in minimizing immunogenicity, so that the activity of mAbs in humans is not exposed to undesirable effects (84-87). Preferably, humanized mAbs exhibit toxin-neutralizing activity that is within two orders of magnitude of the original mouse mAbs. In addition, humanized mAbs optimally demonstrate strong efficacy in a hamster C. difficile infection model.

To obtain humanized forms of mouse mAb to toxin A and / or toxin B, conventional hypervariable region transplantation (CDR) transplantation methods were used as described herein. The humanized mAb was compared with the original mouse mAb (s) for toxin neutralizing in vitro and in vivo activity. In accordance with the present invention, humanized mAbs may retain activity against the toxin of the original mouse mAbs and may be suitable for repeated dosing in humans.

A. Molecular Cloning of Mouse mAb Heavy and Light Chain Genes

Conventional methods have been used to clone antibody genes (88). Briefly, total RNA was purified from 1 × October ⁷ hybridoma cells using TRIzol reagent (Invitrogen) according to manufacturer's recommended protocol. 5 μg of total RNA was reverse transcribed using Superscript II Reverse Transcriptase (Invitrogen) using oligo-dT primer. The resulting cDNA was treated with RNase H to remove the RNA template and then purified using a QIAquick PCR purification kit (Qiagen) to remove free nucleotides and primers. The next step was to add the tail of guanidine nucleotides to the 3'-end of the cDNA using the terminal transferase enzyme (NEB) in the presence of dGTP according to the manufacturer's recommended protocol. The resulting tail cDNA was then subjected to PCR using one primer, which is annealed with a constant region of either the heavy or light chain, and one universal primer, which is annealed with the guanosine tail on the cDNA. For both heavy and light chains, the universal primer 5TATATCTAGAATTCCCCCCCCCCCCCCCCCCCCC3 'SEQ ID NO: 11 was used. For amplification of the light chain, primer 5'TATAGAGCTCAAGCTTGGATGGTGGGAAGATGGATACAGTTGGTGC3 '(SEQ ID NO: 12) was used, while the heavy chain was amplified using primer 5TATAGAGCTCAAGCTTCCAGTGGATAGAC (CAT) GTG (TCGTGT (TCG) TCGT (TCG) TCGT (CTGT) where the sequences in parentheses indicate degeneracy of the bases. The resulting PCR amplified by PCR DNA was purified using a QIAquick PCR purification kit (Qiagen) and sequenced. PCR reactions were performed and sequenced in triplicate to ensure that no errors were made during amplification of DNA fragments of approximately 500 base pairs.

B. Humanization of the variable regions of mAB

To obtain humanized mAb sequences, framework amino acid residues important for the CDR structure were first identified. In parallel, from the known human immunoglobulin sequences, human V _H and V _L sequences with high homology with respect to mouse V _H and V _L were selected, respectively. CDR sequences from mouse mAbs, together with any frame amino acid residues that are important for maintaining the structure of the CDRs, were transplanted, if necessary, into selected human frame sequences. In addition, human framework amino acid residues that were found to be atypical in the corresponding subgroup of the V region were replaced by typical residues in an attempt to reduce the potential immunogenicity of the resulting humanized mAb. These humanized regions of V _H and V _{L were} cloned into expression vectors, such as, but not limited to, pCON Gamma1 and pCON kappa (Lonza Biologies, Berkshire, UK), respectively. These vectors encode the genes of the constant region (s) of the heavy and light chains of human immunoglobulin. 293F cells were transiently transfected with these expression vectors using the Effectene system (Qiagen, Valencia, CA). Cell supernatants containing secreted, humanized mAbs were harvested seven days after transfection and purified using Protein A chromatography.

C. Obtaining clonal stable CHO cells expressing humanized mAB

The preparation of stable CHO cells / cell lines enables the production of sufficient quantities of mAb for testing in both in vitro cell assays and in the C. difficile associated diarrhea (CDAD) model on Syrian golden hamsters. As just one example, CHO K1 SV cells from Lonza Biologies (Berkshire, UK) can be used using a glutamine synthetase selection and amplification (GS) system to produce stably transfected CHO cells. The Lonza GS system typically produces highly productive CHO cell lines that can produce significant amounts of humanized mAb.

CHO K1 SV cells were grown in CD culture medium for CHO cells (Invitrogen) supplemented with IX glutamine (Invitrogen) and IX H / T Supplement (Invitrogen). 1 × 10 ⁷ viable cells were electroporated at 290 W, infinite resistance, and 960 μF using 40 μg of linearized plasmid DNA resuspended in 100 μl of sterile TE buffer. Cells were transferred to a T-150 mattress containing 50 ml of complete CDO medium for CHO and incubated for approximately 48 hours at 37 ° C and 8.0% CO ₂ . Cells were centrifuged and resuspended to a final density of 3.3 × 10 ⁵ cells / ml in GS selective medium (CD for CHO + IX GS Supplement (JRH Biosciences) + IX N / T Supplement) containing 100 μM MSX (Sigma), 5000 were seeded viable cells / well in 96-well plates (Coming) and incubated for approximately 3-4 weeks until colonies of primary cells (clones of transfected cells) began to appear. Approximately 300 cell colonies (clones) were selected for the production of recombinant mAb by carefully removing 20 μl of the supernatant and performing an ELISA assay in a 96-well format. Briefly, 96-well plates were coated with an immobilized antibody (goat anti-human antibody), and then supernatants from cloned CHO transfectants (diluted 1: 800) were added to allow contact with the immobilized antibody bound to the plate wells. After washing, a secondary antibody (goat anti-human antibody conjugated with alkaline phosphatase) was added to the plate and allowed to bind to the human antibody in the sample before washing to remove non-specific binding. The tablet was then analyzed for alkaline phosphatase activity using the 1-Step PNPP kit (Thermo, Rockford, IL) to identify clones producing the highest amount of secreted antibody. Clones producing high amounts of mAb were grown in CD culture medium for CHO cells supplemented with IX glutamine and IX N / T Supplement. Cell supernatants containing secreted, humanized mAbs were harvested and purified using protein A. Clones showing the best productivity were subcloned by limiting dilution and gradually increased to produce gram quantities of a recombinant, humanized, monoclonal antibody.

