Patent Grant
10160797
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on 17 Sep. 2018, is named 0171-0002-US-Substitute-SL and is 219 kilobytes in size.
This application relates generally to antibodies against Clostridium difficile (C. difficile) toxins and methods of using the same to detect or treat C. difficile infections and/or C. difficile-associated disease.
C. difficile is a gram positive, anaerobic bacterium that causes gastrointestinal disease in humans. The bacteria are transmitted through feces and spread to food and other surfaces when people who are infected do not thoroughly wash their hands. C. difficile form spores that can persist outside of a human body for weeks or even months. Symptoms of C. difficile infection can range from diarrhea to life-threatening inflammation of the colon. C. difficile infections are the most common cause of infectious diarrhea in the healthcare setting (Cohen S H et al., Infect Control Hosp Epidemiol 2010; 31:431-55).
C. difficile infections are more frequent in older adults in a hospital or long-term care facility and commonly occur during or following antibiotic treatment, which disrupts the normal flora of the gut and permits the opportunistic C. difficile to colonize the gut. In more severe infections, the colon can become inflamed (colitis) or form patches of raw tissue that can bleed or produce pus (pseudomembranous colitis). Symptoms of severe C. difficile infection include watery diarrhea, abdominal cramping and pain, nausea, fever, dehydration, and weight loss.
C. difficile produces two cytotoxic enterotoxins, toxin A and toxin B, that have been identified as targets for therapeutic intervention. Toxins A and B are released by the bacteria into the gut and believed to be involved in causing C. difficile-associated disease (CDAD) or the symptoms associated with CDAD. Symptoms of CDAD can be reproduced in animal models by transfer of the toxins. Toxins A and B have glucosyl transferase activity, which is capable of transferring glucose residues from UDP-glucose to Rho-GTPases, thereby inactivating the GTPase proteins found inside the target host cell. Inhibition of the Rho-GTPases results in depolymerization of actin filaments within the host cell, leading to dysregulation of actin cytoskeleton and tight junction integrity, which in turn produces increased cell permeability and loss of barrier function, diarrhea, inflammation, and an influx of molecules associated with the innate immune response. Toxins A and B are found in fecal samples and can be used to diagnose C. difficile infection.
Once a C. difficile infection has been identified, it is best, if possible, to stop taking the antibiotic that caused the infection. The typical treatment for C. difficile is another antibiotic, usually metronidazole or fidaxomicin, for mild to moderate illness, or vancomycin for more severe symptoms. If effective, these antibiotics prevent C. difficile from growing and allow the normal flora to return and colonize the gut. However, in recent years, strains resistant to these antibiotics have been identified, as well as higher recurrence or reinfection rates. Another approach is taking probiotics. Probiotics are non-pathogenic microorganisms, such as bacteria or yeast that compete with C. difficile and help restore balance to the intestinal tract. For patients with severe pain or inflammation, another option is surgery to remove the diseased portion of the colon.
Therapeutic antibodies have been a rapidly emerging field in recent years and provide another possible strategy for treating C. difficile infections. Patients infected with C. difficile experience a wide range of symptoms, the reasons for which are not fully understood. However, antibodies may play a role, as patients who experience milder symptoms tend to possess high titers of anti-toxin A antibody serum titers, while patients susceptible to recurring infections have demonstrated low titers of circulating anti-toxin A antibodies (Hussack and Tanha, Toxins, 2010, (2):998-1018). US2012/269841 describes murine antibodies that bind mutant C. difficile toxin-A or anti-toxin B. WO2011/130650 describes murine anti toxin-A and anti-toxin B antibodies that were optionally humanized to reduce their immunogenicity, including the lead anti-toxin A antibody, PA-50, and the lead anti-toxin B antibody, PA-41. U.S. Pat. No. 8,257,709 describes anti toxin-A and anti-toxin B antibodies that were generated in transgenic mice, including the lead anti-toxin A antibody, 3D8, and the lead anti-toxin B antibody, 124-152. The transgenic mice contain human immunoglobulin genes encoding certain unrearranged human heavy chain and kappa light chain sequences and, thus, are less immunogenic than murine antibodies.
There remains an unmet need for effective treatment of C. difficile infection, particularly non-invasive treatments that are effective against antibiotic-resistant strains of C. difficile and/or against high-toxin producing strains, including therapeutic antibodies that present reduced immunogenicity while providing high binding affinity for C. difficile toxin A or toxin B and/or potent neutralization activity.
The present disclosure provides antibodies that bind to C. difficile toxin A or C. difficile toxin B and can be used, for example, in methods of detecting or treating C. difficile infection.
One embodiment is directed to monoclonal antibodies that bind to C. difficile toxin A. The anti-toxin A antibodies are preferably human antibodies. In one embodiment, the anti-toxin A antibodies are recombinant antibodies.
One embodiment is directed to an isolated monoclonal antibody that binds to an epitope in the C-terminal receptor domain (CTD) of C. difficile toxin A, wherein the epitope comprises the amino acid sequence X1TGWQTI (SEQ ID NO:232), where X1 is A or V or the amino acid sequence of X2TGWQTIX3GKX4YYF (SEQ ID NO:233), where X2 is A or V, X3 is N or D and X4 is K or V.
Another embodiment is directed to an isolated monoclonal antibody that binds to C. difficile toxin A, wherein said antibody comprises a heavy chain variable domain and a light chain variable domain, and
(a) wherein the heavy chain variable domain comprises a complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO:6; a CDR2 comprising the amino acid sequence of SEQ ID NO:8; and a CDR3 comprising the amino acid sequence of SEQ ID NO:10; and wherein the light chain variable domain comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO:13; a CDR2 comprising the amino acid sequence of SEQ ID NO:15; and a CDR3 comprising the amino acid sequence of SEQ ID NO:17;
(b) wherein the heavy chain variable domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:24; a CDR2 comprising the amino acid sequence of SEQ ID NO:26; and a CDR3 comprising the amino acid sequence of SEQ ID NO:28; and wherein the light chain variable domain comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO:31; a CDR2 comprising the amino acid sequence of SEQ ID NO:33; and a CDR3 comprising the amino acid sequence of SEQ ID NO:35;
(c) wherein the heavy chain variable domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:42; a CDR2 comprising the amino acid sequence of SEQ ID NO:44; and a CDR3 comprising the amino acid sequence of SEQ ID NO:46; and wherein the light chain variable domain comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO:49; a CDR2 comprising the amino acid sequence of SEQ ID NO:51; and a CDR3 comprising the amino acid sequence of SEQ ID NO:53;
(d) wherein the heavy chain variable domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:60; a CDR2 comprising the amino acid sequence of SEQ ID NO:62; and a CDR3 comprising the amino acid sequence of SEQ ID NO:64; and wherein the light chain variable domain comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO:67; a CDR2 comprising the amino acid sequence of SEQ ID NO:69; and a CDR3 comprising the amino acid sequence of SEQ ID NO:71; or
(e) wherein the heavy chain variable domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:78; a CDR2 comprising the amino acid sequence of SEQ ID NO:80; and a CDR3 comprising the amino acid sequence of SEQ ID NO:82; and wherein the light chain variable domain comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO:85; a CDR2 comprising the amino acid sequence of SEQ ID NO:87; and a CDR3 comprising the amino acid sequence of SEQ ID NO:89.
Another embodiment is directed to an isolated monoclonal antibody that binds to Clostridium difficile toxin A, wherein said antibody comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:38, SEQ ID NO:56, or SEQ ID NO:74 or a light chain variable domain comprising the amino acid sequence of SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:40, SEQ ID NO:58, or SEQ ID NO:76.
Another embodiment is directed to an isolated monoclonal antibody that binds to C. difficile toxin A, wherein said antibody comprises a heavy chain variable domain and a light chain variable domain, and
(a) wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:2 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:4;
(b) wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:20 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:22;
(c) wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:38 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:40;
(d) wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:56 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:58; or
(e) wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:74 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:76.
Yet another embodiment is directed to an isolated, human monoclonal antibody that binds to the same epitope of C. difficile toxin A recognized by:
(a) an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:2 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:4;
(b) an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:20 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:22;
(c) an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:38 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:40;
(d) an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:56 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:58; or
(e) an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:74 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:76.
In another embodiment, the antibody is an isolated, human monoclonal antibody comprising at least one of the following characteristics:
The antibody may have at least two, at least three, or all four of the above-identified characteristics.
Another aspect is drawn to monoclonal antibodies that bind to C. difficile toxin B. The anti-toxin B antibodies are preferably human antibodies. In one embodiment, the anti-toxin B antibodies are recombinant antibodies.
One embodiment is directed to an isolated monoclonal antibody that binds to an epitope in the glucosyl transferase domain of C. difficile toxin B, wherein the epitope comprises the amino acid sequence SGRNK (SEQ ID NO:234), amino acids 56-80 of SEQ ID NO:231, or amino acids 10-520 of SEQ ID NO:231.
Another embodiment is directed to an isolated monoclonal antibody that binds to an epitope in the N-terminal translocation domain of C. difficile toxin B, wherein the epitope comprises amino acids 1110-1530 of SEQ ID NO:231.
Another embodiment is directed to an isolated monoclonal antibody that binds to an epitope in the receptor binding domain of C. difficile toxin B, wherein the epitope comprises amino acids 1750-2360 of SEQ ID NO:231.
Yet another embodiment is directed to an isolated monoclonal antibody that binds to C. difficile toxin B, wherein said antibody comprises a heavy chain variable domain and a light chain variable domain, and
(a) wherein the heavy chain variable domain comprises a complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO:96; a CDR2 comprising the amino acid sequence of SEQ ID NO:98; and a CDR3 comprising the amino acid sequence of SEQ ID NO:100; and wherein the light chain variable domain comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO:103; a CDR2 comprising the amino acid sequence of SEQ ID NO:105; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 107;
(b) wherein the heavy chain variable domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:114; a CDR2 comprising the amino acid sequence of SEQ ID NO:116; and a CDR3 comprising the amino acid sequence of SEQ ID NO:118; and wherein the light chain variable domain comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO:121; a CDR2 comprising the amino acid sequence of SEQ ID NO:123; and a CDR3 comprising the amino acid sequence of SEQ ID NO:125;
(c) wherein the heavy chain variable domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:132; a CDR2 comprising the amino acid sequence of SEQ ID NO:134; and a CDR3 comprising the amino acid sequence of SEQ ID NO:136; and wherein the light chain variable domain comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO:139; a CDR2 comprising the amino acid sequence of SEQ ID NO:141; and a CDR3 comprising the amino acid sequence of SEQ ID NO:143;
(d) wherein the heavy chain variable domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:150; a CDR2 comprising the amino acid sequence of SEQ ID NO:152; and a CDR3 comprising the amino acid sequence of SEQ ID NO:154; and wherein the light chain variable domain comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO:157; a CDR2 comprising the amino acid sequence of SEQ ID NO:159; and a CDR3 comprising the amino acid sequence of SEQ ID NO:161;
(e) wherein the heavy chain variable domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:168; a CDR2 comprising the amino acid sequence of SEQ ID NO:170; and a CDR3 comprising the amino acid sequence of SEQ ID NO:172; and wherein the light chain variable domain comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO:175; a CDR2 comprising the amino acid sequence of SEQ ID NO:177; and a CDR3 comprising the amino acid sequence of SEQ ID NO:179; or
(f) wherein the heavy chain variable domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:186; a CDR2 comprising the amino acid sequence of SEQ ID NO:188; and a CDR3 comprising the amino acid sequence of SEQ ID NO:190; and wherein the light chain variable domain comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO:193; a CDR2 comprising the amino acid sequence of SEQ ID NO:195; and a CDR3 comprising the amino acid sequence of SEQ ID NO:197.
One embodiment is directed to an isolated, monoclonal antibody that binds to Clostridium difficile toxin B, wherein said antibody comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:92, SEQ ID NO:110, SEQ ID NO:128, SEQ ID NO:146, SEQ ID NO:164, or SEQ ID NO:182 or a light chain variable domain comprising the amino acid sequence of SEQ ID NO:94, SEQ ID NO:112, SEQ ID NO:130, SEQ ID NO:148, SEQ ID NO:166, or SEQ ID NO:184.
Another embodiment is directed to an isolated, monoclonal antibody that binds to C. difficile toxin B, wherein said antibody comprises a heavy chain variable domain and a light chain variable domain, and
(a) wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:92 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:94;
(b) wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:110 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:112;
(c) wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:128 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:130;
(d) wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:146 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:148;
(e) wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:164 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:166; or
(f) wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:182 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:184.
Yet another embodiment is directed to an isolated monoclonal antibody that binds to the same epitope of C. difficile toxin B recognized by:
(a) an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:92 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:94;
(b) an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:110 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:112;
(c) an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:128 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:130;
(d) an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:146 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:148;
(e) an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:164 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:166; or
(f) an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:182 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:184.
In another embodiment, the antibody is an isolated, human monoclonal antibody comprising at least one of the following characteristics:
The antibody may have at least two, at least three, or all four of the above-identified characteristics.
In another embodiment, the antibody binds to toxin B produced by at least the strains of toxinotypes 0, III, V, and VIII. In another embodiment, the antibody binds to toxin B produced by at least the strains of toxinotypes 0, III, V, VIII, and XII. In another embodiment, the antibody binds to toxin B produced by at least the strains of toxinotypes 0, III, V, VIII, XII, and XV.
In one embodiment, the antibody is a bispecific antibody, where the bispecific antibody comprises 1) a first antigen binding site comprising the heavy chain variable domain and light chain variable domain of an antibody that binds to C. difficile toxin A, as described herein, and 2) a second antigen binding site comprising the heavy chain variable domain and light chain variable domain of an antibody that binds to C. difficile toxin B, as described herein. In an alternative embodiment, the antibody is a bispecific antibody, where the bispecific antibody comprises two antigen binding sites, each antigen binding site comprising the heavy chain variable domain from an antibody that binds to C. difficile toxin A, as described herein, and the light chain variable domain from an antibody that binds to C. difficile B, as described herein. In a further alternative embodiment, the antibody is a bispecific antibody, where the bispecific antibody comprises two antigen binding sites, each antigen binding site comprising the heavy chain variable domain from an antibody that binds to C. difficile toxin B, as described herein, and the light chain variable domain from an antibody that binds to C. difficile toxin A, as described herein. In a further alternative embodiment, the antibody is a bispecific antibody, where the bispecific antibody comprises two antigen binding sites, each antigen binding site comprising the heavy chain variable domain from an antibody that binds to C. difficile toxin B, such as B1, and as further described herein, and the light chain variable domain from an antibody that binds to a different part of C. difficile toxin B, such as B2, and as further described herein.
In one embodiment of the bispecific antibody, the first antigen binding site comprises the heavy chain variable domain and light chain variable domain of the A1, A2, A3, A4, or A5 antibody. In another embodiment of the bispecific antibody, the second antigen binding site comprises the heavy chain variable domain and light chain variable domain of the B1, B2, B3, B4, B5, or B6 antibody. In yet another embodiment of the bispecific antibody, the first antigen binding site comprises the heavy chain variable domain and light chain variable domain of the A1, A2, A3, A4, or A5 antibody and the second antigen binding site comprises the heavy chain variable domain and light chain variable domain of the B1, B2, B3, B4, B5, or B6 antibody.
In another embodiment, the antibody is a bispecific antibody, where the bispecific antibody comprises 1) a first antigen binding site, wherein the first antigen binding site comprises the heavy chain variable domain and light chain variable domain of an antibody that binds to C. difficile toxin B, as described herein and 2) a second antigen binding site, wherein the second antigen binding site comprises the heavy chain variable domain and light chain variable domain of an antibody that binds to C. difficile toxin B, as described herein, wherein the first and second antigen binding sites are different. Preferably, the first and second antigen binding sites comprise the heavy chain variable domain and light chain variable domain of the B1, B2, B3, B4, B5, or B6 antibody, wherein the first and second antigen binding sites are different. In yet another embodiment of the bispecific antibody, the first antigen binding site comprises the heavy chain variable domain and light chain variable domain of the B1 antibody and the second antigen binding site comprises the heavy chain variable domain and light chain variable domain of the B2 antibody.
In a further embodiment, the antibody is a bispecific antibody, where the bispecific antibody comprises 1) a first antigen binding site, wherein the first antigen binding site comprises the heavy chain variable domain and light chain variable domain of an antibody that binds to C. difficile toxin A, as described herein and 2) a second antigen binding site, wherein the second antigen binding site comprises the heavy chain variable domain and light chain variable domain of an antibody that binds to C. difficile toxin A, as described herein, wherein the first and second antigen binding sites are different. Preferably, the first and second antigen binding sites comprise the heavy chain variable domain and light chain variable domain of the A1, A2, A3, A4, or A5 antibody, wherein the first and second antigen binding sites are different.
Another aspect is related to compositions comprising one or more of the A1, A2, A3, A4, A5, B1, B2, B3, B4, B5, or B6 antibodies, which compositions can be used, by way of example, for treating a C. difficile infection. In certain embodiments, the composition comprises one antibody that binds to C. difficile toxin A and one antibody that binds to C. difficile toxin B, as described herein. In one embodiment, the composition comprises at least one antibody that binds to C. difficile toxin A and at least one antibody that binds to C. difficile toxin B, wherein the at least one antibody that binds to C. difficile toxin A is preferably one or more of the A1, A2, A3, A4, and A5 antibodies. In another embodiment, the composition comprises at least one antibody that binds to C. difficile toxin A and at least one antibody that binds to C. difficile toxin B, wherein the at least one antibody that binds to C. difficile toxin B is preferably one or more of the B1, B2, B3, B4, B5, or B6 antibodies. In yet another embodiment, the at least one antibody that binds to C. difficile toxin A is preferably one or more of the A1, A2, A3, A4, and A5 antibodies and the at least one antibody that binds to C. difficile toxin B is preferably one or more of the B1, B2, B3, B4, B5, or B6 antibodies. These compositions can be used, by way of example, for treating a C. difficile infection. In one embodiment, the at least one antibody that binds to C. difficile toxin A is the A2 antibody and the at least one antibody that binds to C. difficile toxin B is one or more of the B1, B2, B3, B4, B5, or B6 antibodies, preferably one or more of B1, B2, B4 or B6. Thus, in certain embodiments, the composition comprises the A2 and B1 antibodies, the A2 and B2 antibodies, the A2 and B4 antibodies, or the A2 and B6 antibodies. In another embodiment, the at least one antibody that binds to C. difficile toxin A is the A1 antibody and the at least one antibody that binds to C. difficile toxin B is one or more of the B1, B2, B3, B4, B5, or B6 antibodies, preferably one or more of B1, B2, B4 or B6. Thus, in certain other embodiments, the composition comprises the A1 and B1 antibodies, the A1 and B2 antibodies, the A1 and B4 antibodies, or the A1 and B6 antibodies. In other embodiments, the composition further comprises a pharmaceutically acceptable excipient.
In other embodiments, the composition comprises a combination of at least three antibodies. In one embodiment, the composition comprises two antibodies that bind to C. difficile toxin A, as described herein, and one antibody that binds to C. difficile toxin B, as described herein. Alternatively, the composition comprises one antibody that binds to C. difficile toxin A, as described herein, and two antibodies that bind to C. difficile toxin B, as described herein.
In a further embodiment the composition comprises a three antibody combination comprising one antibody that binds to C. difficile toxin A, as described herein, and preferably selected from the A1, A2, A3, A4, and A5 antibodies, and two antibodies that bind to C. difficile toxin B, as described herein, which are preferably selected from the B1, B2, B3, B4, B5, or B6 antibodies. In one embodiment, the composition comprises the A2, B1 and B2 antibodies. In another embodiment, the composition comprises the A2, 82, and B4 antibodies. In another embodiment, the composition comprises the A2, B2, and B6 antibodies.
In another embodiment, the composition comprises a first antibody that binds to C. difficile toxin A, as described herein, which is preferably selected from the A1, A2, A3, A4, and A5 antibodies, more preferably the A2 antibody, and a second antibody, wherein the second antibody is a bispecific antibody that binds to C. difficile toxin B and wherein the bispecific antibody comprises 1) a first antigen binding site, wherein the first antigen binding site comprises the heavy chain variable domain and light chain variable domain of the B1, B2, B3, B4, B5, or B6 antibody and 2) a second antigen binding site, wherein the second antigen binding site comprises the heavy chain variable domain and light chain variable domain of the B1, B2, B3, B4, B5, or B6 antibody, wherein the first and second antigen binding sites are different. In one embodiment, the composition comprises the A2 antibody and a bispecific B1+B2 antibody, a bispecific B2+B4 antibody, or a bispecific B2+B6 antibody. In another embodiment, the composition comprises the A1 antibody and a bispecific B1+B2 antibody, a bispecific B2+B4 antibody, or a bispecific B2+B6 antibody.
Another aspect is directed to methods of using antibodies that bind to C. difficile toxin A and/or C. difficile toxin B to treat C. difficile infection. In one embodiment, the method of treating a C. difficile infection comprises administering to a subject one or more of the A1, A2, A3, A4, A5, B1, B2, B3, B4, B5, or B6 antibodies or a bispecific antibody derived therefrom, including, for example, a bispecific B1+B2 antibody, a bispecific B2+B4 antibody, or a bispecific B2+B6 antibody in an amount effective to treat the C. difficile infection. In another embodiment, the method of treating a C. difficile infection comprises administering a composition to the subject in an amount effective to treat the C. difficile infection, wherein the composition comprises at least one antibody that binds to C. difficile toxin A selected from the A1, A2, A3, A4, and A5 antibodies and at least one antibody that binds to C. difficile toxin B selected from the B1, B2, B3, B4, B5, or B6 antibodies or a bispecific antibody derived therefrom, including, for example, a bispecific B1+B2 antibody, a bispecific B2+B4 antibody, or a bispecific B2+B6 antibody. In one embodiment, the at least one antibody that binds to C. difficile toxin A is the A2 antibody and the at least one antibody that binds to C. difficile toxin B is one or more of the B1, B2, B3, B4, B5, or B6 antibodies or a bispecific antibody derived therefrom, preferably one or more of B1, B2, or B4, or a bispecific antibody selected from B1+B2 or B2+B4.
Another aspect is directed to nucleic acids that encode an antibody of interest, or portion(s) thereof. One embodiment is directed to an isolated nucleic acid that encodes the amino acid sequence of one or more of the CDRs of the light and/or heavy chain variable regions of an A2, A2, A3, A4, A5, B1, B2, B3, B4, B5, or B6 antibody, or a bispecific antibody derived therefrom, including, for example, a bispecific B1+B2 antibody, a bispecific B2+B4 antibody, or a bispecific B2+B6 antibody. Another embodiment is directed to an isolated nucleic acid that encodes an amino acid sequence of the light and/or heavy chain variable regions of the A1, A2, A3, A4, A5, B1, B2, B3, B4, B5, or B6 monoclonal antibody or a bispecific antibody derived therefrom, including, for example, a bispecific B1+B2 antibody, a bispecific B2+B4 antibody, or a bispecific B2+B6 antibody. Other embodiments are directed to a recombinant expression vector comprising the nucleic acid or an isolated host cell comprising the recombinant expression vector.
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain embodiments, and together with the written description, serve to explain certain principles of the antibodies and methods disclosed herein.
Reference will now be made in detail to various exemplary embodiments, examples of which are illustrated in the accompanying drawings. It is to be understood that the following detailed description is provided to give the reader a fuller understanding of certain embodiments, features, and details of aspects of the invention, and should not be interpreted as a limitation of the scope of the invention.
1. Definitions
In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.
The term “antibody” as used in this disclosure refers to an immunoglobulin or an antigen-binding fragment thereof. Unless otherwise specified, the term includes, but is not limited to, polyclonal, monoclonal, monospecific, polyspecific, humanized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies. The antibody can include a constant region, or a portion thereof, such as the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes. For example, heavy chain constant regions of the various isotypes can be used, including: IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE. By way of example, the light chain constant region can be kappa or lambda.
The terms “antigen-binding domain” and “antigen-binding fragment” refer to a part of an antibody molecule that comprises amino acids responsible for the specific binding between antibody and antigen. For certain antigens, the antigen-binding domain or antigen-binding fragment may only bind to a part of the antigen. The part of the antigen that is specifically recognized and bound by the antibody is referred to as the “epitope” or “antigenic determinant.” Antigen-binding domains and antigen-binding fragments include Fab (Fragment antigen-binding); a F(ab′)2 fragment, a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region; Fv fragment; a single chain Fv fragment (scFv) see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883); a Fd fragment having the two VH and CH1 domains; dAb (Ward et al., (1989) Nature 341:544-546), and other antibody fragments that retain antigen-binding function. The Fab fragment has VH-CH1 and VL-CL domains covalently linked by a disulfide bond between the constant regions. The Fv fragment is smaller and has VH and VL domains non-covalently linked. To overcome the tendency of non-covalently linked domains to dissociate, a scFv can be constructed. The scFv contains a flexible polypeptide that links (1) the C-terminus of VH to the N-terminus of VL, or (2) the C-terminus of VL to the N-terminus of VH. A 15-mer (Gly4Ser)3 (SEQ ID NO:331) peptide may be used as a linker, but other linkers are known in the art. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are evaluated for function in the same manner as are intact antibodies.
The terms “(cross)-block,” “(cross)-blocked,” “(cross)-blocking,” “competitive binding,” “(cross)-compete,” “(cross)-competing,” and “(cross)-competition” are used interchangeably herein to mean the ability of an antibody to interfere with the binding of other antibodies to a given target. The extent to which one antibody is able to interfere with the binding of another antibody to the target, and therefore whether it can be said to cross-block, as used herein, can be determined using competition binding assays. One particularly suitable quantitative cross-blocking assay uses a Biacore instrument which can measure the extent of interactions using surface plasmon resonance technology.
The following generally describes a suitable Biacore assay for determining whether an antibody cross-blocks or is capable of cross-blocking. It will be appreciated that the assay can be used with any of antibodies described herein. The Biacore instrument (for example the Biacore 3000) is operated in line with the manufacturer's recommendations. Thus in one cross-blocking assay, the target protein (e.g. toxin A or toxin B) is coupled to a CMS Biacore chip using standard amine coupling chemistry to generate a surface that is coated with the target. Typically 200-800 resonance units of the target would be coupled to the chip (an amount that gives easily measurable levels of binding but that is readily saturable by the concentrations of test reagent being used), Two test binding agents {termed A* and B*} to be assessed for their ability to cross-block each other are mixed at a one to one molar ratio of binding sites in a suitable buffer to create the test mixture. When calculating the concentrations on a binding site basis the molecular weight of a binding agent is assumed to be the total molecular weight of the binding agent divided by the number of target binding sites on that binding agent. The concentration of each binding agent in the test mix should be high enough to readily saturate the binding sites for that binding agent on the target molecules captured on the Biacore chip. The binding agents in the mixture are at the same molar concentration (on a binding basis) and that concentration would typically be between 1.00 and 1.5 micromolar (on a binding site basis). Separate solutions containing A* alone and B* alone are also prepared. A* and B* in these solutions should be in the same buffer and at the same concentration as in the test mix. The test mixture is passed over the target-coated Biacore chip and the total amount of binding recorded. The chip is then treated in such a way as to remove the bound binding agents without damaging the chip-bound target. Typically this is done by treating the chip with 30 mM HCl for 60 seconds. The solution of A* alone is then passed over the target-coated surface and the amount of binding recorded. The chip is again treated to remove all of the bound binding agents without damaging the chip-bound target. The solution of B* alone is then passed over the target-coated surface and the amount of binding recorded. The maximum theoretical binding of the mixture of A* and B* is next calculated, and is the sum of the binding of each binding agent when passed over the target surface a lone. If the actual recorded binding of the mixture is less than this theoretical maximum then the two binding agents are said to cross-block each other. Thus, in general, a cross-blocking antibody is one which will bind to the target in the above Biacore cross-blocking assay such that during the assay and in the presence of a second antibody the recorded binding is between 80% and 0.1% of maximum theoretical binding {as defined above) of the two antibodies in combination. Other affinity assays may also be used, including the Octet assay, as described in the examples that follow.
As used herein, a “therapeutically effective amount” of an antibody refers to an amount of an antibody that is effective, upon single or multiple dose administration to a subject (such as a human patient) at treating C. difficile infection.
The terms “treatment of C. difficile infection” or “treating C. difficile infection” and the like refer to any treatment of any disease (e.g., CDAD) or condition in a subject caused by C. difficile infection and includes inhibiting a disease, condition, or symptom of a C. difficile infection, e.g., arresting its development and/or delaying or preventing its onset or manifestation in the subject; relieving a disease, condition, or symptom of a C. difficile infection, e.g., causing regression of the condition or disease and/or one or more of its symptoms (e.g., diarrhea, colitis, and/or abdominal pain); or preventing or reducing the recurrence or relapse of a disease, condition, or symptom of a C. difficile infection.
The terms “subject,” “host,” “patient,” and “individual” are used interchangeably herein to refer to any mammalian subject for whom diagnosis or therapy is desired, particularly humans.
The term “pharmaceutically acceptable excipient” means solvents, diluents, dispersion media, coatings, antibacterial agents and antifungal agents, isotonic agents, solid and liquid fillers, and absorption delaying agents, and the like, that are suitable for administration into a human. The use of such media and agents for pharmaceutically active substances is well known in the art.
The term “human antibody” refers to an antibody having variable and constant regions corresponding substantially to human germline immunoglobulin sequences. A human antibody may also include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs, and in particular, CDR3.
The term “recombinant antibody” refers to an antibody produced or expressed using a recombinant expression vector, where the expression vector comprises a nucleic acid encoding the recombinant antibody, such that introduction of the expression vector into an appropriate host cell results in the production or expression of the recombinant antibody.
The term “bispecific” or “bifunctional antibody” refers to an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab′ fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992). For example, the bispecific antibody can comprises a first antigen binding site, such as a Fab′ fragment, that binds to C. difficile toxin A and a second antigen binding site, such as a Fab′ fragment, that binds to C. difficile toxin B. The first and second antigen binding site may be linked using any available technique, including, for example, an immunoglobulin constant region.
The term “neutralizing antibody” refers to an antibody whose binding an antigen results in inhibition of the biological activity of that antigen, respectively. For example, “toxin A neutralizing antibody” or “toxin B neutralizing antibody” (or an “antibody that neutralizes toxin A or toxin B activity”) refers to an antibody whose binding to toxin A or toxin B results in the inhibition of the biological activity of toxin A or toxin B. This inhibition of the biological activity of toxin A or toxin B can be assessed by measuring one or more indicators of toxin A or toxin B biological activity, such as toxin A- or toxin B-induced cytotoxicity or loss of transepithelial electrical resistance (TEER), as demonstrated in the examples.
The term “isolated antibody,” refers to an antibody that is substantially free of its natural environment, including other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds C. difficile toxin A is substantially free of antibodies that specifically bind antigens other than C. difficile toxin A, unless the isolated antibody is combined with one or more isolated antibodies of interest, such as an antibody that specifically binds C. difficile toxin B).
The term “Isolated nucleic acid,” as used in the context of a nucleic acid encoding an antibody, or antigen-binding fragment thereof, refers to a nucleic acid molecule in which the nucleotide sequences encoding the antibody or antibody, or antigen-binding fragment thereof, are free of other nucleotide sequences encoding antibodies or portions thereof that bind antigens other than C. difficile toxin A or toxin B, which other sequences may naturally flank the nucleic acid in human genomic DNA. Thus, for example, an isolated nucleic acid encoding a VH region of an anti-toxin A antibody contains no other sequences encoding other VH regions that bind antigens other than C. difficile toxin A.
The term “identity,” as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as determined by the match between strings of such sequences. “Identity” and “similarity” can be readily calculated by known methods, including, but not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., Siam J. Applied Math., 48:1073 (1988). In addition, values for percentage identity can be obtained from amino acid and nucleotide sequence alignments generated using the default settings for the AlignX component of Vector NTI Suite 8.0 (Informax, Frederick, Md.).
Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec. Biol. 215:403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894: Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). The well-known Smith Waterman algorithm may also be used to determine identity.
2. Overview
The present application provides monoclonal antibodies that bind to either C. difficile toxin A or C. difficile toxin B with high affinity and exhibit potent neutralizing activity in both in vitro assays (Vero cell-based toxin neutralization assay and T-84 cell-based TEER assay) and in an art-recognized, in vivo animal model for C. difficile infection. Using a unique antibody discovery strategy, tens of millions of antibody producing B lymphocytes from selected human subjects were screened for binding to and/or neutralizing activity against C. difficile toxin A or B, selected for cloning and recombinant expression, and further characterization to identify specific human antibodies with high binding affinity for and strong neutralizing activity against either C. difficile toxin A or C. difficile toxin B, preferably with a broad spectrum of binding to various C. difficile toxinotypes, such as 0, III, V, VIII, XII, and XV. The human A1, A2, A3, A4, and A5 antibodies bind and neutralize C. difficile toxin A of toxinotype 0 and also recognize toxin A from toxinotypes III, V, XII, and XV. The human B1, B2, B3, B4, B5, and B6 antibodies bind and neutralize C. difficile toxin B from toxinotype 0 and also recognize toxin B from at least toxinotype Ill, and, in some instances, toxin B from at least toxinotypes, III, V, and VIII. These antibodies have therapeutic activity against active disease caused by or associated with C. difficile and can be used either singularly, or in combination, to treat C. difficile infections and/or to protect against the illness.
3. Antibodies
Antibodies, also known as immunoglobulins, are typically tetrameric glycosylated proteins composed of two light (L) chains of approximately 25 kDa each and two heavy (H) chains of approximately 50 kDa each. Two types of light chain, termed lambda and kappa, may be found in antibodies. Depending on the amino acid sequence of the constant domain of heavy chains, immunoglobulins can be assigned to five major classes: A, D, E, G, and M, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Each light chain includes an N-terminal variable (V) domain (VL) and a constant (C) domain (CL). Each heavy chain includes an N-terminal V domain (VH), three or four C domains (CHs), and a hinge region. The CH domain most proximal to VH is designated as CH1. The VH and VL domains consist of four regions of relatively conserved sequences called framework regions (FR1, FR2, FR3, and FR4), which form a scaffold for three regions of hypervariable sequences (complementarity determining regions, CDRs). The CDRs contain most of the residues responsible for specific interactions of the antibody with the antigen. CDRs are referred to as CDR1, CDR2, and CDR3. Accordingly, CDR constituents on the heavy chain are referred to as H1, H2, and H3, while CDR constituents on the light chain are referred to as L1, L2, and L3. Identification and numbering of framework and CDR residues is as described by Chothia et al., Structural determinants in the sequences of immunoglobulin variable domain, J Mol Biol 1998, 278:457-79, which is hereby incorporated by reference in its entirety.