D. Humanized mAb hPA-39, hPA-41 and hPA-50

Molecularly cloned, humanized mAbs were isolated as described above and characterized (see section E below). The constant (CL) region of the light (L) chain of each humanized antibody is classified as kappa (κ); the constant region (CH) of the heavy (H) chain of each humanized antibody is of the IgG1 isotype. Humanized mAbs containing unique variable (V) regions have been found to bind and neutralize the activity of either toxin A or toxin B of C. difficile. The V regions of the L and H chains of humanized mAbs can form part of a complete immunoglobulin (Ig) or antibody molecule composed of two H chain polypeptides and two L chain polypeptides typically linked by disulfide bonds, or they can be separate parts or fragments of antibodies, in particular parts or fragments of antibodies that bind toxin A and / or toxin B and / or which neutralize the activity of toxins. Non-limiting examples of suitable fragments or parts of an immunoglobulin containing a V region include F (ab), F (ab ') or F (ab') ₂ fragments.

Humanized mAbs to Toxin A and Toxin B. C. difficile were prepared according to the methods described above. The humanization process yielded several humanized mAb (hmAb) to C. difficile toxin A, which bound toxin A and neutralized the activity of toxin A on susceptible cells. Examples of such hmAb include a humanized C. difficile toxin A mAb comprising an H chain polypeptide sequence including the VH region of SEQ ID NO: 1 (Fig. 32A) and a human IgG1 C region and an L chain polypeptide sequence including VL -region of SEQ ID NO: 3 (Fig. 33A) and to the C region of a person; hmAb to C. difficile toxin A comprising the H chain polypeptide sequence including the VH region of SEQ ID NO: 2 (Fig. 32B) and human IgG1 C region and the L chain polypeptide sequence comprising the VL region of SEQ ID NO : 3 (Fig. 33A) and to the C-region of a person; hmAb to C. difficile toxin A comprising the H chain polypeptide sequence including the VH region of SEQ ID NO: 1 (Fig. 32A) and human IgG1 C region and the L chain polypeptide comprising the VL region of SEQ ID NO: 4 (Fig. 33B) and to the C region of a person; and hmAb to C. difficile toxin A comprising an H chain polypeptide sequence including the VH region of SEQ ID NO: 2 (Fig. 32B) and a human IgG1 C region and an L chain polypeptide sequence including the VQ region of SEQ ID NO: 4 (Fig. 33B) and to the C region of a person. Such humanized C. difficile toxin A mAbs encompass the hmAb PA-39 (hPA-39) of the present invention. A complete hPA-39 immunoglobulin with two L chains and two H chains can be produced in a host cell that coexpresses and secrets the hPA-39 H chain polypeptide composed of the VH region of the present invention (eg, SEQ ID NO: 1; SEQ ID NO: 2) and a suitable CH region, for example an IgG1 isotype, such as that contained under Genbank accession number NW_001838121, and an hPA-39 L chain polypeptide composed of the hPA-39 VL region of the present invention (e.g. SEQ ID NO : 3; SEQ ID NO: 4) and a suitable CL region, for example a subtype of, such as is contained under Genbank Accession No. NW_001838785.

Other examples of humanized C. difficile toxin A mAb of the present invention include a humanized C. difficile toxin A mAb comprising the H chain polypeptide sequence including the VH region of SEQ ID NO: 5 (Fig. 34A) and the human IgG1 C region, and the polypeptide sequence of the L chain, including the VL region of SEQ ID NO: 7 (Fig. 35) and to the C region of a person; and a humanized C. difficile toxin A mAb comprising an H chain polypeptide sequence including the VH region of SEQ ID NO: 6 (Fig. 34B) and a human IgG1 C region and an L chain polypeptide sequence including the SEQ VL region ID NO: 7 (Fig. 35) and to the C region of a person. Such humanized C. difficile toxin A mAbs encompass the humanized PA-50 (hPA-50) mAb of the present invention. A complete hPA-50 immunoglobulin with two L chains and two H chains can be produced in a host cell that coexpresses and secrets the hPA-50 H chain polypeptide consisting of the VH region of the present invention (for example, SEQ ID NO: 5; SEQ ID NO: 6) and a suitable CH region, for example an IgG1 isotype, such as that contained under Genbank accession number NW_001838121, and an hPA-50 L chain polypeptide consisting of the VL region of the present invention (SEQ ID NO: 7) and CL -regions, for example, a subtype of, such that is contained within the access number Genbank NW_001838785.

The humanization process further provided humanized mAbs to C. difficile Toxin B, which bound Toxin B and neutralized the activity of Toxin B on susceptible cells in vitro. Examples of such hmAb include a humanized C. difficile Toxin B mAb comprising an H chain polypeptide sequence including the VH region of SEQ ID NO: 8 (Fig. 36A) and a human IgG1 C region and an L chain polypeptide sequence comprising VL -region of SEQ ID NO: 10 (Fig.37) and to the C-region of a person; and a humanized C. difficile toxin B mAb comprising an H chain polypeptide sequence including the VH region of SEQ ID NO: 9 (Fig. 36B) and a human IgG1 C region and an L chain polypeptide sequence including the SEQ VL region ID NO: 10 (Fig. 37) and to the C-region of a person. Such humanized C. difficile toxin B mAbs encompass the humanized PA-41 (hPA-41) mAb of the present invention. A complete hPA-41 immunoglobulin with two L chains and two H chains can be produced in a suitable host cell that coexpresses and secrets the hPA-41 H chain polypeptide consisting of the hPA-41 VH region of the present invention (e.g., SEQ ID NO: 8; SEQ ID NO: 9) and a suitable CH region, for example an IgG1 isotype, such as that contained under Genbank accession number NW_001838121, and an hPA-41 L chain polypeptide consisting of the hPA-41 VL region of the present invention (e.g. , SEQ ID NO: 10) and a CL region, for example a subtype κ, such as that contained under Genbank Accession No. NW_00183 8785.