CDR3 is typically the greatest source of molecular diversity within the antibody-binding site. H3, for example, can be as short as two amino acid residues or greater than 26 amino acids. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known in the art. For a review of the antibody structure, see Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, eds. Harlow et al., 1988. One of skill in the art will recognize that each subunit structure, e.g., a CH, VH, CL, VL, CDR, FR structure, comprises active fragments, e.g., the portion of the VH, VL, or CDR subunit the binds to the antigen, i.e., the antigen-binding fragment, or, e.g., the portion of the CH subunit that binds to and/or activates, e.g., an Fc receptor and/or complement. The CDRs typically refer to the Kabat CDRs, as described in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services (1991), eds. Kabat et al. Another standard for characterizing the antigen binding site is to refer to the hypervariable loops as described by Chothia. See, e.g., Chothia, D. et al. (1992) J. Mol. Biol. 227:799-817; and Tomlinson et al. (1995) EMBO J. 14:4628-4638. Still another standard is the AbM definition used by Oxford Molecular's AbM antibody modeling software. See, generally, e.g., Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). Embodiments described with respect to Kabat CDRs can alternatively be implemented using similar described relationships with respect to Chothia hypervariable loops or to the AbM-defined loops. Another standard for residue numbering that can be used is IMGT (Lefranc et al., Dev & Comp Immunol, 27(1):55-77 (2003).
The Fab fragment (Fragment antigen-binding) consists of VH-CH1 and VL-CL domains covalently linked by a disulfide bond between the constant regions. The Fv fragment is smaller and consists of VH and VL domains non-covalently linked. To overcome the tendency of non-covalently linked domains to dissociate, a single chain Fv fragment (scFv) can be constructed. The scFv contains a flexible connector, usually a polypeptide, that links (1) the C-terminus of VH to the N-terminus of VL, or (2) the C-terminus of VL to the N-terminus of VH. A 15-mer (Gly4Ser)3 (SEQ ID NO:331) peptide may be used as a linker, but other linkers are known in the art.
It is possible to modify an antibody to increase productivity and/or when relevant, to decrease possible immunogenicity. In addition, monoclonal antibodies may be modified at either the DNA sequence level to improve expression by removing hairpins or other secondary structure, by optimizing codon utilization, or at the amino acid level to improve expression or stability. For example, it is possible to remove residues such as unpaired cysteines to reduce aggregation, to alter glycosylation sites, or to substitute residues prone to deamidation or oxidization.
It may also be desirable to modify an antibody to improve effector function, e.g., so as to enhance antigen-dependent cell-mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) of the antagonist. One or more amino acid substitutions or the introduction of cysteine in the Fc region may be made, thereby improving internalization capability and/or increased complement-mediated cell killing and ADCC. See Caron et al., J. Ex. Med. 176:1191-1195 (1991) and Shopes, B J. Immunol. 148:2918-2022 (1992), incorporated herein by reference in their entirety. An antibody fusion protein may be prepared that has dual Fc regions with both enhanced complement lysis and ADCC capabilities. Typical Fc receptors that bind to an Fc region of an antibody (e.g., an IgG antibody) include, but are not limited to, receptors of the FcγRI, FcγRII, and FcγRIII and FcRn subclasses, including allelic variants and alternatively spliced forms of these receptors. Fe receptors are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92, 1991; Capel et al., Immunomethods 4:25-34, 1994; and de Haas et al., J. Lab. Clin. Med. 126:330-41, 1995). It is also possible to couple or join an antibody to another agent, such as a cytotoxic agent, drug, or therapeutic.
Anti-toxin A or anti-toxin B antibodies described in this application may optionally comprise antibody constant regions or parts thereof. For example, a VL domain may be attached at its C-terminal end to a light chain constant domain like Cκ or Cλ. Similarly, a VH domain or portion thereof may be attached to all or part of a heavy chain like IgA, IgD, IgE, IgG, and IgM, and any isotype subclass. Constant regions are known in the art (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Publications, Bethesda, Md. (1991)).
VHH molecules (or nanobodies), as known to the skilled artisan, are heavy chain variable domains derived from immunoglobulins naturally devoid of light chains, such as those derived from Camelidae as described in WO 9404678, incorporated herein by reference. Such a VHH molecule can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco and is sometimes called a camelid or camelized variable domain. See e.g., Muyldermans., J. Biotechnology (2001) 74(4):277-302, incorporated herein by reference. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain. VHH molecules are about 10 times smaller than IgG molecules. They are single polypeptides and very stable, resisting extreme pH and temperature conditions. Moreover, they are resistant to the action of proteases which is not the case for conventional antibodies. Furthermore, in vitro expression of VHHs produces high yield, properly folded functional VHHs. In addition, antibodies generated in Camelids will recognize epitopes other than those recognized by antibodies generated in vitro through the use of antibody libraries or via immunization of mammals other than Camelids (see WO 9749805, which is incorporated herein by reference).
The disclosed antibodies can be modified to alter their glycosylation; that is, at least one carbohydrate moiety can be deleted or added to the antibody. Deletion or addition of glycosylation sites can be accomplished by changing amino acid sequence to delete or create glycosylation consensus sites, which are well known in the art. Another means of adding carbohydrate moieties is the chemical or enzymatic coupling of glycosides to amino acid residues of the antibody (see WO 87/05330 and Aplin et al. (1981) CRC Crit. Rev. Biochem., 22: 259-306). Removal of carbohydrate moieties can also be accomplished chemically or enzymatically (see Hakimuddin et al. (1987) Arch. Biochem. Biophys, 259: 52; Edge et al. (1981) Anal. Biochem., 118: 131; Thotakura et al. (1987) Meth. Enzymol., 138: 350).
The antibodies of this invention may be tagged with a detectable or functional label. These labels include radiolabels (e.g., 131I or 99Tc), enzymatic labels (e.g., horseradish peroxidase or alkaline phosphatase), fluorescent labels, chemiluminescent labels, bioluminescent labels, and other chemical moieties (e.g., streptavidin/biotin, avidin/biotin).
4. C. difficile Toxin A and Toxin B
C. difficile produces two cytotoxic enterotoxins, toxins A and toxin B that are released by the bacteria into the gut and believed to be involved in causing the symptoms associated with C. difficile infection. The genes encoding toxins A and B, tcdA and tcdB, respectively, are located in the 19.6 kb C. difficile pathogenicity locus (PaLoc). Toxins A and B are high molecular weight proteins (about 308 and 270 kDa, respectively) consisting of four major structural domains, the N-terminal glucosyl transferase domain, a protease domain, a central, hydrophobic translocation domain, and a C-terminal receptor binding domain. The C terminus is responsible for toxin binding to the surface of epithelial cells and contains repeating oligopeptides that mediate binding to sugar moieties on the surface of target cells. After binding the cell surface receptor, the toxins enter the target cell via receptor-mediate endocytosis. The amino terminal domain contains the glucosyl transferase active site that modifies and inactivates the Rho/Ras superfamily of GTPase proteins found inside the target host cell. Inhibition of the Rho-GTPases results in depolymerization of actin filaments within the host cell, leading to dysregulation of actin cytoskeleton and tight junction integrity, which in turn produces increased cell permeability and loss of barrier function, resulting in diarrhea, inflammation, and an influx of innate immune response molecules.
The amino acid sequences of C. difficile toxin A are known. For example, the amino acid sequence of toxin A from Strain VPI10463 is set forth below.
Similarly, the amino acid sequences of C. difficile toxin B are known. For example, the amino acid sequence of toxin B from Strain VPI10463 is set forth below.
C. difficile strains are classified into variant toxinotypes according to variations in restriction sites within the DNA sequence of the PaLoc encoding toxins A and B. Currently 27 such variant toxinotypes are recognized (I to XXVII). Toxinotype 0 includes strains with restriction patterns identical to the reference laboratory strain VPI 10463 (ATCC43255) and is the most prevalent toxinotype. In a survey of strains in various culture collections worldwide, the toxinotypes 0, III, V, and VIII (toxin B only) arc the most common. (Rupnik, FEMS Microbiol Rev 32 (2008) 541-555.)
5. Anti-Toxin A Antibodies
This disclosure provides antibodies that bind to C. difficile toxin A, including human, monoclonal antibodies having 1) high binding affinity, 2) potent in vitro neutralization activity, and 3) optionally with a broad spectrum of binding to various toxinotypes. Thus, in one embodiment, the antibody has at least one of the following characteristics:
The antibody may have at least two, at least three, or all 4 of the above-identified characteristics.
In one embodiment, the human, monoclonal antibody binds to C. difficile toxin A with a dissociation constant (KD) equal to or less than 500 pM, 250 pM, 200 pM, 150 pM, 100 pM (10−10 M) 10 pM (10−11 M), 1 pM (10−12 M), 0.1 pM (10−13 M), 0.01 pM (10−14 M), or 0.001 pM (10−15 M). The dissociation constant may be measured using techniques known in the art. In one embodiment, the dissociation constant is measured using biolayer interferometry, as described in the examples of this application.
In another embodiment, the human, monoclonal antibody neutralizes the in vitro cytotoxicity of C. difficile toxin A at 2.4 ng/mL in the Vero monkey kidney cell line with an NT50 equal to or less than 3000 pM, 2000 pM, 1000 pM, 100 pM, 60 pM, or 50 pM. For the sake of consistency, when measuring the neutralizing activity in the Vero monkey kidney cell line, Vero cells (2.5×104 cells/well with 5% heat-inactivated FBS) are seeded in a 96-well tissue culture microtiter plates and incubated 37° C. overnight. An equal volume (80 μl) of 4.8 ng/mL (8×MC50) C. difficile toxin A solution and individual dilutions of the antibody solutions (80 μl) in Vero cell medium are combined in a new 96-well plate, and incubated at 37° C., 5% CO2 for 1 hour before 100 μl of the toxin/antibody solutions are added to the Vero cells, and incubated at 37° C. for 72 hours. After incubating for 72 hours, the cells are washed twice with 120 μl each of MEM medium that does not contain phenol, L-glutamine and FBS before adding 100 μl MEM medium that does not contain phenol, L-glutamine and FBS and 10 μl of Alamar Blue® (Life Technologies) to each well. The plates are lightly mixed and incubated at 37° C. for 4 hours before reading fluorescence at 560-590 nm with a cut off at 590 nm.
In yet another embodiment, the human, monoclonal antibody neutralizes the C. difficile toxin A (at 200 ng/mL applied apically) induced loss of transepithelial electrical resistance (TEER) in the T-84 cell line with an NT50 equal to or less than 6 nM, 5 nM, 2 nM, or 1.5 nM. For the sake of consistency, when measuring TEER, T-84 cells are seeded into 0.4 micron polyester transwell plates at a seeding density of 3.6×105 cells/cm2 and maintained at 37° C., 5% CO2 in 10% heat-inactivated FBS in DMEM/F12 culture media for 10-12 days until stable TEER achieved and media is replaced in both apical and basolateral compartments of the transwells daily from day 6 and on the day of assay. The C. difficile toxin A (final concentration of 200 ng/mL) is combined 1:1 with an antibody and incubated at 37° C. with gentle rocking for 30 minutes before replacing the media in the apical compartment with the toxin/antibody samples. Transepithelial electrical resistance of the T-84 cells is measured at To immediately before sample addition and after 2.5 hours (T150) incubation at 37° C. 5% CO2.
In another embodiment, the human, monoclonal antibody binds to toxin A produced by the strains of toxinotypes 0, III, V, XII, and XV. Toxinotype binding may be measured using techniques known in the art, including the techniques described in the examples of this application, such as Western analysis. In another embodiment, for antibodies that bind to an epitope in the C-terminal domain (CTD) of toxin A or toxin B, the toxinotype can be measured using a CTD competition assay, as described in the examples of this application.
In another embodiment, the human, monoclonal anti-toxin A antibody has an on rate constant (Kon) to toxin A of at least 105 M−1 s−1. In another embodiment, the human, monoclonal anti-toxin A antibody has an off rate constant (Koff) to toxin A of 10−4 s−1, 10−5 s−1, 10−6 s−1, 10−7 s−1, or 10−8 s−1, or less. The Kon and Koff may be measured using techniques known in the art. In one embodiment, the dissociation constant is measured using biolayer interferometry, as described in the examples of this application. In one embodiment, the antibody is an isolated A1 antibody. As used herein, the term “A1” refers to a monoclonal antibody, or antigen-binding fragment thereof, that binds to C. difficile toxin A, wherein the antibody comprises 1) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:20 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:22; or 2) a heavy chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:24, a CDR2 comprising the amino acid sequence of SEQ ID NO:26, and a CDR3 comprising the amino acid sequence of SEQ ID NO:28 and a light chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:31, a CDR2 comprising the amino acid sequence of SEQ ID NO:33, and a CDR3 comprising the amino acid sequence of SEQ ID NO:35. In one embodiment, the monoclonal antibody comprises a heavy chain variable domain comprising a FR1 comprising the amino acid sequence SEQ ID NO:23, a CDR1 comprising the amino acid sequence of SEQ ID NO:24, a FR2 comprising the amino acid sequence SEQ ID NO:25, a CDR2 comprising the amino acid sequence of SEQ ID NO:26, a FR3 comprising the amino acid sequence SEQ ID NO:27, a CDR3 comprising the amino acid sequence of SEQ ID NO:28, and a FR4 comprising the amino acid sequence SEQ ID NO:29 and a light chain variable domain comprising a FR1 comprising the amino acid sequence SEQ ID NO:30, a CDR1 comprising the amino acid sequence of SEQ ID NO:31, a FR2 comprising the amino acid sequence SEQ ID NO:32, a CDR2 comprising the amino acid sequence of SEQ ID NO:33, a FR3 comprising the amino acid sequence SEQ ID NO:34, a CDR3 comprising the amino acid sequence of SEQ ID NO:35, and a FR4 comprising the amino acid sequence SEQ ID NO:36. In yet another embodiment, the antibody is a monoclonal antibody that binds to a C. difficile toxin A epitope that is recognized by the A1 antibody, such that the monoclonal antibody competitively inhibits, or cross-blocks, the binding of the A1 antibody to C. difficile toxin A.
In another embodiment, the antibody is an isolated A2 antibody. As used herein, the term “A2” refers to a monoclonal antibody, or antigen-binding fragment thereof that binds to C. difficile toxin A, wherein the antibody comprises 1) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:2 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:4; or 2) a heavy chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:6, a CDR2 comprising the amino acid sequence of SEQ ID NO:8, and a CDR3 comprising the amino acid sequence of SEQ ID NO:10 and a light chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:13, a CDR2 comprising the amino acid sequence of SEQ ID NO:15, and a CDR3 comprising the amino acid sequence of SEQ ID NO:17. In one embodiment, the monoclonal antibody comprises a heavy chain variable domain comprising a FR1 comprising the amino acid sequence SEQ ID NO:5, a CDR1 comprising the amino acid sequence of SEQ ID NO:6, a FR2 comprising the amino acid sequence SEQ ID NO:7, a CDR2 comprising the amino acid sequence of SEQ ID NO:8, a FR3 comprising the amino acid sequence SEQ ID NO:9, a CDR3 comprising the amino acid sequence of SEQ ID NO:10, and a FR4 comprising the amino acid sequence SEQ ID NO:11 and a light chain variable domain comprising a FR1 comprising the amino acid sequence SEQ ID NO:12, a CDR1 comprising the amino acid sequence of SEQ ID NO:13, a FR2 comprising the amino acid sequence SEQ ID NO:14, a CDR2 comprising the amino acid sequence of SEQ ID NO:15, a FR3 comprising the amino acid sequence SEQ ID NO:16, a CDR3 comprising the amino acid sequence of SEQ ID NO:17, and a FR4 comprising the amino acid sequence SEQ ID NO:18. In yet another embodiment, the antibody is a monoclonal antibody that binds to a C. difficile toxin A epitope that is recognized by the A2 antibody, such that the monoclonal antibody competitively inhibits, or cross-blocks, the binding of the A2 antibody to C. difficile toxin A.
The A2 antibody binds to an epitope in the C-terminal receptor domain of C. difficile toxin A that comprises the amino acid sequence of X1TGWQTI (SEQ ID NO:232), where X1 is A or V or the amino acid sequence of X2TGWQTIX3GKX4YYF (SEQ ID NO:233), where X2 is A or V, X3 is N or D and X4 is K or V. Thus, another embodiment is directed to an isolated monoclonal antibody that binds to an epitope in the C-terminal receptor domain of C. difficile toxin A that comprises the amino acid sequence of X1TGWQTI (SEQ ID NO:232), where X1 is A or V or the amino acid sequence of X2TGWQTIX3GKX4YYF (SEQ ID NO:233), where X2 is A or V, X3 is N or D and X4 is K or V.