In addition, humanized, cloned mAbs (hmAb) were prepared that bind toxin A toxin B and C. difficile B and strongly neutralize the activity of toxins. hmAb PA-50 to C. difficile toxin A consists of two heavy chain polypeptides, each heavy chain containing a human VH region and a CH region, and two light chain polypeptides, each light chain containing a VL region and CL area of man. 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. 38 B); 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). hmAb PA-39 to C. difficile toxin A consists of two heavy chain polypeptides, each heavy chain containing a human VH region and a CH region, and two light chain polypeptides, each light chain containing a VL region and CL area of man. 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. 39B); the nucleic acid sequence (or cDNA) encoding the amino acid sequence of the hPA-39 light chain polypeptide of SEQ ID NO: 20 is set forth in SEQ ID NO: 21 (Fig. 39A). hmAb PA-41 to toxin B. C. difficile consists of two heavy chain polypeptides, each heavy chain containing a human VH region and a CH region, and two light chain polypeptides, each light chain containing a VL region and CL area of man. 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. 40 B); 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).

For use in comparison mAbs, monoclonal antibodies CDA-1 and CDB1 were obtained (7, 89). DNA sequences encoding the variable regions of the heavy and light chains of Ig 3D8 and 124 (WO 2006/121422 and US 2005/0287150) were synthesized (DNA 2.0) and cloned into pCON-gamma1 and pCON-kappa vectors. IgG1, κ full-length mAbs were expressed in stably transfected CHO-K1SV cells and purified as described above. When testing for binding affinity for toxins A and B using Biacore, suppression of toxin-mediated cytopathic effects and hemagglutination according to published methods (7), the CDA1 and CDB1 preparations showed the expected levels of activity.

E. In Vitro Characterization of Humanized mAbs

C. difficile toxin neutralization experiments were performed in vitro to compare the functional activity of humanized mAbs with that of the original mouse mAbs. As shown in Figure 29, a humanized mAb PA-41 (hPA-41) effectively neutralized toxin B cytotoxicity (EC ₅₀ - 6 pM) compared to the EC ₅₀ - 9 pM in the case of mAb PA-41 (mPA-41) mouse . Similarly, humanized PA-39 mAb (hPA-39) and PA-50 humanized mAb (hPA-50) were found to be equally effective compared to their original mouse mAbs in neutralizing toxin A using CHO-K1 cells or T-84 cells, respectively, as shown in FIG. 30 for hPA-39 and in FIG. 31 for hPA-50. These results demonstrate that the original mouse mAbs were successfully humanized and that the humanized mAbs were functional and efficient.

From the mAb to the C. difficile toxin studied in these studies, RA-50 showed a different dose-response neutralization curve with Hill coefficients that were typically more than two, indicating cooperative suppression. Cooperative interactions are often found in nature and are often characterized by steep dose-response curves and Hill coefficients> 1. Medicines that exhibit cooperative activity have been associated with increased clinical activity in the treatment of viral infections. In addition, RA-50 binds toxin A in a multivalent manner, a condition that is often necessary but not sufficient for cooperativeness.

Example 11

Obtaining Fab fragments from murine mAb to toxin of the present invention

A. Preparation of Fab Fragments

Fab fragmentation was carried out using Mouse IgG1 Fab and F (ab ') ₂ Preparation Kit (Pierce) in accordance with the manufacturer's instructions and reagents provided in the kit. The same fragmentation protocol was used for all mAbs; PA-39, PA-41 and RA-50. Briefly, the immobilized suspension of ficin (750 μl) was washed with cleavage buffer (75 mM 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 rotation and inversion. After cleavage was completed, the suspension was centrifuged, and the cleavage product was collected. The suspension was washed three times with protein A binding buffer, and the washed material was added to the complete cleavage product. Protein A NAb column was equilibrated with protein A binding buffer and a digested antibody sample was added. The column and sample were incubated at room temperature for 10 minutes. The column was centrifuged at 1000g for one minute to collect the eluate that contained Fab fragments. The column was washed three times with protein A binding buffer. The eluate was collected, the buffer was replaced with PBS and concentrated.

B. SDS-PAGE Fab Fragments

Samples were analyzed by SDS-PAGE using the Novex gel system (Invitrogen), and all of the reagents listed below were from Invitrogen, unless otherwise indicated. Samples were mixed with NuPage sample buffer and restored using DTT. Reduced and unreduced samples were incubated at 100 ° C for 10 minutes. After loading samples (4 μg) into a 4-12% Bis Tris NuPage gel, electrophoresis was carried out using a movable MOPS buffer at 180V for 60 minutes. After electrophoresis, the gel was incubated with a fixative (40% methanol, 10% acetic acid) for 20 minutes, rinsed with water and stained with Simply Blue dye overnight with constant rotation. C. In Vitro Fab Characterization

In vitro experiments to neutralize C. difficile toxin were performed to compare the functional activity of Fab (▲) with that of whole mAb (■), based on the number of binding sites. Fab PA-39 strongly neutralize toxin A cytotoxicity on CHO-K1 cells compared with whole PA-39 (EC ₅₀ - 880 pM and the EC ₅₀ -200 pM, respectively) (41A). It was found that Fab PA-41 was equally effective with whole PA-41 in neutralizing toxin B activity on CHO-K1 cells (EC ₅₀ - 88 pM and EC ₅₀ - 80 pM, respectively) (Fig. 41B). Fab PA-50 had an EC ₅₀ value of 1.8 nM compared to an EC ₅₀ value of 100 pM whole RA-50 mAb when neutralizing toxin A on T-84 cells (Fig. 41C).