In another embodiment, the antibody is an isolated A3 antibody. As used herein, the term “A3” refers to a monoclonal antibody, or antigen-binding fragment thereof, that binds to C. difficile toxin A, wherein the antibody comprises 1) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:38 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:40; or 2) a heavy chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:42, a CDR2 comprising the amino acid sequence of SEQ ID NO:44, and a CDR3 comprising the amino acid sequence of SEQ ID NO:46 and a light chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:49, a CDR2 comprising the amino acid sequence of SEQ ID NO:51, and a CDR3 comprising the amino acid sequence of SEQ ID NO:53. In one embodiment, the monoclonal antibody comprises a heavy chain variable domain comprising a FR1 comprising the amino acid sequence SEQ ID NO:41, a CDR1 comprising the amino acid sequence of SEQ ID NO:42, a FR2 comprising the amino acid sequence SEQ ID NO:43, a CDR2 comprising the amino acid sequence of SEQ ID NO:44, a FR3 comprising the amino acid sequence SEQ ID NO:45, a CDR3 comprising the amino acid sequence of SEQ ID NO:46, and a FR4 comprising the amino acid sequence SEQ ID NO:47 and a light chain variable domain comprising a FR1 comprising the amino acid sequence SEQ ID NO:48, a CDR1 comprising the amino acid sequence of SEQ ID NO:49, a FR2 comprising the amino acid sequence SEQ ID NO:50, a CDR2 comprising the amino acid sequence of SEQ ID NO:51, a FR3 comprising the amino acid sequence SEQ ID NO:52, a CDR3 comprising the amino acid sequence of SEQ ID NO:53, and a FR4 comprising the amino acid sequence SEQ ID NO:54. In yet another embodiment, the antibody is a monoclonal antibody that binds to a C. difficile toxin A epitope that is recognized by the A3 antibody, such that the monoclonal antibody competitively inhibits, or cross-blocks, the binding of the A3 antibody to C. difficile toxin A.
In another embodiment, the antibody is an isolated A4 antibody. As used herein, the term “A4” refers to a monoclonal antibody, or antigen-binding fragment thereof, that binds to C. difficile toxin A, wherein the antibody comprises 1) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:56 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:58; or 2) a heavy chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:60, a CDR2 comprising the amino acid sequence of SEQ ID NO:62, and a CDR3 comprising the amino acid sequence of SEQ ID NO:64 and a light chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:67, a CDR2 comprising the amino acid sequence of SEQ ID NO-69, and a CDR3 comprising the amino acid sequence of SEQ ID NO:71. In one embodiment, the monoclonal antibody comprises a heavy chain variable domain comprising a FR1 comprising the amino acid sequence SEQ ID NO:59, a CDR1 comprising the amino acid sequence of SEQ ID NO:60, a FR2 comprising the amino acid sequence SEQ ID NO:61, a CDR2 comprising the amino acid sequence of SEQ ID NO:62, a FR3 comprising the amino acid sequence SEQ ID NO:63, a CDR3 comprising the amino acid sequence of SEQ ID NO:64, and a FR4 comprising the amino acid sequence SEQ ID NO:65 and a light chain variable domain comprising a FR1 comprising the amino acid sequence SEQ ID NO:66, a CDR1 comprising the amino acid sequence of SEQ ID NO:67, a FR2 comprising the amino acid sequence SEQ ID NO:68, a CDR2 comprising the amino acid sequence of SEQ ID NO:69, a FR3 comprising the amino acid sequence SEQ ID NO:70, a CDR3 comprising the amino acid sequence of SEQ ID NO:71, and a FR4 comprising the amino acid sequence SEQ ID NO:72. In yet another embodiment, the antibody is a monoclonal antibody that binds to a C. difficile toxin A epitope that is recognized by the A4 antibody, such that the monoclonal antibody competitively inhibits, or cross-blocks, the binding of the A4 antibody to C. difficile toxin A.
In another embodiment, the antibody is an isolated A5 antibody. As used herein, the term “A5” refers to a monoclonal antibody, or antigen-binding fragment thereof, that binds to C. difficile toxin A, wherein the antibody comprises 1) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:74 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:76; or 2) a heavy chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:78, a CDR2 comprising the amino acid sequence of SEQ ID NO:80, and a CDR3 comprising the amino acid sequence of SEQ ID NO:82 and a light chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:85, a CDR2 comprising the amino acid sequence of SEQ ID NO:87, and a CDR3 comprising the amino acid sequence of SEQ ID NO:89. In one embodiment, the monoclonal antibody comprises a heavy chain variable domain comprising a FR1 comprising the amino acid sequence SEQ ID NO:77, a CDR1 comprising the amino acid sequence of SEQ ID NO:78, a FR2 comprising the amino acid sequence SEQ ID NO:79, a CDR2 comprising the amino acid sequence of SEQ ID NO:80, a FR3 comprising the amino acid sequence SEQ ID NO:81, a CDR3 comprising the amino acid sequence of SEQ ID NO:82, and a FR4 comprising the amino acid sequence SEQ ID NO:83 and a light chain variable domain comprising a FR1 comprising the amino acid sequence SEQ ID NO:84, a CDR1 comprising the amino acid sequence of SEQ ID NO:85, a FR2 comprising the amino acid sequence SEQ ID NO:86, a CDR2 comprising the amino acid sequence of SEQ ID NO:87, a FR3 comprising the amino acid sequence SEQ ID NO:88, a CDR3 comprising the amino acid sequence of SEQ ID NO:89, and a FR4 comprising the amino acid sequence SEQ ID NO:90. In yet another embodiment, the antibody is a monoclonal antibody that binds to a C. difficile toxin A epitope that is recognized by the A5 antibody, such that the monoclonal antibody competitively inhibits, or cross-blocks, the binding of the A5 antibody to C. difficile toxin A.
Whether an antibody competitively inhibits the binding of an antibody to C. difficile toxin A can be assessed using routine methods in the art, including, for example, the Octet methods described in the examples of this application and other routine quantitative methods, such as the Biacore assay. In one embodiment, competitive binding is measured using biolayer interferometry.
The amino acid and nucleotide sequences for the VH and VL domains of the A1 antibody are as follows:
The amino acid sequences for the FR and CDR sequences of the A1 antibody are as follows:
One aspect is directed to an isolated polypeptide comprising one or more of the CDR sequences of the A1 antibody (i.e., one or more of SEQ ID NOs. 24, 26, 28, 31, 33, or 35). In certain embodiments, the isolated polypeptide further comprises additional amino acid sequences, or other molecules, to form a molecule that binds toxin A, including, but not limited to an anti-toxin A antibody.
The amino acid and nucleotide sequences for the VH and VL domains of the A2 antibody are as follows:
The amino acid sequences for the FR and CDR sequences of the A2 antibody are as follows:
One aspect is directed to an isolated polypeptide comprising one or more of the CDR sequences of the A2 antibody (i.e., one or more of SEQ ID NOs. 6, 8, 10, 13, 15, or 17). In certain embodiments, the isolated polypeptide further comprises additional amino acid sequences, or other molecules, to form a molecule that binds toxin A, including, but not limited to an anti-toxin A antibody.
The amino acid and nucleotide sequences for the VH and VL domains of the A3 antibody are as follows:
The amino acid sequences for the FR and CDR sequences of the A3 antibody are as follows:
One aspect is directed to an isolated polypeptide comprising one or more of the CDR sequences of the A3 antibody (i.e., one or more of SEQ ID NOs. 42, 44, 46, 49, 51, or 53).
In certain embodiments, the isolated polypeptide further comprises additional amino acid sequences, or other molecules, to form a molecule that binds toxin A, including, but not limited to an anti-toxin A antibody.
The amino acid and nucleotide sequences for the VH and VL domains of the A4 antibody are as follows:
The amino acid sequences for the FR and CDR sequences of the A4 antibody are as follows:
One aspect is directed to an isolated polypeptide comprising one or more of the CDR sequences of the A4 antibody (i.e., one or more of SEQ ID NOs. 60, 62, 64, 67, 69, or 71). In certain embodiments, the isolated polypeptide further comprises additional amino acid sequences, or other molecules, to form a molecule that binds toxin A, including, but not limited to an anti-toxin A antibody.
The amino acid and nucleotide sequences for the VH and VL domains of the A5 antibody are as follows:
The amino acid sequences for the FR and CDR sequences of the A5 antibody are as follows:
One aspect is directed to an isolated polypeptide comprising one or more of the CDR sequences of the A5 antibody (i.e., one or more of SEQ ID NOs. 78, 80, 82, 85, 87, or 89). In certain embodiments, the isolated polypeptide further comprises additional amino acid sequences, or other molecules, to form a molecule that binds toxin A, including, but not limited to an anti-toxin A antibody.
The SEQ ID NOs corresponding to the sequences of the A1, A2, A3, A4, and A5, antibodies are listed in Table 1.
6. Anti-Toxin B Antibodies
This disclosure provides antibodies that bind to C. difficile toxin B, including human, monoclonal antibodies having 1) high binding affinity, 2) potent in vitro neutralization activity, and 3) optionally a broad spectrum of binding to the toxins of various toxinotypes. Thus, in one embodiment, the antibody has at least one of the following characteristics:
The antibody may have at least two, at least three, or all 4 of the above-identified characteristics.
In one embodiment, the human, monoclonal antibody binds to C. difficile toxin B with a dissociation constant (KD) equal to or less than 500 pM, 250 pM, 200 pM, 150 pM, 100 pM (10−10 M), 50 pM, 30 pM, 10 pM (10−11 M), or 1 pM (10−12 M). The dissociation constant may be measured using techniques known in the art. In one embodiment, the dissociation constant is measured using biolayer interferometry, as described in the examples of this application.
In another embodiment, the human, monoclonal antibody neutralizes the in vitro cytotoxicity of C. difficile toxin B at 17 pg/mL in the Vero monkey kidney cell line with an NT50 of equal to or less than 1000 pM, 500 pM, 100 pM, 60 pM, or 50 pM. For the sake of consistency, when measuring the neutralizing activity in the Vero monkey kidney cell line, Vero cells (2.5×104 cells/well with 5% heat-inactivated FBS) are seeded in 96-well tissue culture microtiter plates and incubated 37° C. overnight. An equal volume (80 μl) of 34.4 pg/mL (8×MC50) C. difficile toxin B solution and individual dilutions of the antibody solutions (80 μl) in Vero cell medium are combined in a new 96-well plate, and incubated at 37° C., 5% CO2 for 1 hour before 100 μl of the toxin/antibody solutions are added to the Vero cells, and incubated at 37° C. for 72 hours. After incubating for 72 hours, the cells are washed twice with 120 μl/each of MEM medium that does not contain phenol, L-glutamine and FBS before adding 100 μl MEM medium that does not contain phenol, L-glutamine and FBS and 10 μl of Alamar Blue® (Life Technologies) to each well. The plates are lightly mixed and incubated at 37° C. for 4 hours before reading fluorescence at 560-590 nm with a cut off at 590 nm.
In yet another embodiment, the human, monoclonal antibody neutralizes the C. difficile toxin B (at 75 ng/mL, applied basolaterally) induced loss of transepithelial electrical resistance (TEER) in the T-84 cell line with an NT50 equal to or less than 200 pM, 150 pM, 100 pM, or 70 pM. For the sake of consistency, when measuring TEER, T-84 cells are seeded into 0.4 micron polyester transwell plates at a seeding density of 3.6×105 cells/cm2 and maintained at 37° C., 5% CO2 in 10% heat-inactivated FBS in DMEM/F12 culture media for 10-12 days until stable TEER is achieved and media is replaced in both apical and basolateral compartments of the transwells daily from day 6 and on the day of assay. The C. difficile toxin B (final concentration of 75 ng/mL) is combined 1:1 with an antibody and incubated at 37° C. with gentle rocking for 30 minutes before replacing the media in the basolateral compartment with the toxin/antibody samples. Transepithelial electrical resistance of the T-84 cells is measured at T0 immediately before sample addition and after 2.5 hours (T150) incubation at 37° C. 5% CO2.
In another embodiment, the human, monoclonal anti-toxin B antibody binds to toxin B produced by strains of at least toxinotypes 0, III, and V, toxin B produced by strains of at least toxinotypes 0, III, V, and VIII, toxin B produced by the strains of at least toxinotypes 0, III, V, VIII, and XII, or toxin B produced by the strains of at least toxinotypes 0, III, V, VIII, XII, and XV. Toxinotype binding may be measured using techniques known in the art, including the techniques described in the examples of this application, such as Western analysis. In another embodiment, for antibodies that bind to an epitope in the C-terminal domain (CTD) of toxin A or toxin B, the toxinotype can be measured using a CTD competition assay, as described in the examples of this application.
In another embodiment, the human, monoclonal anti-toxin B antibody has an on rate constant (Kon) to toxin B of at least 105 M−1 s−1. In another embodiment, the human, monoclonal anti-toxin B antibody has an off rate constant (Koff) to toxin B of 10−4 s−1, 10−5 s−1, 10−6 s−1, 10−7 s−1, or 10−8 s−1, or less. The Kon and Koff may be measured using techniques known in the art. In one embodiment, the dissociation constant is measured using biolayer interferometry, as described in the examples of this application.
In one embodiment, the antibody is an isolated B1 antibody. As used herein, the term “B1” refers to a monoclonal antibody, or antigen-binding fragment thereof; that binds to C. difficile toxin B, wherein the antibody comprises 1) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:110 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:112; or 2) a heavy chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:114, a CDR2 comprising the amino acid sequence of SEQ ID NO:116, and a CDR3 comprising the amino acid sequence of SEQ ID NO:118 and a light chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:121, a CDR2 comprising the amino acid sequence of SEQ ID NO:123, and a CDR3 comprising the amino acid sequence of SEQ ID NO:125. In one embodiment, the monoclonal antibody comprises a heavy chain variable domain comprising a FR1 comprising the amino acid sequence SEQ ID NO:113, a CDR1 comprising the amino acid sequence of SEQ ID NO: 114, a FR2 comprising the amino acid sequence SEQ ID NO:115, a CDR2 comprising the amino acid sequence of SEQ ID NO:116, a FR3 comprising the amino acid sequence SEQ ID NO: 117, a CDR3 comprising the amino acid sequence of SEQ ID NO:118, and a FR4 comprising the amino acid sequence SEQ ID NO:119 and a light chain variable domain comprising a FR1 comprising the amino acid sequence SEQ ID NO:120, a CDR1 comprising the amino acid sequence of SEQ ID NO:121, a FR2 comprising the amino acid sequence SEQ ID NO:122, a CDR2 comprising the amino acid sequence of SEQ ID NO:123, a FR3 comprising the amino acid sequence SEQ ID NO:124, a CDR3 comprising the amino acid sequence of SEQ ID NO:125, and a FR4 comprising the amino acid sequence SEQ ID NO:126. In yet another embodiment, the antibody is a monoclonal antibody that binds to a C. difficile toxin B epitope that is recognized by the B1 antibody, such that the monoclonal antibody competitively inhibits, or cross-blocks, the binding of the B1 antibody to C. difficile toxin B.
In another embodiment, the antibody is an isolated B2 antibody. As used herein, the term “B2” refers to a monoclonal antibody, or antigen-binding fragment thereof, that binds to C. difficile toxin B, wherein the antibody comprises 1) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:92 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:94; or 2) a heavy chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:96, a CDR2 comprising the amino acid sequence of SEQ ID NO:98, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 100 and a light chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ 11 NO:103, a CDR2 comprising the amino acid sequence of SEQ ID NO:105, and a CDR3 comprising the amino acid sequence of SEQ ID NO:107. In one embodiment, the monoclonal antibody comprises a heavy chain variable domain comprising a FR1 comprising the amino acid sequence SEQ ID NO:95, a CDR1 comprising the amino acid sequence of SEQ ID NO:96, a FR2 comprising the amino acid sequence SEQ ID NO:97, a CDR2 comprising the amino acid sequence of SEQ ID NO:98, a FR3 comprising the amino acid sequence SEQ ID NO:99, a CDR3 comprising the amino acid sequence of SEQ ID NO:100, and a FR4 comprising the amino acid sequence SEQ ID NO:101 and a light chain variable domain comprising a FR1 comprising the amino acid sequence SEQ ID NO:102, a CDR1 comprising the amino acid sequence of SEQ ID NO:103, a FR2 comprising the amino acid sequence SEQ ID NO:104, a CDR2 comprising the amino acid sequence of SEQ ID NO:105, a FR3 comprising the amino acid sequence SEQ ID NO:106, a CDR3 comprising the amino acid sequence of SEQ ID NO: 107, and a FR4 comprising the amino acid sequence SEQ ID NO:108. In yet another embodiment, the antibody is a monoclonal antibody that binds to a C. difficile toxin B epitope that is recognized by the B2 antibody, such that the monoclonal antibody competitively inhibits, or cross-blocks, the binding of the B2 antibody to C. difficile toxin B.