Example 12

Immunohistochemical analysis of humanized mAb to C. difficile toxin in human tissue samples

The value of immunohistochemistry (IHC) in the study of the expression of this antigen is that it allows you to evaluate the details of microanatomy and heterogeneity in normal and tumor tissues. IHC has an advantage over other methods of analysis because it can directly locate proteins in individual cell types. One can detect differences in gene expression in normal and tumor tissue, while at the same time noting changes in the number and composition of cells. Limitations of this technique include possible false negative results due to low expression levels of the test molecule, as well as false positive results (cross-reactivity) due to the binding of antibodies to similar epitopes or epitopes that are common with other antigens. To eliminate these limitations, this study was performed at the lowest possible concentration of each of the antibodies, which showed strong specific staining on positive toxin-specific control samples from injected mice.

Humanized mAb PA-41 and PA-50 were biotinylated to determine the immunohistochemical binding pattern in a sample of frozen human tissues, including the adrenal gland, bladder, bone marrow, mammary gland, cerebellum, cerebral cortex, cervix, large intestine, esophagus, eye , fallopian tube, heart, ileum, jejunum, kidney, liver, lung, lymph node, muscle, ovary, peripheral nerve, pancreas, parathyroid gland, pituitary, placenta, prostate, skin, small intestine, spinal cord, spleen , Stomach, testis, thymus, thyroid gland, ureter, uterus, and white blood cells. Each tissue from the above 37 different types of human tissue was stained with each antibody. Working IHC assays were developed for both antibodies. An inappropriate control IgG1 isotype antibody, human κ was included for all samples.

For tissue preparations, frozen samples embedded in an OST Compound (Optimal Cutting Temperature embedding compound; Sakura, Torrance, CA) were cut in 5 micron sections and placed on positively charged glass slides. Methods and conditions for IHC staining for each antibody and tissue sample were developed, tested and optimized. The direct biotinylation IHC procedure was carried out using only sliced frozen non-fixed tissue sections. Slides were removed from the cryostat, allowed to dry in air for 10 minutes at room temperature, fixed in 95% ethanol for 5 minutes at room temperature, and then washed in three successive cuvettes with wash buffer Tris-buffered saline / 0.1 % Tween-20 (TBST; DakoCytomation) for 3 minutes. All subsequent washing was carried out in this way. Endogenous peroxidase activity was blocked by 5-minute incubation at room temperature with ready-to-use Peroxidase Block. After washing with a buffer, the activity of endogenous biotin was also blocked by 15-minute incubations, each with avidin, followed by biotin, with each step followed by washing with a buffer. In the case of RA-41, slides were also incubated with the protein blocking reagent Background Sniper for 10 minutes at room temperature without subsequent washing with buffer. Slides were incubated with the test product or reagent for negative control (1.25 μg / ml for RA-41 and 10 μg / ml for RA-50) for 30 minutes at room temperature. RA-50 primary antibody was diluted 1: 350 in Dako diluent, while RA-41 primary antibody was diluted 1: 3520 in Dako diluent with proline (250 mM, 0.576 g, Genzyme, CA) and histidine (15 mM, 0.046 g , Genzyme, CA) added to 20 ml of diluent (pH 7.7). Following washes in TBST, ABC detection reagent (1:50 in TBST) was applied to tissue sections for analysis of both antibodies and incubated for 30 minutes at room temperature, followed by washing with buffer. The immune response was visualized by incubation with a solution of 3,3'-diaminobenzidine tetrahydrochloride (DAB) for 5 minutes at room temperature. The slides were rinsed with deionized (DI) water 3 times, each for 30-60 seconds, contrast stained with Mayer hematoxylin (DakoCytomation), blued in 0.2% ammonia, dehydrated with a set of alcohol of increasing concentration, clarified in xylene and covered with a coverslip glass. Interpretation of stained slides was carried out using microscopic examination. Typically, morphologically, analysis of tissue on a glass slide after staining with an antibody determined whether an adequate amount of tissue is present and whether appropriately labeled elements of normal tissue are present. Samples that do not meet the above standards were discarded from the analysis by the pathologist conducting the study.

The rating system included a semi-quantitative analysis of the staining intensity. The staining intensity of the test product was evaluated compared to the intensity of a slide with a control tissue containing an adjacent slice stained with a negative control antibody. Staining of a slice labeled with a negative control reagent was considered as “background” staining. Ball "0" indicates the absence of staining compared to the background; “1+” indicates weak staining; “2+” indicates moderate staining and “3+” indicates strong staining. In accordance with standard pathological practice, staining intensity was described at the highest level of intensity observed in all tissue elements.

The results of the IHC analysis for both humanized PA-50 and PA-41 mAbs were as follows; positive staining (0%) was not evident in any of the tested human tissue samples. Sustained strong staining (e.g., 3+) was observed in control tissues of the leg muscles of mice injected with toxin (toxin A for RA-50 and toxin B for RA-41) throughout the study. In the case of RA-50, no true positive staining was observed for any tissue sample (i.e. 100% of the cells showed 0% staining). In the case of RA-41, true positive staining did not appear in 37 human tissues tested, however, weak (1+) positive staining was observed as the highest staining intensity in normal liver (due to lipochromic pigment), normal lung (pulmonary macrophages with foreign body) and normal muscle (reaction corresponding to artifact staining). Such values of weak staining for RA-41 were considered as insignificant relative to all controls, and due to the minimal change in staining during the analysis.

Example 13

Pharmacokinetic analysis of humanized mAb to C. difficile toxin in primates other than humans

A pharmacokinetic (PK) study was performed on previously exposed Javanese macaques using purified, humanized mAb RA-41 or mAb RA-50. In this study, male cynomolgus monkeys (Masas fascicularis), previously treated with the drug, were injected intravenously with 1 mg / kg / animal or 5 mg / kg / animal purified humanized mAb RA-41 or mAb RA-50. The study was carried out in accordance with the principles and methods of the Committee for the maintenance and use of laboratory animals (IACUC).

Table 8 presents the parameters of PK studies showing that each mAb (at a concentration of 10 mg / kg) was administered intravenously at two dose levels to animals previously exposed.

Table 8 PK study of humanized mAbs on primates other than humans

Group Treatment Way Dose Level (mg / kg / day) Concentration (mg / kg) Dose volume (ml / kg / day) Number of monkeys (males) one PA-41 IV one 10 0.1 3 2 PA-41 IV 5 10 0.5 3 3 PA-50 IV one 10 0.1 3 four PA-50 IV 5 10 0.5 3

Animals received a single intravenous injection of the test antibody at the start of the study. After that, blood samples were obtained using venipuncture of peripheral vessels from each animal at 14 separate points in time for 29 days (i.e., before drug administration; after 0.5, 2, 6, 12 and 24 hours on day 1 (after administration ); and on days 3, 4, 7, 9, 12, 15, 22, and 29). Blood samples were collected in serum separation tubes and stored on water ice until coagulation. After coagulation, blood samples were centrifuged at 1800 g for 15 minutes at 4 ° C to obtain serum. Serum samples were stored at -70 ° C until use.

Serum mAb concentration was determined by ELISA. Ninety-six-well ELISA plates (Thermo Fisher Scientific, Rochester, NY) were coated overnight at 4 ° C. with Toxin A (Techlab) or Toxin B (Techlab) at 100 ng / well. The plates were washed three times with PBS / 0.05% Tween-20® (PBS-T) and blocked with 200 μl blocking buffer (PBS without calcium or magnesium, 0.1% Tween 20®, 1% casein) for one hour at room temperature. The reference standard antibody (purified mAb PA-41 or mAb PA-50) was diluted in 1% pooled serum of previously unexposed cynomolgus monkeys (Bioreclamation) to obtain a standard curve in the range of 0.3-4000 ng / ml. Diluted test samples and standards were tested in triplicate and incubated for one hour at room temperature.

The plates were washed six times with PBS-T and incubated for one hour at room temperature with HRP-conjugated goat anti-human IgG1 antibodies (The Binding Site, San Diego, CA). Plates were developed using 1 component SureBlue TMB peroxidase substrate (KPL), blocked 1n. hydrochloric acid (Thermo Fisher Scientific) and read on a SpectraMax plate reader (Molecular Devices) at 450 nm. The concentration of mAb in each monkey at different points in time was calculated using standard curves. Non-compartmental pharmacokinetic analysis was performed using WinNonLin, version 4.0 (Pharsight Corp., Mountain View, CA). The PK results for the humanized PA-50 mAb are shown in Fig. 42A; the results for the RA-41 humanized mAb are shown in Fig. 42B. For RA-50 at doses of 1 mg / kg and 5 mg / kg, the average T _1/2 (days) was 14.5 ± 0.3 and 12.3 ± 1.5, respectively. For RA-41 at doses of 1 mg / kg and 5 mg / kg, the average T _1/2 (days) was 8.9 ± 1.3 and 9.2 ± 3.3, respectively.

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Although the present invention has been described in detail for the purpose of illustration, it should be understood that such detail is intended solely for this purpose and those skilled in the art can make changes without departing from the spirit and scope of the present invention, which are defined by the following claims.

The contents of all references, patents, and published patent applications cited throughout this application are hereby incorporated by reference.

Inclusion of any link in the list does not constitute recognition that the link is prior art.

Claims (81)