In another embodiment, the antibody is an isolated B3 antibody. As used herein, the term “B3” refers to a monoclonal antibody, or antigen-binding fragment thereof, that binds to C. difficile toxin B, wherein the antibody comprises 1) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:164 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:166; or 2) a heavy chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:168, a CDR2 comprising the amino acid sequence of SEQ ID NO:170, and a CDR3 comprising the amino acid sequence of SEQ ID NO:172 and a light chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:175, a CDR2 comprising the amino acid sequence of SEQ ID NO:177, and a CDR3 comprising the amino acid sequence of SEQ ID NO:179. In one embodiment, the monoclonal antibody comprises a heavy chain variable domain comprising a FR1 comprising the amino acid sequence SEQ ID NO:167, a CDR1 comprising the amino acid sequence of SEQ ID NO:168, a FR2 comprising the amino acid sequence SEQ ID NO:169, a CDR2 comprising the amino acid sequence of SEQ ID NO:170, a FR3 comprising the amino acid sequence SEQ ID NO:171, a CDR3 comprising the amino acid sequence of SEQ ID NO:172, and a FR4 comprising the amino acid sequence SEQ ID NO:173 and a light chain variable domain comprising a FR1 comprising the amino acid sequence SEQ ID NO:174, a CDR1 comprising the amino acid sequence of SEQ ID NO:175, a FR2 comprising the amino acid sequence SEQ ID NO:176, a CDR2 comprising the amino acid sequence of SEQ ID NO:177, a FR3 comprising the amino acid sequence SEQ ID NO:178, a CDR3 comprising the amino acid sequence of SEQ ID NO:179, and a FR4 comprising the amino acid sequence SEQ ID NO:180. In yet another embodiment, the antibody is a monoclonal antibody that binds to a C. difficile toxin B epitope that is recognized by the B3 antibody, such that the monoclonal antibody competitively inhibits, or cross-blocks, the binding of the B3 antibody to C. difficile toxin B.
In another embodiment, the antibody is an isolated B4 antibody. As used herein, the term “B4” refers to a monoclonal antibody, or antigen-binding fragment thereof, that binds to C. difficile toxin B, wherein the antibody comprises 1) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:146 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:148; or 2) a heavy chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:150, a CDR2 comprising the amino acid sequence of SEQ ID NO:152, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 154 and a light chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:157, a CDR2 comprising the amino acid sequence of SEQ ID NO:159, and a CDR3 comprising the amino acid sequence of SEQ ID NO:161. In one embodiment, the monoclonal antibody comprises a heavy chain variable domain comprising a FR1 comprising the amino acid sequence SEQ ID NO:149, a CDR1 comprising the amino acid sequence of SEQ ID NO:150, a FR2 comprising the amino acid sequence SEQ ID NO:151, a CDR2 comprising the amino acid sequence of SEQ ID NO:152, a FR3 comprising the amino acid sequence SEQ ID NO: 153, a CDR3 comprising the amino acid sequence of SEQ ID NO:154, and a FR4 comprising the amino acid sequence SEQ ID NO:155 and a light chain variable domain comprising a FR1 comprising the amino acid sequence SEQ ID NO:156, a CDR1 comprising the amino acid sequence of SEQ ID NO:157, a FR2 comprising the amino acid sequence SEQ ID NO:158, a CDR2 comprising the amino acid sequence of SEQ ID NO:159, a FR3 comprising the amino acid sequence SEQ ID NO:160, a CDR3 comprising the amino acid sequence of SEQ ID NO:161, and a FR4 comprising the amino acid sequence SEQ ID NO:162. In yet another embodiment, the antibody is a monoclonal antibody that binds to a C. difficile toxin B epitope that is recognized by the B4 antibody, such that the monoclonal antibody competitively inhibits, or cross-blocks, the binding of the B4 antibody to C. difficile toxin B.
In another embodiment, the antibody is an isolated B5 antibody. As used herein, the term “B5” refers to a monoclonal antibody, or antigen-binding fragment thereof; that binds to C. difficile toxin B, wherein the antibody comprises 1) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:182 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:184; or 2) a heavy chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:186, a CDR2 comprising the amino acid sequence of SEQ ID NO:188, and a CDR3 comprising the amino acid sequence of SEQ ID NO:190 and a light chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:193, a CDR2 comprising the amino acid sequence of SEQ ID NO:195, and a CDR3 comprising the amino acid sequence of SEQ ID NO:197. In one embodiment, the monoclonal antibody comprises a heavy chain variable domain comprising a FR1 comprising the amino acid sequence SEQ ID NO:185, a CDR1 comprising the amino acid sequence of SEQ ID NO:186, a FR2 comprising the amino acid sequence SEQ ID NO:187, a CDR2 comprising the amino acid sequence of SEQ ID NO:188, a FR3 comprising the amino acid sequence SEQ ID NO:189, a CDR3 comprising the amino acid sequence of SEQ ID NO:190, and a FR4 comprising the amino acid sequence SEQ ID NO:191 and a light chain variable domain comprising a FR1 comprising the amino acid sequence SEQ ID NO:192, a CDR1 comprising the amino acid sequence of SEQ ID NO:193, a FR2 comprising the amino acid sequence SEQ ID NO:194, a CDR2 comprising the amino acid sequence of SEQ ID NO:195, a FR3 comprising the amino acid sequence SEQ ID NO:196, a CDR3 comprising the amino acid sequence of SEQ ID NO:197, and a FR4 comprising the amino acid sequence SEQ ID NO:198. In yet another embodiment, the antibody is a monoclonal antibody that binds to a C. difficile toxin B epitope that is recognized by the B5 antibody, such that the monoclonal antibody competitively inhibits, or cross-blocks, the binding of the B5 antibody to C. difficile toxin B.
In another embodiment, the antibody is an isolated B6 antibody. As used herein, the term “B6” refers to a monoclonal antibody, or antigen-binding fragment thereof, that binds to C. difficile toxin B, wherein the antibody comprises 1) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:128 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:130; or 2) a heavy chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:132, a CDR2 comprising the amino acid sequence of SEQ ID NO:134, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 136 and a light chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:139, a CDR2 comprising the amino acid sequence of SEQ ID NO:141, and a CDR3 comprising the amino acid sequence of SEQ ID NO:143. In one embodiment, the monoclonal antibody comprises a heavy chain variable domain comprising a FR1 comprising the amino acid sequence SEQ ID NO:131, a CDR1 comprising the amino acid sequence of SEQ ID NO:132, a FR2 comprising the amino acid sequence SEQ ID NO:133, a CDR2 comprising the amino acid sequence of SEQ ID NO:134, a FR3 comprising the amino acid sequence SEQ ID NO:135, a CDR3 comprising the amino acid sequence of SEQ ID NO:136, and a FR4 comprising the amino acid sequence SEQ ID NO:137 and a light chain variable domain comprising a FR1 comprising the amino acid sequence SEQ ID NO:138, a CDR1 comprising the amino acid sequence of SEQ ID NO:139, a FR2 comprising the amino acid sequence SEQ ID NO: 140, a CDR2 comprising the amino acid sequence of SEQ ID NO:141, a FR3 comprising the amino acid sequence SEQ ID NO:142, a CDR3 comprising the amino acid sequence of SEQ ID NO:143, and a FR4 comprising the amino acid sequence SEQ ID NO:144. In yet another embodiment, the antibody is a monoclonal antibody that binds to a C. difficile toxin B epitope that is recognized by the B6 antibody, such that the monoclonal antibody competitively inhibits, or cross-blocks, the binding of the B6 antibody to C. difficile toxin B.
Whether an antibody competitively inhibits the binding of an antibody to C. difficile toxin B can be assessed using routine methods in the art, including, for example, the Octet methods described in the examples of this application or other routine quantitative binding assays, such as the Biacore assay. In one embodiment, competitive binding is measured using biolayer interferometry.
The B1, B2, and B3 antibodies bind to an epitope within amino acids 10-520 of SEQ ID NO:231. Thus, one embodiment is directed to an isolated monoclonal antibody that binds to an epitope in the glucosyl transferase domain of C. difficile toxin B, wherein the epitope comprises amino acids 10-520 of SEQ ID NO:231. More specifically, the B1 and B3 antibodies bind to an epitope comprising the amino acid sequence SGRNK (SEQ ID NO:234) or amino acids 56-80 of SEQ ID NO:231. Thus, another embodiment is directed to an isolated monoclonal antibody that binds to an epitope in the glucosyl transferase domain of C. difficile toxin B, wherein the epitope comprises the amino acid sequence SGRNK (SEQ ID NO:234) or amino acids 56-80 of SEQ ID NO:231.
The B4 antibody binds to an epitope in the N-terminal translocation domain of C. difficile toxin B, wherein the epitope comprises amino acids 1110-1530 of SEQ ID NO:231. Thus, another embodiment is directed to an isolated monoclonal antibody that binds to an epitope in the N-terminal translocation domain of C. difficile toxin B, wherein the epitope comprises amino acids 1110-1530 of SEQ ID NO:231.
The B6 antibody binds to an epitope in the receptor binding domain of C. difficile toxin B, wherein the epitope comprises amino acids 1750-2360 of SEQ ID NO:231. Thus, another embodiment is directed to an isolated monoclonal antibody that binds to an epitope in the receptor binding domain of C. difficile toxin B, wherein the epitope comprises amino acids 1750-2360 of SEQ ID NO:231.
The amino acid and nucleotide sequences for the VH and VL domains of the B1 antibody are as follows:
The amino acid sequences for the FR and CDR sequences of the B1 antibody are as follows:
One aspect is directed to an isolated polypeptide comprising one or more of the CDR sequences of the B1 antibody (i.e., one or more of SEQ ID NOs. 114, 116, 118, 121, 123, or 125). In certain embodiments, the isolated polypeptide further comprises additional amino acid sequences, or other molecules, to form a molecule that binds toxin B, including, but not limited to an anti-toxin B antibody.
The amino acid and nucleotide sequences for the VH and VL domains of the B2 antibody are as follows:
The amino acid sequences for the FR and CDR sequences of the B2 antibody are as follows:
One aspect is directed to an isolated polypeptide comprising one or more of the CDR sequences of the B2 antibody (i.e., one or more of SEQ ID NOs.96, 96, 100, 103, 105, or 107). In certain embodiments, the isolated polypeptide further comprises additional amino acid sequences, or other molecules, to form a molecule that binds toxin B, including, but not limited to an anti-toxin B antibody.
The amino acid and nucleotide sequences for the VH and VL domains of the B3 antibody are as follows:
The amino acid sequences for the FR and CDR sequences of the B3 antibody are as follows:
One aspect is directed to an isolated polypeptide comprising one or more of the CDR sequences of the B3 antibody (i.e., one or more of SEQ ID NOs. 168, 170, 172, 175, 177, or 179). In certain embodiments, the isolated polypeptide further comprises additional amino acid sequences, or other molecules, to form a molecule that binds toxin B, including, but not limited to an anti-toxin B antibody.
The amino acid and nucleotide sequences for the VH and VL domains of the B4 antibody are as follows:
The amino acid sequences for the FR and CDR sequences of the B4 antibody are as follows:
One aspect is directed to an isolated polypeptide comprising one or more of the CDR sequences of the B4 antibody (i.e., one or more of SEQ ID NOs. 150, 152, 154, 157, 159, or 161). In certain embodiments, the isolated polypeptide further comprises additional amino acid sequences, or other molecules, to form a molecule that binds toxin B, including, but not limited to an anti-toxin B antibody.
The amino acid and nucleotide sequences for the VH and VL domains of the B5 antibody are as follows:
The amino acid sequences for the FR and CDR sequences of the B5 antibody are as follows:
One aspect is directed to an isolated polypeptide comprising one or more of the CDR sequences of the B5 antibody (i.e., one or more of SEQ ID) NOs. 186, 188, 190, 193, 195, or 197). In certain embodiments, the isolated polypeptide further comprises additional amino acid sequences, or other molecules, to form a molecule that binds toxin B, including, but not limited to an anti-toxin B antibody.
The amino acid and nucleotide sequences for the VH and VL domains of the B6 antibody are as follows:
The amino acid sequences for the FR and CDR sequences of the B6 antibody are as follows:
One aspect is directed to an isolated polypeptide comprising one or more of the CDR sequences of the B6 antibody (i.e., one or more of SEQ ID NOs.132, 134, 136, 139, 141, or 143). In certain embodiments, the isolated polypeptide further comprises additional amino acid sequences, or other molecules, to form a molecule that binds toxin B, including, but not limited to an anti-toxin B antibody.
The SEQ ID NOs corresponding to the sequences of the B1, B2, B3, B4, B5, and B6 antibodies are listed in Table 2.
7. Modified Antibodies
Modified versions of the A1, A2, A3, A4, A5, B1, B2, B3, B4, B5, and B6 antibodies are also provided. Typically modifications to an antibody can be introduced through the nucleic acids that encode the heavy or light chain variable domains of the antibody. These modifications can include deletions, insertions, point mutations, truncations, and amino acid substitutions and addition of amino acids or non-amino acid moieties. For example, random mutagenesis of the disclosed VH or VL sequences can be used to generate variant VH or VL domains still capable of binding C. difficile toxin A or B. A technique using error-prone PCR is described by Gram et al. (Proc. Nat. Acad. Sci. U.S.A. (1992) 89: 3576-3580). Another method uses direct mutagenesis of the disclosed VH or VL sequences. Such techniques are disclosed by Barbas et al. (Proc. Nat. Acad. Sci. U.S.A. (1994) 91: 3809-3813) and Schier et al. (J. Mol. Biol. (1996) 263: 551-567). Modifications can also be made directly to the amino acid sequence, such as by cleavage, addition of a linker molecule or addition of a detectable moiety, such as biotin, addition of a fatty acid, and the like.
In one embodiment, the antibody is a monoclonal antibody that binds to C. difficile toxin A and comprises 1) a heavy chain variable domain that is at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98% identical, or 100% identical to the amino acid sequence of the heavy chain variable domain of the A1, A2, A3, A4, or A5 antibody, and 2) a light chain variable domain that is at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98% identical, or 100% identical to the amino acid sequence of the light chain variable domain of the A1, A2, A3, A4, or A5 antibody, wherein the heavy chain and light chain variable domains from the same antibody are combined as shown in Table 3.
C. difficile Toxin A Antibody
In another embodiment, the antibody is a monoclonal antibody binds to C. difficile toxin B and comprises 1) a heavy chain variable domain that is at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98% identical, or 100% identical to the amino acid sequence of the heavy chain variable domain of the B1, B2, B3, B4, B5, or B6 antibody, and 2) a light chain variable domain that is at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98% identical, or 100% identical to the amino acid sequence of the light chain variable domain of the B1, B2, B3, B4, B5, or B6 antibody, wherein the heavy chain and light chain variable domains from the same antibody are combined as shown in Table 4.
C. difficile Toxin B Antibody
In another embodiment, the monoclonal antibody binds to C. difficile toxin A and comprises six CDRs (H1, H2, H3, L1, L2, and L3) that are at least about 70%, at least about 80%, at least about 90%, at least about 95% or at least about 98% identical to the amino acid sequences of the six CDRs (H1, H2, H3, L1, L2, and L3) of the heavy and light chain variable domains of the A1, A2, A3, A4, or A5 antibody.
In yet another embodiment, the monoclonal antibody binds to C. difficile toxin B and comprises six CDRs (H1, H12, L3, L1, L2, and L3) that are at least about 70%, at least about 80%, at least about 90%, at least about 95% or at least about 98% identical to the amino acid sequences of the six CDRs (H1, H2, H3, L1, L2, and L3) of the heavy and light chain variable domains of the B1, B2, B3, B4, B5, or B6 antibody.
In another embodiment, the monoclonal antibody binds to C. difficile toxin A and comprises a heavy chain variable domain identical to SEQ ID NO:20 (A1), SEQ ID NO:2(A2), SEQ ID NO:38 (A3), SEQ ID NO:56 (A4), or SEQ ID NO:74 (A5) except for 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, and in certain cases, up to 10 amino acid substitutions in the CDR sequences. In another embodiment, the monoclonal antibody binds to C. difficile toxin A and comprises a light chain variable domain identical to SEQ ID NO:22 (A1), SEQ ID NO:4 (A2), SEQ ID NO:40 (A3), SEQ ID NO:58 (A4), or SEQ ID NO:76 (A5) except for 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, and in certain cases, up to 10 amino acid substitutions in the CDR sequences.
In yet another embodiment, the monoclonal antibody binds to C. difficile toxin B and comprises a heavy chain variable domain identical to SEQ ID NO:110 (B1), SEQ ID NO:92 (B2), SEQ ID NO:164 (B3), SEQ ID NO:146 (B4), SEQ ID NO:182 (B5), or SEQ ID NO:128 (B6) except for 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, and in certain cases, up to 10 amino acid substitutions in the CDR sequences. In another embodiment, the monoclonal antibody binds to C. difficile toxin A and comprises a light chain variable domain identical to SEQ ID NO:112 (B1), SEQ ID NO:94 (B2), SEQ ID NO:166 (B3), SEQ ID NO:148 (B4), SEQ ID NO:184 (B5), or SEQ ID NO:130 (B6) except for 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, and in certain cases, up to 10 amino acid substitutions in the CDR sequences.