1. The isolated antibody or antigen binding fragment, where the antibody or antigen binding fragment specifically binds C. difficile Toxin B, is a monoclonal antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9693 (RA-41), its chimeric or the humanized form or antigen binding fragment thereof, and wherein the antibody or antigen binding fragment specifically binds C. difficile Toxin with a K _{D of} about 0.59 nM, determined by Biacore analysis, neutralizes the cytotoxicity of the toxin and B in CHO-K1 cells with an EC ₅₀ of about 9.2 pM, and / or specifically binds 63 kDa N-terminal enzymatic domain processed caspase-1 toxin B. 2. The selected antibody or its antigennegative fragment, which: (a) specifically binds C. difficile toxin A and is a monoclonal antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9694 (RA-50), its chimeric or humanized form or antigen binding fragment thereof, and wherein the antibody or its antigen binding the fragment specifically binds C. difficile toxin A with a K _{D of} about 0.16 nM, determined by Biacore analysis, neutralizes the toxin A cytotoxicity on T-84 cells with an EC _{50 of} about 146 pM and / or specifically binds to the C-terminal binding cell receptor ( CRB) toxin A domain; or (b) specifically binds C. difficile toxin A and is a monoclonal antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9692 (RA-39), its chimeric or humanized form or antigen binding fragment thereof, and where the antibody or antigen binding fragment specifically binds C. difficile toxin a with a K _D of about 0.16 nM as determined by Biacore analysis, neutralizes toxin a cytotoxicity on CHO-K1 cells with an EC ₅₀ of about 93 pM, and / or specifically binds to the C-terminal cell binding receptors (CRB) domain of toxin A; or (c) specifically binds C. difficile toxin A and is a monoclonal antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9888 (PA-38), its chimeric or humanized form or antigen binding fragment thereof, and where the antibody or antigen binding thereof the fragment specifically binds C. difficile toxin A with K _{D of} about 1.0 nM as determined by Biacore analysis and / or neutralizes the cytotoxicity of toxin A on T-84 cells with an EC _{50 of} about 175 pM. 3. The selected antibody or antigen binding fragment, where the antibody or antigen binding fragment specifically binds C. difficile Toxin B and VH includes CDR1, CDR2 and CDR3 of SEQ ID NO: 8 or SEQ ID NO: 9, and VL includes CDR1, CDR2 and CDR3 of SEQ ID NO: 10. 4. The isolated antibody or antigen binding fragment, where the antibody or antigen binding fragment: (a) specifically binds C. difficile toxin A, and VH includes CDR1, CDR2 and CDR3 of SEQ ID NO: 1 or SEQ ID NO: 2, and VL includes CDR1, CDR2 and CDR3 of SEQ ID NO: 3 or SEQ ID NO : four; or (b) specifically binds C. difficile toxin A, and VH includes CDR1, CDR2 and CDR3 of SEQ ID NO: 5 or SEQ ID NO: 6, and VL includes CDR1, CDR2 and CDR3 of SEQ ID NO: 7. 5. The isolated antibody or antigen binding fragment, where the antibody or antigen binding fragment specifically binds C. difficile toxin B and VH includes CDR1 comprising amino acid residues 31-36 of SEQ ID NO: 8, CDR2 including amino acid residues 50-66 of SEQ ID NO: 8, and CDR3 including amino acid residues 99-108 sequences of SEQ ID NO: 8, and VL includes CDR1 comprising amino acid residues 24-34 of the sequence SEQ ID NO: 10, CDR2 comprising amino acid residues 50-56 of the sequence SEQ ID NO: 10, and CDR3 comprising amino acid residues 89-97 of the sequence SEQ ID NO: 10. 6. The selected antibody or antigen binding fragment, where the antibody or antigen binding fragment: (a) specifically binds C. difficile toxin A, and VH includes CDR1 comprising amino acid residues 31-35 of the sequence SEQ ID NO: 1, CDR2 including amino acid residues 50-65 of the sequence SEQ ID NO: 1, and CDR3 including amino acid residues 95-102 of the sequence SEQ ID NO: 1, and VL includes CDR1 including amino acid residues 24-34 of the sequence SEQ ID NO: 3, CDR2 including amino acid residues 50-56 of the sequence SEQ ID NO: 3, and CDR3 including amino acid residues 89 -97 sequences of SEQ ID NO: 3; or (b) specifically binds C. difficile toxin A, and VH includes CDR1 comprising amino acid residues 31-35 of SEQ ID NO: 5, CDR2 including amino acid residues 50-65 of SEQ ID NO: 5, and CDR3 including amino acid residues 95-102 of the sequence SEQ ID NO: 5, and VL includes CDR1 including amino acid residues 24-33 of the sequence SEQ ID NO: 7, CDR2 including amino acid residues 49-55 of the sequence SEQ ID NO: 7, and CDR3 including amino acid residues 88 -94 sequences of SEQ ID NO: 7. 7. The selected antibody, which is an antibody to C. difficile Toxin B, or an antigen binding fragment thereof according to any one of paragraphs. 1, 3 or 5, where the antibody or its antigennegative fragment specifically binds toxin C. difficile and: The V region of the L chain contains the amino acid sequence set forth in SEQ ID NO: 10; and / or The V region of the H chain contains an amino acid sequence selected from SEQ ID NO: 8 or SEQ ID NO: 9. 8. The selected antibody, which is an antibody to C. difficile toxin A, or an antigen-binding fragment thereof according to any one of paragraphs. 2, 4 or 6, where the antibody or antigen binding fragment thereof specifically binds C. difficile toxin A and: (a) the V region of the L chain contains an amino acid sequence selected from SEQ ID NO: 3 or SEQ ID NO: 4, and / or the V region of the H chain contains an amino acid sequence selected from SEQ ID NO: 1 or SEQ ID NO: 2; or (b) the V region of the L chain contains the amino acid sequence of SEQ ID NO: 7 and / or the V region of the H chain contains the amino acid sequence selected from SEQ ID NO: 5 or SEQ ID NO: 6. 9. The selected antibody or antigennegative fragment according to any one of paragraphs. 1-6, wherein the antibody or antigen binding fragment thereof inhibits, blocks or prevents the toxicity of C. difficile toxin A by inhibiting, blocking or preventing the internalization and cytotoxicity of Toxin A and / or inhibits, blocks or prevents the toxicity of C. difficile B toxin by binding to epitope site in the N-terminal enzyme region of toxin B. 10. The selected antibody or antigennegative fragment according to any one of paragraphs. 1-6, where the antibody or antigen binding fragment neutralizes the toxicity in vivo of toxin A or toxin B. C. difficile or both. 11. The selected antibody or antigennegative fragment according to any one of paragraphs. 3-6, where the antibody or its antigennegative fragment: (i) is a monoclonal antibody; (ii) is in humanized form; (iii) is human; or (iv) is in chimeric form. 12. The antibody or antigennegative fragment according to any one of paragraphs. 1-6, including the CH region selected from IgG1, IgG2a, IgG2b, IgG3, IgG4, IgA, IgE or IgM. 13. The antibody or antigen binding fragment of claim 12, wherein the CH region includes IgG1. 14. The antibody or its antigennegative fragment according to any one of paragraphs. 1-6, 13, including the CL region of a person selected from the isotype κ or λ. 15. The selected antibody or antigennegative fragment according to any one of paragraphs. 1-6, 13, where the antibody or antigen binding fragment thereof contains a single chain antibody. 16. The antibody or its antigennegative fragment according to any one of paragraphs. 1-6, 13, where the antigen binding fragment is selected from a Fab fragment, F (ab ') ₂ fragment or Fv fragment. 17. A kit for treating an infection of Clostridium difficile (C. difficile) and disease states and pathologies associated with C. difficile, comprising an antibody to toxin B or an antigen-binding fragment thereof according to any one of claims. 1, 3, 5, 7 and 9-16 and instructions for use. 18. A kit for treating infection of Clostridium difficile (C. difficile) and disease states and pathologies associated with C. difficile, comprising an antibody to toxin A or an antigen binding fragment thereof according to any one of claims. 2, 4, 6, 8 and 9-16 and instructions for use. 19. A kit for treating an infection of Clostridium difficile (C difficile) and C. difficile-associated disease states and pathologies, comprising an antibody to toxin B or an antigen-binding fragment thereof according to any one of claims. 1, 3, 5, 7, and 9-16, and an anti-toxin A antibody or antigen binding fragment thereof according to any one of claims. 2, 4, 6, 8 and 9-16 and instructions for use. 20. A composition for treating Clostridium difficile infection (C. difficile) and C. difficile associated disease states and pathologies, comprising a therapeutically effective amount of an anti-toxin B antibody or antigen-binding fragment thereof according to any one of claims. 1, 3, 5, 7, and 9-16; and a pharmaceutically acceptable carrier, excipient, or diluent. 21. A composition for treating infection of Clostridium difficile (C. difficile) and disease states and pathologies associated with C. difficile, comprising a therapeutically effective amount of an anti-toxin A antibody or antigen-binding fragment thereof according to any one of claims. 2, 4, 6, 8, and 9-16; and a pharmaceutically acceptable carrier, excipient, or diluent. 22. A composition for treating Clostridium difficile infection (C. difficile) and C. difficile associated disease states and pathologies, comprising a therapeutically effective amount of an anti-toxin B antibody or antigen-binding fragment thereof according to any one of claims. 1, 3, 5, 7, and 9-16, and antibodies to toxin A or an antigen binding fragment thereof according to any one of claims. 2, 4, 6, 8, and 9-16; and a pharmaceutically acceptable carrier, excipient, or diluent. 23. The composition of claim 20, further comprising at least one additional therapeutic agent. 24. The composition of claim 23, wherein the at least one additional therapeutic agent is metronidazole, vancomycin, fidaxomycin, nitazoxanide, rifaximin, ramoplanin, or a combination thereof. 25. A composition for treating Clostridium difficile infection (C. difficile) and disease states and pathologies associated with C. difficile, comprising (i) an antibody or antigen-binding fragment thereof that specifically binds C. difficile Toxin B; and (ii) an antibody or antigen binding fragment thereof that specifically binds C. difficile toxin A; where the antibody or its antigennegative fragment that specifically binds toxin C. difficile selected from: (a) a monoclonal antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9693 (RA-41), its chimeric or humanized form, or its antigen-binding fragment, and where the antibody or its antigen-binding fragment specifically binds C. difficile toxin B with K _{A D of} about 0.59 nM, determined by Biacore analysis, neutralizes the cytotoxicity of Toxin B on CHO-K1 cells with an EC _{50 of} about 9.2 pM and / or specifically binds to the 63 kDa N-terminal enzymatic domain of caspase-1-treated toxin B; (b) an antibody or antigen-binding fragment thereof, in which the variable region of the heavy chain (VH) includes CDR1, CDR2 and CDR3 of the VH region of the monoclonal antibody produced by the hybridoma cell line deposited under accession number ATCC PTA-9693, and the variable region of the light chain ( VL) includes CDR1, CDR2 and CDR3 of the VL region of a monoclonal antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9693; (c) an antibody or antigen binding fragment thereof, in which VH includes CDR1, CDR2 and CDR3 of SEQ ID NO: 8 or SEQ ID NO: 9 and VL includes CDR1, CDR2 and CDR3 of SEQ ID NO: 10; (d) an antibody or antigen binding fragment thereof, in which VH includes CDR1 comprising amino acid residues 31-36 of the sequence SEQ ID NO: 8, CDR2 including amino acid residues 50-66 of the sequence SEQ ID NO: 8, and CDR3 including amino acid residues 99 -108 of SEQ ID NO: 8, and VL includes CDR1 including amino acid residues 24-34 of SEQ ID NO: 10, CDR2 including amino acid residues 50-56 of SEQ ID NO: 10, and CDR3 including amino acid residues 89- 97 sequences of SEQ ID NO: 10; (e) an antibody or antigen binding fragment thereof in which the L chain V region contains an amino acid sequence as set forth in SEQ ID NO: 10 and the H chain V region contains an amino acid sequence selected from SEQ ID NO: 8 or SEQ ID NO: 9. 26. The composition of claim 25, wherein the antibody or antigen binding fragment thereof that specifically binds C. difficile toxin A includes: (a) CDR1, CDR2 and CDR3 of the variable region of the heavy chain (VH) and CDR1, CDR2 and CDR3 of the variable region of the light chain (VL) of a monoclonal antibody produced by a hybridoma cell line deposited under accession number ATCC PTA-9692; or (b) CDR1, CDR2 and CDR3 of the VH region and CDR1, CDR2 and CDR3 of the VL region of the monoclonal antibody produced by the hybridoma cell line deposited under accession number ATCC PTA-9694; or (c) CDR1, CDR2 and CDR3 of the VH region and CDR1, CDR2 and CDR3 of the VL region of the monoclonal antibody produced by the hybridoma cell line deposited under accession number ATCC PTA-9888. 27. The composition according to any one of paragraphs. 