The specific amino acid positions that can be substituted in a CDR, as well as the donor amino acid that can be substituted into those positions can be readily determined by one of skill in the art using known methods, such as those disclosed in published U.S. Application 2006/0099204, the disclosure of which is hereby incorporated by reference in its entirety. Typically, this involves substitution of an amino acid with an amino acid having similar charge, hydrophobic, or stereochemical characteristics. More drastic substitutions in FR regions, in contrast to CDR regions, may also be made as long as they do not adversely affect (e.g., reduce affinity by more than 50% as compared to unsubstituted antibody) the binding properties of the antibody.
Modified versions of the A1, A2, A3, A4, A5, B1, B2, B3, B4, B5, and B6 antibodies can also be screened to identify which mutation provides a modified antibody that retains a desired property, such as high affinity binding of the parent antibody for either C. difficile toxin A or B and/or potent in vitro neutralizing activity.
Thus, in one embodiment, the modified antibody, including those described in Table 3, binds to C. difficile toxin A with a dissociation constant (KD) equal to or less than 10 pM (10−11 M), 1 pM (10−12 M), 0.1 pM (10−14 M), 0.01 pM (10−14 M), or 0.001 pM (10−15 M). In another embodiment, the modified antibody, including those described in Table 4, binds to C. difficile toxin B with a dissociation constant (KD) equal to or less than 250 pM, 200 pM, 150 pM, 100 pM, 50 pM, 30 pM, 10 pM, 1 pM (10−12 M), or 0.1 pM (10−13 M). The dissociation constant may be measured using techniques known in the art, including biolayer interferometry, as described in the examples of this application.
In one embodiment, the modified antibody, including those described in Table 3, neutralizes the in vitro cytotoxicity of C. difficile toxin A at 2.4 ng/mL in the Vero monkey kidney cell line with an NT50 equal to or less than 3000 pM, 2000 pM, 1000 pM, 100 pM, 60 pM, or 50 pM. In another embodiment, the modified antibody, including those described in Table 4, neutralizes the in vitro cytotoxicity of C. difficile toxin B at 17 pg/mL in the Vero monkey kidney cell line with an NT50 of equal to or less than 1000 pM, 100 pM, 60 pM, or 50 pM. For the sake of consistency, when measuring the neutralizing activity in the Vero monkey kidney cell line, Vero cells (2.5×104 cells/well with 5% heat-inactivated FBS) are seeded in a 96-well tissue culture microtiter plates and incubated 37° C., 5% CO2 overnight. An equal volume (80 μl) of 4.8 ng/mL (8×MC50) C. difficile toxin A solution or 34.4 pg/mL (8×MC50) C. difficile toxin B solution and individual dilutions of the antibody solutions (80 μl) in Vero cell medium are combined in a new 96-well plate, and incubated at 37° C., 5% CO2 for 1 hour before 100 μl of the toxin/antibody solutions are added to the Vero cells, and incubated at 37° C. for 72 hours. After incubating for 72 hours, the cells are washed twice with 120 μl/each of MEM medium that does not contain phenol, L-glutamine and FBS before adding 100 μl MEM medium that does not contain phenol, L-glutamine and FBS and 10 μl of Alamar Blue® (Life Technologies) to each well. The plates are lightly mixed and incubated at 37° C. for 4 hours before reading fluorescence at 560-590 nm with a cut off at 590 nm.
In one embodiment, the modified antibody, including those described in Table 3, neutralizes the C. difficile toxin A (at 200 ng/mL, applied apically) induced loss of transepithelial resistance (TEER) in the T-84 cell line with an NT50 equal to or less than 6 nM, 5 nM, 2 nM, or 1.5 nM. In another embodiment, the modified antibody, including those described in Table 4, neutralizes the C. difficile toxin B (at 75 ng/mL, applied basolaterally) induced loss of TEER in the T-84 cell line with an NT50 equal to or less than 200 pM, 150 pM, 100 pM, or 70 pM. For the sake of consistency, when measuring TEER, T-84 cells are seeded into 0.4 micron polyester transwell plates at a seeding density of 3.6×105 cells/cm2 and maintained at 37° C., 5% CO2 in 10% heat-inactivated FBS in DMEM/F12 culture media for 10-12 days until stable TEER is achieved and media is replaced in both apical and basolateral compartments of the transwells daily from day 6 and on the day of assay. The C. difficile toxin A (final concentration 200 ng/mL) or toxin B (final concentration of 75 ng/mL) is combined 1:1 with an antibody and incubated at 37° C. with gentle rocking for 30 minutes before replacing the media in the apical compartment with the toxin A/antibody samples or the media in the basolateral compartment with the toxin B/antibody samples. Transepithelial electrical resistance of the T-84 cells is measured at T0 immediately before sample addition and after 2.5 hours (To) incubation at 37° C. 5% CO2.
In one embodiment, the modified antibody is a monoclonal antibody, including, but not limited to, a bispecific antibody, that cross-blocks the binding of at least one of the A1, A2, A3, A4, or A5 antibodies to toxin A, using a routine quantitative cross-blocking assay, such as the Biacore assay discussed above. In one embodiment, the modified antibody is a monoclonal antibody, including, but not limited to, a bispecific antibody, that cross-blocks the binding of the A1 antibody to toxin A. In another embodiment, the modified antibody is a monoclonal antibody, including, but not limited to, a bispecific antibody, that cross-blocks the binding of the A2 antibody to toxin A. In another embodiment, the modified antibody is a monoclonal antibody, including, but not limited to, a bispecific antibody, that cross-blocks the binding of the A3 antibody to toxin A. In another embodiment, the modified antibody is a monoclonal antibody, including, but not limited to, a bispecific antibody, that cross-blocks the binding of the A4 antibody to toxin A. In another embodiment, the modified antibody is a monoclonal antibody, including, but not limited to, a bispecific antibody, that cross-blocks the binding of the A5 antibody to toxin A.
In one embodiment, the modified antibody is a monoclonal antibody, including, but not limited to, a bispecific antibody, that cross-blocks the binding of at least one of the B1, B2, B3, B4, B5, or B6 antibodies to toxin B, using a routine quantitative cross-blocking assay, such as the Biacore assay discussed above. In another embodiment, the modified antibody is a monoclonal antibody, including, but not limited to, a bispecific antibody, that cross-blocks the binding of the B1 antibody to toxin B. In another embodiment, the modified antibody is a monoclonal antibody, including, but not limited to, a bispecific antibody, that cross-blocks the binding of the B2 antibody to toxin B. In another embodiment, the modified antibody is a monoclonal antibody, including, but not limited to, a bispecific antibody, that cross-blocks the binding of the B3 antibody to toxin B. In another embodiment, the modified antibody is a monoclonal antibody, including, but not limited to, a bispecific antibody, that cross-blocks the binding of the B4 antibody to toxin B. In another embodiment, the modified antibody is a monoclonal antibody, including, but not limited to, a bispecific antibody, that cross-blocks the binding of the B5 antibody to toxin B. In another embodiment, the modified antibody is a monoclonal antibody, including, but not limited to, a bispecific antibody, that cross-blocks the binding of the B6 antibody to toxin B.
8. Nucleic Acids, Cloning and Expression Systems
The present disclosure further provides isolated nucleic acids encoding the A1, A2, A3, A4, A5, B1, B2, B3, B4, B5, and B6 antibodies or portions thereof. The nucleic acids may comprise DNA or RNA and may be wholly or partially synthetic or recombinant. Reference to a nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence, and encompasses a RNA molecule with the specified sequence in which U is substituted for T, unless context requires otherwise.
The nucleic acids provided herein encode at least one CDR, all six CDRs (i.e., H1, H2, H3, L1, L2, and L3), a VH domain, and/or a VL domain of one of the A1, A2, A3, A4, A5, B1, B2, B3, B4, B5, or B6 antibodies.
The present disclosure also provides expression vectors (or plasmids) comprising at least one nucleic acid encoding a CDR, all six CDRs (i.e., H1, H2, H3, L1, L2, and L3), a VH domain, and/or a VL domain of one of the A1, A2, A3, A4, A5, B1, B2, B3, B4, B5, or B6 antibodies, as well as other nucleic acid sequences useful for regulating polypeptide expression. Suitable expression vectors can be chosen or constructed, so that they contain appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
The expression vectors can be introduced into a host cell to produce the desired antibody. Systems for cloning and expression of a polypeptide in a variety of different host cells are well known in the art. For cells suitable for producing antibodies, see Gene Expression Systems, Academic Press, eds. Fernandez et al., 1999. Any protein compatible expression system may be used to produce the disclosed antibodies. Suitable expression systems include transgenic animals described in Gene Expression Systems, Academic Press, eds. Fernandez et al., 1999.
A further aspect of the disclosure provides an isolated host cell comprising a nucleic acid (or expression vector) as disclosed herein. A still further aspect provides a method comprising introducing such nucleic acid (or expression vector) into a host cell. The introduction may employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g., vaccinia or, for insect cells, baculovirus. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage. The introduction of the nucleic acid into the cells may be followed by causing or allowing expression from the nucleic acid, e.g., by culturing host cells under conditions for expression of the gene. Following production by expression an antibody may be isolated and/or purified using any suitable technique, then used as appropriate.
9. Methods of Making Antibodies
Numerous methods known to those skilled in the art are available for obtaining antibodies or antigen-binding fragments thereof. Antibodies can also be produced using recombinant DNA methods. See, e.g., U.S. Pat. No. 4,816,567, EPO 8430268.0; EPO 85102665.8; EPO 85305604.2; PCT/GB 85/00392; EPO 85115311.4; PCT/US86/002269; and Current Trends in Monoclonal Antibody Development (Steven Shire et al., Eds. Springer, 2010), the disclosures of which are incorporated herein by reference in their entirety. Given the disclosure in this application of specific nucleic acid sequences and the VH and VL (or CDR) amino acid sequences encoded thereby, it is possible, using recombinant DNA techniques, to insert a nucleic acid of interest into an expression vector or otherwise express the nucleic acid of interest in a host cell to produce the desired antibody. In addition, as disclosed elsewhere in this application, modified versions of the antibodies described herein can be produced using known techniques, including, for example, random mutagenesis, error-prone PCR, and direct mutagenesis.
Monoclonal antibodies may also be produced by preparing immortalized cell lines capable of producing antibodies having desired specificity, for example against an antigen expressing a desired epitope, such as the specific C. difficile toxin A and B epitopes disclosed in this application. Such immortalized cell lines may be produced in a variety of ways. Conveniently, a small non-human animal, such as a mouse, is hyperimmunized with the desired immunogen. The vertebrate is then sacrificed, usually several days after the final immunization, the spleen cells removed, and the spleen cells immortalized. The most common technique is fusion with a myeloma cell fusion partner, as first described by Kohler and Milstein (1975) Nature 256:495-497. Other techniques, including EBV transformation, transformation with bare DNA, e.g., oncogenes, retroviruses, etc., or any other method which provides for stable maintenance of the cell line and production of monoclonal antibodies. Specific techniques for preparing monoclonal antibodies are described in Antibodies: A Laboratory Manual, Harlow and Lane, eds., Cold Spring Harbor Laboratory, 1988, the full disclosure of which is incorporated herein by reference.
In one embodiment, the non-human animal includes at least a part of a human immunoglobulin gene. For example, it is possible to engineer transgenic mouse strains that express human heavy and light chain genes, but are incapable of expressing the endogenous mouse immunoglobulin heavy and light chain genes. Using the hybridoma technology, antigen-specific monoclonal antibodies derived from the genes with the desired specificity may be produced and selected. See, e.g., XENOMOUSE™, Green et al. (1994) Nature Genetics 7:13-21, US 2003-0070185, U.S. Pat. No. 5,225,539, WO 96/34096, published Oct. 31, 1996, and PCT Application No. PCT/US96/05928, filed Apr. 29, 1996, the disclosures of which are incorporated herein by reference in their entirety.
Immortalized cell lines can be screened using standard methods, such as enzyme-linked immunosorbent assay (ELISA) or surface plasmon resonance analysis, to identify one or more hybridomas that produce an antibody that specifically binds with a specified antigen and/or epitope. Any form of the specified antigen may be used as the immunogen, e.g., recombinant antigen, naturally occurring forms, any variants or fragments thereof, as well as antigenic peptide thereof.
Another exemplary method of making antibodies includes screening protein expression libraries, e.g., phage or ribosome display libraries. Phage display technology mimics the mammalian immune system by cloning large libraries of antibody genes and selecting for binding to a desired target, such as the specific C. difficile toxin A and B epitopes disclosed in this application. Phage display is described, for example, in Ladner et al., U.S. Pat. No. 5,223,409; Smith (1985) Science 228:1315-1317; Clackson et al. (1991) Nature, 352: 624-628; Marks et al. (1991) J. Mol. Biol., 222: 581-597WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; and WO 90/02809, the disclosures of which are incorporated herein by reference in their entirety. It is also possible to produce antibodies that bind a specific antigen, such as one of the specific C. difficile epitopes disclosed in this application, by using a variable heavy domain (e.g., SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:40, SEQ ID NO:58, SEQ ID NO:76, SEQ ID NO:94, SEQ ID NO:112, SEQ ID NO:130, SEQ ID NO:148, SEQ ID NO:166, or SEQ ID NO:184) and screening a library of complimentary variable domains to identify antibodies that retain the desired binding specificity. See Portolano et al., The Journal of Immunology (1993) 150:880-887 and Clarkson et al., Nature (1991) 352:624-628, the disclosures of which are incorporated herein by reference in their entirety.
10. Methods of Use
The antibodies described in this application that bind to C. difficile toxin A or toxin B can be used in a variety of research and medical applications. In one aspect, the disclosure provides a method of treating a C. difficile infection in a subject, comprising administering to the subject one or more of the A1, A2, A3, A4, A5, B1, B2, B3, B4, B5, or B6 antibodies in an amount effective to treat the C. difficile infection. In another embodiment, the method of treating a C. difficile infection in a subject, comprises administering at least one antibody that binds to C. difficile toxin A selected from the A1, A2, A3, A4, and A5 antibodies and at least one antibody that binds to C. difficile toxin B selected from the B1, B2, B3, B4, B5, and B6 antibodies. In one embodiment, the at least one antibody that binds to C. difficile toxin A is the A2 antibody and the at least one antibody that binds to C. difficile toxin B is one or more of the B1, B2, B3, B4, B5, and B6 antibodies, preferably one or more of B1, B2, or B4. In another embodiment, the method comprises administering the A2 antibody and at least two antibodies that binds to C. difficile toxin B, wherein the at least two antibodies that binds to C. difficile toxin B are the B1 and B2 antibodies, the B2 and B4 antibodies, or the B2 and B6 antibodies. In another embodiment, the method comprises administering the 1) A2 antibody and 2) a bispecific B1+B2 antibody, a bispecific B2+B4 antibody, or a bispecific B2+B6 antibody. The antibodies may be administered at the same time or sequentially.
In another embodiment, the method of treating a C. difficile infection comprises administering a composition to the subject in an amount effective to treat the C. difficile infection, wherein the composition comprises at least one antibody that binds to C. difficile toxin A selected from the A1, A2, A3, A4, and A5 antibodies and at least one antibody that binds to C. difficile toxin B selected from the B1, B2, B3, B4, B5, and B6 antibodies. In another embodiment, the composition comprises at least one antibody that binds to C. difficile toxin A and at least one antibody that binds to C. difficile toxin B selected from the B1, B2, B3, B4, B5, and B6 antibodies. In another embodiment, the composition comprises at least one antibody that binds to C. difficile toxin A selected from the A1, A2, A3, A4, and A5 antibodies and at least one antibody that binds to C. difficile toxin B.
In one embodiment, the at least one antibody that binds to C. difficile toxin A is the A2 antibody and the at least one antibody that binds to C. difficile toxin B is one or more of the B1, B2, B3, B4, B5, and B6 antibodies, preferably one or more of B1, B2, or B4. In one embodiment, the composition comprises the A2 antibody and the B4 antibody. In another embodiment, the composition comprises the A2 antibody and the B2 antibody. In another embodiment, the composition comprises the A2 antibody and the B1 antibody.
In another embodiment, the composition comprises the A2 antibody and two antibodies that bind to C. difficile toxin B, wherein the two antibodies that bind to C. difficile toxin B are selected from the B1, B2, B3, B4, B5, and B6 antibodies. In one embodiment, the composition comprises the A2, B1, and B2 antibodies. In another embodiment, the composition comprises the A2, B2, and B4 antibodies. In another embodiment the composition comprises the A2, B2, and B6 antibodies.
In yet another embodiment, the composition comprises the 1) A2 antibody and 2) a bispecific B1+B2 antibody, a bispecific B2+B4 antibody, or a bispecific B2+B6 antibody.
Subjects that can be treated with the antibodies disclosed in this application include humans and non-human mammals, including, but not limited to, non-human primates, dogs, cats, horses, cows, sheep, pigs, goats, mice, rats, hamsters, and guinea pigs.