20-26 to inhibit or neutralize toxicity caused by Toxin A or C. difficile Toxin B, for the cell. 28. The composition according to any one of paragraphs. 20-26 to neutralize toxins produced by the hypervirulent C. difficile strain. 29. The composition according to any one of paragraphs. 20-26 for treating a subject who is symptomatic but who is susceptible to or has an increased risk of contracting C. difficile infection, C. difficile disease associated with or C. difficile diarrhea (CDAD). 30. A hybridoma cell line deposited under accession number ATCC PTA-9693, where the hybridoma produces a monoclonal antibody that specifically binds C. difficile toxin B. 31. A hybridoma cell line deposited under accession number ATCC PTA-9692, where the hybridoma produces a monoclonal antibody that specifically binds C. difficile toxin A. 32. A hybridoma cell line deposited under accession number ATCC PTA-9694, where the hybridoma produces a monoclonal antibody that specifically binds C. difficile toxin A. 33. A hybridoma cell line deposited under accession number ATCC PTA-9888, where the hybridoma produces a monoclonal antibody that specifically binds C. difficile toxin A. 34. A bispecific antibody or antigen binding fragment thereof that specifically binds to toxin B and toxin A of Clostridium difficile (C. difficile) and contains: (i) a first antigen binding region comprising an antibody or antigen binding fragment thereof that specifically binds C. difficile Toxin B according to any one of claims. 1, 3, 5 and 7; and (ii) a second antigen binding region comprising an antibody or antigen binding fragment thereof that specifically binds C. difficile toxin A according to any one of claims. 2, 4, 6 and 8. 35. The bispecific antibody or antigen binding fragment of claim 34, wherein the second antigen binding region comprises: (a) an antibody or antigen binding fragment thereof that specifically binds C. difficile toxin A and includes CDR1, CDR2 and CDR3 in the variable region of the heavy chain (VH) and CDR1, CDR2 and CDR3 in the variable region of the light chain (VL) of a monoclonal antibody produced a hybridoma cell line deposited under accession number ATCC PTA-9692; or (b) an antibody or antigen binding fragment thereof which specifically binds C. difficile toxin A and includes CDR1, CDR2 and CDR3 in the VH region and CDR1, CDR2 and CDR3 in the VL region of the monoclonal antibody produced by the hybridoma cell line deposited under accession No. ATCC RTA-9694; or (c) an antibody or antigen binding fragment thereof that specifically binds to difficile Toxin A and includes CDR1, CDR2 and CDR3 in the VH region and CDR1, CDR2 and CDR3 in the VL region of the monoclonal antibody produced by the hybridoma cell line deposited under accession ATCC PTA-9888. 36. A composition comprising a therapeutically effective amount of a bispecific antibody or antigen binding fragment thereof according to claim 34 or 35, and a pharmaceutically acceptable carrier, excipient or diluent, for treating C. difficile infection associated with C. difficile disease or associated with C. difficile diarrhea ( CDAD). 37. A composition comprising a bispecific antibody or antigen binding fragment thereof according to claim 34 or 35 and a pharmaceutically acceptable carrier, excipient or diluent, for inhibiting or neutralizing toxicity caused by C. difficile toxin A, for a cell. 38. A composition comprising a bispecific antibody or antigen binding fragment thereof according to claim 34 or 35 and a pharmaceutically acceptable carrier, excipient or diluent, for inhibiting or neutralizing toxicity caused by C. difficile Toxin B, for a cell. 39. A composition comprising a bispecific antibody or antigen binding fragment thereof according to claim 34 or 35 and a pharmaceutically acceptable carrier, excipient or diluent, to neutralize toxins produced by the hypervirulent C. difficile strain. 40. A composition comprising a bispecific antibody or antigen binding fragment thereof according to claim 34 or 35, and a pharmaceutically acceptable carrier, excipient or diluent, for treating a subject who is symptomatic but who is sensitive to or has an increased risk of infection with C. difficile infection, C. difficile-associated disease or C. difficile-associated diarrhea (CDAD). 41. The composition according to any one of paragraphs. 36-40, further comprising an additional therapeutic agent. 42. The composition according to any one of paragraphs. 36-40, where the hypervirulent C. difficile strain is selected from one or more of BI / NAP1 / 027, CCL678, HMS553, Pitt45, CD196, montreal 5, montreal 7.1, MH5, Pitt2, CCL14137, UVA17, UVA30 / TL42 and Pitt7. 43. The use of a composition according to any one of paragraphs. 20-26 for the treatment of C. difficile infection associated with C. difficile disease or C. difficile associated diarrhea (CDAD). 44. An isolated nucleic acid that encodes an antibody to toxin B or an antigen binding fragment thereof according to any one of claims. 1, 3, 5, 7, 9-16. 45. The selected nucleic acid that encodes an antibody to toxin A or its antigennegative fragment according to any one of paragraphs. 2, 4, 6, 8-16. 46. An expression vector containing a nucleic acid molecule according to claim 44 or 45. 47. A host cell transformed or transfected with an expression vector according to claim 46, wherein the host cell expresses a nucleic acid. 48. An ex vivo method for inhibiting or neutralizing toxicity caused by C. difficile toxin A, for a cell, which comprises exposing the cell to an effective C. difficile toxin A inhibiting or neutralizing dose of an antibody or antigen-binding fragment thereof according to any one of claims. 2, 4, 6 or 8. 49. An ex vivo method of inhibiting or neutralizing toxicity caused by C. difficile Toxin B, for a cell, which comprises exposing the cell to an effective C. difficile Toxin B inhibiting or neutralizing dose of an antibody or antigen binding fragment thereof according to any one of claims. 1, 3, 5 or 7. 50. An ex vivo method for inhibiting or neutralizing toxicity caused by C. difficile toxin A and Toxin B, for a cell, which comprises exposing the cell to an effective C. difficile toxin A inhibiting or neutralizing dose of an antibody or antigen binding fragment thereof according to any one of claims. 2, 4, 6 or 8 and an effective inhibitory or neutralizing toxin C. difficile dose of the antibody or antigen binding fragment thereof according to any one of paragraphs. 1, 3, 5 or 7.

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