In addition, one or more of the A1, A2, A3, A4, and A5 antibodies can be used to detect C. difficile toxin A in a sample, while one or more of the B1, B2, B3, B4, B5, or B6 antibodies can be used to detect C. difficile toxin B in a sample. In one embodiment, the method comprises contacting one or more of the A1, A2, A3, A4, A5, B1, B2, B3, B4, B5, or B6 antibodies with the sample and analyzing the sample to detect binding of the antibody to toxin A or toxin B in the sample, wherein binding of the antibody to toxin A or toxin B in the sample indicates the presence of C. difficile in the biological sample. In one embodiment, the sample comprises a non-biological sample, such as soil, water, or food products such as meat. In other embodiments, the sample comprises a biological sample, such as blood, serum, tissue, or stool. Such methods can be used to detect a C. difficile infection in a patient, wherein binding of the antibody to toxin A or toxin B in a sample from the patient indicates the presence of the C. difficile infection in the patient.
Any appropriate label may be used in the detection methods and compositions described herein. A label is any molecule or composition bound to an antibody, or a secondary molecule that is conjugated thereto, and that is detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Examples of labels, including enzymes, colloidal gold particles, colored latex particles, have been disclosed (U.S. Pat. Nos. 4,275,149; 4,313,734; 4,373,932; and 4,954,452, each incorporated by reference herein). Additional examples of useful labels include, without limitation, haptens (e.g., biotin, digoxigenin (DIG), dintrophenol (DNP), etc.), radioactive isotopes, co-factors, ligands, chemiluminescent or fluorescent agents, protein-adsorbed silver particles, protein-adsorbed iron particles, protein-adsorbed copper particles, protein-adsorbed selenium particles, protein-adsorbed sulphur particles, protein-adsorbed tellurium particles, protein-adsorbed carbon particles, and protein-coupled dye sacs. The attachment of a compound to a label can be through any means, including covalent bonds, adsorption processes, hydrophobic and/or electrostatic bonds, as in chelates and the like, or combinations of these bonds and interactions and/or may involve a linking group.
11. Formulations and Administration
The disclosure provides compositions comprising an antibody described herein that binds to C. difficile toxin A or toxin B. In certain embodiments, the compositions are suitable for pharmaceutical use and administration to patients. These compositions comprise one or more of the A1, A2, A3, A4, A5, B1, B2, B3, B4, B5, or B6 antibodies and a pharmaceutically acceptable excipient. In one embodiment, the composition comprises at least one antibody that binds to C. difficile toxin A selected from the A1, A2, A3, A4, and A5 antibodies and at least one antibody that binds to C. difficile toxin B. In another embodiment, the composition comprises at least one antibody that binds to C. difficile toxin A and at least one antibody that binds to C. difficile toxin B, wherein the at least one antibody that binds to C. difficile toxin B is preferably one or more of the B1, B2, B3, B4, B5, and B6 antibodies.
In one embodiment, the at least one antibody that binds to C. difficile toxin A is the A2 antibody or A1 antibody and the at least one antibody that binds to C. difficile toxin B is one or more of the B1, B2, B3, B4, B5, or B6 antibodies, preferably one or more of B1, B2, or B4. In one embodiment, the composition comprises the A2 antibody and the B4 antibody. In another embodiment, the composition comprises the A2 antibody and the B2 antibody. In another embodiment, the composition comprises the A2 antibody and the B1 antibody. In another embodiment, the composition comprises the A1 antibody and the B1 antibody. In another embodiment, the composition comprises the A1 antibody and the B2 antibody. In another embodiment, the composition comprises the A1 antibody and the B4 antibody. In another embodiment, the composition comprises the A1 antibody and the B6 antibody.
In another embodiment, the composition comprises the A2 antibody or A1 antibody and two antibodies that bind to C. difficile toxin B, wherein the two antibodies that bind to C. difficile toxin B are selected from the B1, B2, B3, B4, B5, and B6 antibodies. In one embodiment, the composition comprises the A2, B1, and B2 antibodies. In another embodiment, the composition comprises the A2, B2, and B4 antibodies. In another embodiment the composition comprises the A2, B2, and B6 antibodies. In yet another embodiment, the composition comprises the 1) A2 antibody and 2) a bispecific B1+B2 antibody, a bispecific B2+B4 antibody, or a bispecific B2+B6 antibody. In one embodiment, the composition comprises the A1, B1, and B2 antibodies. In another embodiment, the composition comprises the A1, B2, and B4 Antibodies. In another embodiment the composition comprises the A1, B2, and B6 antibodies. In yet another embodiment, the composition comprises the 1) A1 antibody and 2) a bispecific B1+B2 antibody, a bispecific B2+B4 antibody, or a bispecific B2+B6 antibody.
In one embodiment, the composition comprises one or more of the A1, A2, A3, A4, A5, B1, B2, B3, B4, B5, or B6 antibodies for use in treating a C. difficile infection. Preferably, the composition comprises at least one antibody that binds to C. difficile toxin A selected from the A1, A2, A3, A4, and A5 antibodies and at least one antibody that binds to C. difficile toxin B selected from the B1, B2, B3, B4, B5, and B6 antibodies for use in treating a C. difficile infection. In one embodiment, the composition comprises the A2 antibody or A1 antibody and the at least one antibody that binds to C. difficile toxin B selected from the B1, B2, B3, B4, B5, or B6 antibodies, preferably one or more of B1, B2, or B4 for use in treating a C. difficile infection. In one embodiment, the composition comprises the A2 antibody and the B4 antibody for use in treating a C. difficile infection. In another embodiment, the composition comprises the A2 antibody and the B2 antibody for use in treating a C. difficile infection. In another embodiment, the composition comprises the A2 antibody and the B1 antibody for use in treating a C. difficile infection. In another embodiment, the composition comprises the A1 antibody and the B4 antibody for use in treating a C. difficile infection. In another embodiment, the composition comprises the A1 antibody and the B2 antibody for use in treating a C. difficile infection. In another embodiment, the composition comprises the A1 antibody and the B1 antibody for use in treating a C. difficile infection. In another embodiment, the composition comprises the A1 antibody and the B6 antibody for use in treating a C. difficile infection.
In yet another embodiment, the composition comprises the A2 or A1 antibody and two antibodies that bind to C. difficile toxin B for use in treating a C. difficile infection, wherein the two antibodies that bind to C. difficile toxin B are selected from the B1, B2, B3, B4, B5, and B6 antibodies. In one embodiment, the composition comprises the A2 antibody, the B1 antibody, and the B2 antibody for use in treating a C. difficile infection. In another embodiment, the composition comprises the A2 antibody, the B2 antibody, and the B4 antibody for use in treating a C. difficile infection. In another embodiment, the composition comprises the A2 antibody, the B2 antibody, and the B6 antibody for use in treating a C. difficile infection. In yet another embodiment, the composition comprises the 1) A2 antibody and 2) a bispecific B1+B2 antibody, a bispecific B2+B4 antibody, or a bispecific B2+B6 antibody for use in treating a C. difficile infection. In one embodiment, the composition comprises the A1 antibody, the B1 antibody, and the B2 antibody for use in treating a C. difficile infection. In another embodiment, the composition comprises the A1 antibody, the B2 antibody, and the B4 antibody for use in treating a C. difficile infection. In another embodiment, the composition comprises the A1 antibody, the B2 antibody, and the B6 antibody for use in treating a C. difficile infection. In yet another embodiment, the composition comprises the 1) A1 antibody and 2) a bispecific B1+B2 antibody, a bispecific B2+B4 antibody, or a bispecific B2+B6 antibody for use in treating a C. difficile infection.
The compositions may also contain other active compounds providing supplemental, additional, or enhanced therapeutic functions. In one embodiment, the other active compound is an antibiotic, including, but not limited to, metronidazole, fidaxomicin, or vanomycin. The pharmaceutical compositions may also be included in a container, pack, or dispenser together with instructions for administration.
Pharmaceutically acceptable excipients include, but are not limited to a carrier or diluent, such as a gum, a starch (e.g. corn starch, progeletanized starch), a sugar (e.g. lactose, mannitol, sucrose, dextrose), a cellulosic material (e.g. microcrystalline cellulose), an acrylate (e.g. polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof; a binder (e.g. acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone); a disintegrating agent (e.g. cornstarch, potato starch, alginic acid, silicon dioxide, croscarmelose sodium, crospovidone, guar gum, sodium starch glycolate), a buffer (e.g. Tris-HCl, acetate, phosphate) of various pH and ionic strength; and additive such as albumin or gelatin to prevent absorption to surfaces; a detergent (e.g. Tween 20, Tween 80, Pluronic F68, bile acid salts); a protease inhibitor, a surfactant (e.g. sodium lauryl sulfate); a permeation enhancer; a solubilizing agent (e.g. glycerol, polyethylene glycerol); an anti-oxidants (e.g. ascorbic acid, sodium metabisulfite, butylated hydroxyanisole); a stabilizer (e.g. hydroxypropyl cellulose, hydroxypropylmethyl cellulose); a viscosity increasing agent (e.g. carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum); a sweetener (e.g. aspartame, citric acid); a preservative (e.g. Thimerosal, benzyl alcohol, parabens); a lubricant (e.g. stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate); a flow-aid (e.g. colloidal silicon dioxide), a plasticizer (e.g. diethyl phthalate, triethyl citrate); an emulsifier (e.g. carbomer, hydroxypropyl cellulose, sodium lauryl sulfate); a polymer coating (e.g. poloxamers or poloxamines); a coating and film forming agent (e.g. ethyl cellulose, acrylates, polymethacrylates); an adjuvant; a pharmaceutically acceptable carrier for liquid formulations, such as an aqueous (water, alcoholic/aqueous solution, emulsion or suspension, including saline and buffered media) or non-aqueous (e.g., propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate) solution, suspension, emulsion or oil; and a parenteral vehicle (for subcutaneous, intravenous, intraarterial, or intramuscular injection), including but not limited to, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils.
Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Examples are sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants. In general, water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycols or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions. Examples of oils are those of animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, olive oil, sunflower oil, fish-liver oil, another marine oil, or a lipid from milk or eggs.
A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Methods to accomplish the administration are known to those of ordinary skill in the art. This includes, for example, injections, by parenteral routes such as intravenous, intravascular, intraarterial, subcutaneous, intramuscular, intraperitoneal, intraventricular, intraepidural, or others as well as oral, nasal, ophthalmic, rectal, or topical.
Sustained release administration is also specifically contemplated, by such means as depot injections or erodible implants. Localized delivery is particularly contemplated, by such means as delivery via a catheter to one or more arteries, such as the renal artery or a vessel supplying a localized site of interest.
In one embodiment a subject antibody is administered to a patient by intravenous, intramuscular or subcutaneous injection. An antibody may be administered within a dose range between about 1 μg/kg to about 100 mg/kg. A therapeutically effective amount of antibody may include, but is not limited to, dosage ranges of about 0.1 mg/kg to about 100 mg/kg; 0.1 mg/kg to about 10 mg/kg; about 0.5 mg/kg to 75 mg/kg; 1 mg/kg to about 50 mg/kg; 1 mg/kg to about 10 mg/kg; 0.5 mg/kg to about 25 mg/kg; or about 1 mg/kg to about 5 mg/kg. The antibody may be administered, for example, by bolus injunction or by slow infusion. The dosage may depend on the type and severity of the infection and/or on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs and should be adjusted, as needed, according to individual need and professional judgment. The dosage may also vary depending upon factors, such as route of administration, target site, or other therapies administered. The skilled artisan will be able to determine appropriate doses depending on these and other factors.
Toxicity and therapeutic efficacy of the composition can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Antibodies that exhibit large therapeutic indices may be less toxic and/or more therapeutically effective.
12. Kits
In some embodiments, at least one antibody described herein that binds to C. difficile toxin A or toxin B is supplied in the form of a kit useful, for example, for performing the treatment or diagnostic methods described in this application. In one embodiment, an appropriate amount of one or more of the A1, A2, A3, A4, A5, B1, B2, B3, B4, B5, or B6 antibodies is provided in one or more containers. In other embodiments, one or more of the A1, A2, A3, A4, A5, B1, B2, B3, B4, B5, or B6 antibodies is provided suspended in an aqueous solution or as a freeze-dried or lyophilized powder, for instance. The container(s) in which the at least one antibody is supplied can be any conventional container that is capable of holding the supplied form, for instance, microfuge tubes, ampoules, or bottles. The amount of antibody supplied can be any appropriate amount.
Other kit embodiments include means for detecting one or more of the A1, A2, A3, A4, A5, B1, B2, B3, B4, B5, or B6 antibodies, such as secondary antibodies. In some such instances, the secondary antibody is directly labeled with a detectable moiety (as described elsewhere in this disclosure). In other instances, the primary or secondary (or higher-order) antibody is conjugated to a hapten (such as biotin, DNP, DIG, etc.), which is detectable by a detectably labeled cognate hapten-binding molecule (e.g., streptavidin (SA)-horse radish peroxidase, SA-alkaline phosphatase, SA-QDot® (Invitrogen, Carlsbad, Calif.), etc.). In some embodiments, the primary or secondary antibody in conjugated with a fluorescent detection moiety (e.g., FITC, rhodamine, ALEXA FLUOR® (Invitrogen, Carlsbad, Calif.) dyes, Cy designated fluorophores, etc.). Some kit embodiments may include colorimetric reagents (e.g., DAB, AEC, etc.) in suitable containers to be used in concert with primary or secondary (or higher-order) antibodies that are labeled with enzymes for the development of such colorimetric reagents.
In one embodiment, a kit includes instructional materials disclosing methods of use of the kit contents (e.g., an antibody described herein that binds to C. difficile toxin A or toxin B) in a disclosed method. The instructional materials may be provided in any number of forms, including, but not limited to, written form (e.g., hardcopy paper, etc.), in an electronic form (e.g., computer diskette or compact disk) or may be visual (e.g., video files). The kits may also include additional components to facilitate the particular application for which the kit is designed. Thus, for example, the kits may additionally include buffers and other reagents routinely used for the practice of a particular method. Such kits and appropriate contents are well known to those of skill in the art.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Blood Donor Selection. Serum was collected from 3000 healthy donors and assessed for its capacity to neutralize C. difficile toxin A and/or toxin B by cytotoxicity assay on IMR90 cells as described by Babcock et al. (Infection and Immunity, November 2006, p. 6339-6347). Briefly, IMR90 cells were seeded in a 96 well plate (half size well plates) at a cell density of 1×104 cells/well in a 50 μl volume. The plates were incubated for 24 hours at 37° C., 5% CO2, before removing the supernatant from the wells. Sera were diluted in IMR90 cell culture medium 1/25 and 1/100 for toxin A and 1/100 and 1/500 for toxin B and incubated for 60 minutes with either 4×MC50 of toxin A or 2×MC50 of toxin B. This mixture was then added to the wells of the 96 well plate and incubated for 16-24 hours at 37° C., 5% CO2 before assessing the cytopathic effect. The cytopathic effect was determined microscopically and scored as 0 (0% rounded cells), 1 (25% rounded cells), 2 (50% rounded cells), 3 (75% rounded cells), 4 (100% rounded cells). Sera exhibiting a neutralizing activity were further tested with the same assay in a series of dilutions ranging from 1/25 to 1/3200. In parallel, neutralizing sera were also tested by ELISA to determine their titers against both toxins, as well as their cross-reactivity for the toxinotypes 0, III, V, VIII, XII, XIV, and XV. Peripheral Blood Mononuclear Cells (PBMCs) from the 12 best donors were used for the screening campaigns to maximize the probability to isolate B cells secreting high quality antibodies.
Antigens. Untoxoided C. difficile toxins A and B were purified from the supernatant of a culture of strain VPI10463 (ATCC 43255).
Antibody Screening. The A2, A4, A5, B6, B4 antibodies were obtained by implementing the method described in Jin et al. (2011, Nature Protocols Vol.6, N° 5 pp668-676) and named ISAAC (ImmunoSpot Array Assay on a Chip). In brief, C. difficile toxin A or B antigen was coated on the chip. The anti-toxin A or B human antibodies, secreted from wells containing one human B cell, diffuse onto the chip surface and bind to the antigen coated on the chip surface. Bound antibodies were visualized using Cyanine 3anti-human IgG monoclonal antibody. The B cells secreting specific anti-toxin A or B antibodies were isolated and the heavy and light chains of human monoclonal antibodies were obtained by single cell reverse transcriptase polymerase chain reaction (RT-PCR). The amplified VH and VL fragments were subsequently cloned into expression vectors for production in Human Embryonic Kidney (HEK) 293T cells or Chinese Hamster Ovary Cells and recombinant antibody testing.
The A1, A3, B1, B2, B3, B5 antibodies were obtained by implementing the method described in the patent application WO2013/000982 named VIVASCREEN. B-lymphocytes from the best donors were isolated, activated and expanded in vitro. Supernatants from expanded B-lymphocyte pools were then screened using a binding assay against C. difficile toxin A or toxin B. B-cell pools that secreted antibodies against toxin A or B were further screened with a functional assay. Notably, the functional hits were identified using a cytotoxicity assay on IMR90 cells as described by Babcock et al. 2006. Briefly, IMR90 cells were seeded in a 96 well plate (half size well plates) at a cell density of 1×10E4 cells/well in a 50 μl volume. The plates were incubated during 24 hours at 37° C., 5% CO2, before removing the supernatant from the wells. 150 μl of supernatants from each of the expanded B cell pools were incubated for 60 minutes with either 8×MC50 of toxin A or 2×MC50 of B. This mixture was then added to the wells of the 96 well plate and incubated for 16-24 hours at 37° C.+CO2 before assessing the cytopathic effect. The cytopathic effect was determined microscopically and scored as 0 (0% rounded cells), 1 (25% rounded cells), 2 (50% rounded cells), 3 (75% rounded cells), 4 (100% rounded cells).
The single B cells secreting specific anti-toxin A or B antibodies from each relevant B-cell pool were further isolated by the ISAAC (ImmunoSpot Array Assay on a Chip) method on toxin A- or toxin B-coated chips, and the heavy and light chains of the monoclonal antibodies were obtained by single cell reverse transcriptase polymerase chain reaction (RT-PCR). The amplified VH and VL fragments were subsequently cloned into expression vectors for production and further recombinant antibody characterization. Using these processes, tens of millions of antibody producing B lymphocytes were screened and characterized to identify extremely rare antibodies having high binding affinity for and strong neutralizing activity against either C. difficile toxin A or C. difficile toxin B, and preferably with a broad spectrum of binding to strains of various C. difficile toxinotypes, such as 0, III, V, VIII, XII, and XV.
The binding affinities of all antibodies were determined by Bio-Layer Interferometry using a Octet® Red96 (FortéBio). Bio-Layer Interferometry is an optical analytical technique that analyzes the interference pattern of white light reflected from two surfaces: a layer of immobilized protein (e.g., antibody) on the biosensor tip, and an internal reference layer. Any change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern that can be measured in real-time. Abdiche et al., Analytical Biochemistry, 2009, 386(2), 172-180.
Sensors coated with Protein A were first wet for 10 minutes in FortéBio kinetics buffer (KB), PBS pH 7.4 containing 0.002% Tween-20 and 0.1 mg/ml BSA. The sensors were then transferred to wells containing antibody at a concentration of 10 μg/ml in KB and the accumulation of antibody on the sensors was measured for 300 seconds. The sensors were then transferred to wells containing KB for 300 seconds to wash off contaminants. Binding of toxin to the antibody was initiated by transferring the antibody-coated sensors into wells containing C. difficile toxin A or B at concentrations ranging from 0.7 to 20 μg/ml. After 400 seconds, the sensors were moved into KB buffer for 900 seconds to monitor the dissociation of the bound toxin. Throughout the experiment, samples were agitated at 30° C. and 1000 rpm. The changes in thickness of the protein layer over time at four different concentrations of toxin were fit to the equations of equilibrium kinetics to calculate association and dissociation constants. The dissociation constants, on rate constants, and off rate constants of the anti-toxin A and anti-toxin B antibodies are provided in Table 5.
The KD of the A3 and A5 antibodies for toxin A was 461 pM and 144 pM, respectively. The KD of the A1, A2, and A4 antibodies for toxin A are shown in Table 5. All were less than 10 pM. The lower limit of quantification by the Octet® Red96 (FortéBio) system is about 10 pM. These results demonstrate that the A1, A2, A3, A4, and A5 antibodies bound C. difficile toxin A with at least picomolar affinities. Some of the antibody affinities may be in the subpicomolar range if measured with a more sensitive system.
The KD of the B1, B2, B3, B4, and B5 antibodies for toxin B was 48, 80, 487, 121, and 202 pM, respectively. The KD of the B6 antibody for toxin B is shown in Table 5 and was less than the lower limit of quantification by the Octet® Red96 (FortéBio) system, which is about 10 pM. These results demonstrate that the B1, B2, B3, B4, B5, and B6 antibodies bound C. difficile toxin B with at least picomolar affinities. Some of the antibody affinities may be in the subpicomolar range if measured with a more sensitive system.
Cell-based neutralization assays in either Vero monkey kidney cells or T-84 human colon epithelial cell monolayer were used to evaluate the ability of the anti-toxin A and anti-toxin B antibodies to neutralize the activity of toxin A or toxin B. The first assay uses Vero cells, a cell line which was derived from the kidney of a normal adult African green monkey. The Vero cell assay assesses the ability of anti-toxin A or anti-toxin B antibodies to inhibit toxin A or toxin B induced killing of Vero cells. This assay uses an Alamar Blue® (Life Technologies) readout to assess cell viability. Resazurin, the active ingredient of Alamar Blue® (Life Technologies), is a non-toxic, cell permeable blue compound. Only living cells are able to reduce Resazurin to a red fluorescent compound, consequently viable cell number is directly proportional red fluorescence. Therefore the lower the fluorescence reading, the fewer viable cells present.
Vero cells (2.5×104 cells/well with 5% heat-inactivated FBS) were seeded in a 96-well tissue culture microtiter plates and incubated 37° C., 5% CO2 overnight. Stock solutions of 8×MC50 (concentration inducing 50% of the maximum response) C. difficile toxin A or B were prepared in Vero cell medium. One MC50 dose was 0.6 ng/mL and 4.3 pg/mL for toxin A and B, respectively.
Various dilutions of the A1, A2, A3, A4, A5, B1, B2, B3, B4, B5, or B6 antibodies were prepared in Vero cell medium and added to a 96-well tissue culture plate. An equal volume (80 μl) of 8×MC50 C. difficile toxin A or toxin B solution and individual dilutions of the antibody solutions (80 μl) were combined in a new 96-well tissue culture plate, and incubated at 37° C. with 5% CO2 and humidity for 1 hour with appropriate controls (toxin A or B without antibody or media). The resulting toxin/antibody solution has a toxin A or B concentration of 4×MC50. After incubating for 1 hour, 100 μl of the toxin/antibody solutions was added to the Vero cells in 96-well tissue culture microtiter plates. The Vero cells were incubated with the toxin/antibody solution at 37° C. for 72 hours.
After incubating for 72 hours of incubation, the cells were washed twice with 120 μl/each of MEM medium that does not contain phenol, L-glutamine and FBS. Next 100 μl MEM medium that does not contain phenol, L-glutamine & FBS and 10 μl of Alamar Blue® (Life Technologies) was added to each well. The plates were lightly mixed and incubated at 37° C. for 4 hours before reading fluorescence at 560-590 nm with a cut off at 590 nm.
Percent survival was plotted over antibody concentration. Cell survival in toxin/antibody treated cells was compared to cells treated with toxin A or B without antibody and NT50 was calculated for each antibody. NT50 is the concentration of antibody that results in 50% reduction in survival as compared to control cells treated with toxin A or B but no antibody.
The results obtained with the A1, A2, A3, A4, A5, B1, B2, B3, B4, B5, and B6 antibodies are summarized in Table 6, depicting both the potency of the neutralizing activity (Cytotox NT50) and the percent completion of the antibody-induced neutralization (% completion cytotox).
All five anti-toxin A antibodies exhibited a cytotoxic NT50 of less than 3500 pM. The A1, A2, A3, and A5 exhibited a cytotoxic NT50 of less than 3000 pM, and remarkably A1, A2, and A3 exhibited a cytotoxic NT50 of less than 1000 pM, with A2 and A1 showing the greatest potency at 55 pM and 48 pM, respectively. All five anti-toxin A antibodies also showed a high completion percentage of at least 75%, with A2 and A3 showing the greatest percent completion of 100% and 90%, respectively.
The anti-toxin B antibodies similarly exhibited high potency in the Vero cell neutralization assay, with all but the B5 antibody having a cytotoxic NT50 of 100 pM or less (B6 and B4 were tested as a combination). All six anti-toxin B antibodies also showed a high completion percentage of at least 75%, with B1, B3, B5, and B6 and B4 (tested as a combination) having a percent completion of at least 95%. The results of the Vero cell neutralization assays for the anti-toxin A and anti-toxin B antibodies are also graphically illustrated in
The second cell-based neutralization assay uses a T-84 human carcinoma cell line derived from a lung metastasis of a colon carcinoma (ATCC CCL-248). This assay assesses the ability of anti-toxin A or anti-toxin B antibodies to inhibit toxin A or toxin B induced loss of transepithelial electrical resistance (TEER) in T-84 cells.
T-84 cells were seeded into 0.4 micron polyester transwell plates at a seeding density of 3.6×105 cells/cm2 and maintained at 37° C., 5% CO2 in 10% heat-inactivated FBS in DMEM/F12 culture media for 10-12 days until stable TEER was achieved. Transepithelial electrical resistance was measured using Millipore Millicell® ERS-2 Volt-Ohm Meter. Media was replaced in both apical and basolateral compartments daily from day 6 and on the day of assay. Final concentration of toxin A used for challenge dose was equivalent to 6 times challenge dose required to produce loss of transepithelial resistance of 50% (TER50). One TER50 dose was 33 ng/mL and 15 ng/mL for toxin A and toxin B, respectively. Toxin A challenge was performed in the apical compartment of the transwell. The final concentration of toxin B used for challenge dose was equivalent to 5 times TER50. Toxin B challenge was performed in basolateral compartment.
Toxin A or toxin B and one of the A1, A2, A3, A4, A5, B1, B2, B3, B4, B5, or B6 antibodies were combined at 1:1 ratio and incubated at 37° C. with gentle rocking for 30 minutes, with appropriate controls (toxin A or B without antibody or media). Media was removed from the appropriate apical or basolateral compartment and the toxin/antibody samples were added to the T-84 cells in the transwell plates. Transepithelial resistance of the T-84 cells is measured at T0 immediately before sample addition and after 2.5 hours (T150) incubation at 37° C. 5% CO2.
Percent TEER loss is calculated for each sample using the following equation: % TEER loss=[(T0−T150)÷T0]*100%−% TEER loss Negative well. Percent protection for antibody is calculated for each treatment using the following equation: % Protection=[(% TEER loss Toxin Challenge)−(% TEER loss Toxin with Treatment)].
Percent TEER loss was plotted over antibody concentration. TEER loss in toxin/antibody treated cells was compared to cells treated with toxin A or B without antibody and NT50 was calculated for each antibody. NT50 is the concentration of antibody that results in 50% reduction in TEER loss as compared to control cells treated with toxin A or B but no antibody.
The results obtained with the A1, A2, A3, A4, A5, B1, B2, B3, B4, B5, and B6 antibodies are summarized in Table 7, depicting both the potency of the neutralizing activity (TEER NT50) and the percent completion of the antibody-induced neutralization (% completion TEER).
All five anti-toxin A antibodies exhibited a TEER NT50 of less than 10 nM. The A1, A2, A3, and A4 antibodies exhibited a TEER NT50 of less than 5 nM, and remarkably A1, A2, and A3 exhibited a TEER NT50 of less than 2 nM. The A1, A2, and A3 antibodies also showed a high completion percentage (TEER) of at least 75%. The plateau for the A4 and A5 antibodies was never reached.
The anti-toxin B antibodies similarly exhibited high potency in the TEER neutralization assay, with all but the B3 antibody having a TEER NT50 of 300 pM or less, and the B2, B4, B5, and B6 antibodies having a TEER NT50 of 200 pM or less, with B6, B4, and B2 showing the greatest potency at 100 pM, 130 pM, and 70 pM, respectively. All 6 anti-toxin B antibodies also showed a high completion percentage of at least 70%, with B1, B2, B3, B4, and B6 having a percent completion of at least 90%. The results of the TEER neutralization assays for the anti-toxin A and anti-toxin B antibodies are also graphically illustrated in
The breadth of protection against various C. difficile toxinotypes was assessed using two different assays. In the first, toxinotype binding was measured using Western analysis. C. difficile strains representative of toxinotypes 0, III, V, VIII, XII, and XV were grown anaerobically at 250 ml scale. The representative strain of toxinotype 0 is VPI10463 or ATCC 43255, the C. difficile reference strain. The representative toxinotype III strain (CDC#2005099) is a hypervirulent NAP1/027 strain isolated from an outbreak in Montreal. The representative toxinotype V strain is CDC#2004255. The representative toxinotype VIII strain is CDC#2005195. The representative toxinotype XII strain is CDC#2004097. The representative toxinotype XV strain is CDC#2004012.
The supernatants were recovered by tangential flow filtration through a 0.2 μm membrane and adjusted to 0.4 M ammonium sulfate using a 3.7 M stock solution. The supernatant was loaded on a 1 ml Phenyl Sepharose FF (hi-sub) column (GE Healthcare) and the column was washed with Buffer A (45 mM Tris-HCl, 45 mM NaCl, 0.4 M NHL4SO4, 1 mM DTT, 0.2 mM EDTA, pH 7.5). The crude toxins were eluted using a 200 ml gradient to Buffer B (50 mM Tris-HCl, 50 mM NaCl, 1 mM DTT, 0.2 mM EDTA, pH 7.5). Fractions containing toxins were identified by SDS-PAGE. Fractions were stored in SDS-PAGE loading buffer to prevent autoproteolysis prior to Western blot analysis.
Purified toxins (about 20 ng) were analyzed using SDS-PAGE on a NuPAGE® (Life Technologies) 4-12% polyacrylamide gel run at 200V using SeeBlue2® standards (Invitrogen). The proteins in the gel were transferred to a nitrocellulose membrane in 6 min, using the iBlot® (Invitrogen) gel blotting system. The blot was blocked with PBST (10 mM sodium phosphate, 2 mM potassium phosphate, 2.7 mM potassium chloride, 137 mM sodium chloride, 0.1% Tween 20) containing 5% nonfat dry milk (NFDM) for 1 h at room temperature. The blot was probed with the mAb diluted 1:5000 in 2.5% NFDM/PBST for 1 h at RT, then washed 3×5 min with PBST. The blot was incubated with goat anti-human Alkaline Phosphatase conjugate (Sigma, A1543) [1: 6600 in 2.5% NFDM/PBST,] for 1 h at RT. The blot was washed 3×5 min with PBST and developed with 1 BCIP/NBT tablet (Sigma, B5655) in 10 ml water. Development was stopped by putting the blot into deionized water.
Each of the anti-toxin A antibodies, A1, A2, A3, A4, and A5, demonstrated binding to toxinotypes 0, III, V, XII, and XV by Western analysis. Of the anti-toxin B antibodies, B3 and B6 bound to toxinotypes 0, III, V, VIII, XII, and XV, while B1 bound to at least toxinotypes 0, III, V, VIII, XII, and B2 bound to toxinotypes 0, III, V, and VIII. B4 bound to toxinotypes 0, III, and V, while B5 bound to toxinotype 0 and III. The toxinotype binding results for the anti-toxin A and anti-toxin B antibodies are summarized below in Table 8.
Toxinotyping by Western analysis can be biased by low toxin production in some strains. Therefore a more sensitive CTD (C Terminal Domain) competition assay was developed. For the CTD competition assay, the CTDs of C. difficile toxin A and toxin B from genomic DNA of toxinotypes 0, III, V, VIII, XII and XV were cloned, expressed, and purified and combined with anti-toxin A or anti-toxin B antibodies to measure the effect on cytotoxicity or TEER in cell based neutralization assays (as described in previous example). The CTD competition assay only works for antibodies that recognize an epitope in the CTD of toxin A or toxin B.
Briefly, for toxin A, a QuickExtract™ DNA Extraction Kit (Epicentre) was used to isolate genomic DNA from 1 ml samples of cultures of each of the six C. difficile strains representing five toxinotypes (0, III, V, XII and XV). The following primers were used for amplification of the toxin A C-terminal domains (CTDs):
The primers were designed to amplify the last 900 amino acids (amino acids 1811-2710 in the VPI10463 reference sequence), or 2700 bp of the toxin A toxinotype 0 CTD. Amplification was performed using Pfx50 DNA Polymerase and a standard touchdown PCR protocol. In the case where multiple bands were amplified, the band of the correct size (about 2700 bp for toxinotypes 0, Ill, V, XII and XV was purified by excision of the band of the correct size from an agarose gel followed by gel extraction. Purified or unpurified PCR product was directionally cloned into the expression plasmid pET101-D-Topo, using a ligation-independent cloning strategy, as per the manufacturer's instructions (Invitrogen, Champion™ pET Directional TOPO® Expression Kit).
Directionality and sequence were confirmed by traditional DNA sequencing, using the forward and reverse cloning primers. Due to the highly repetitive nature of the intervening sequences the sequence of the entire CTD was not confirmed. The translation start site is at the ATG in the forward primer sequence. Expression continues through the reverse primer sequence and the C-terminal tags encoded by the expression plasmid. Recombinant expression of these proteins yields a protein of the following sequence as previously described: Met-GFKIIDNKTYY-[toxinotype-specific A CTD a's 1823-2704]-APGIYG-[V5 epitope]-RTG-[6×His](SEQ ID NO:237).
For toxin B, DNA samples were isolated from the six C. difficile strains representing six toxinotypes (0, III, V, VIII, XII and XV). The following primers were used for amplification of the toxin B CTD's:
The primers were designed to amplify the last 615 amino acids excluding the final 6 amino acids of the Toxin B CTD (as 1752-2360), or 1827 bp of the toxin B toxinotype 0 CTD.
Amplification was performed using TaKaRa LA Taq DNA Polymerase and a standard touchdown PCR protocol. In the case where multiple bands were amplified, the band of the correct size (about 1827 bp for all toxinotypes) was purified by excision of the band of the correct size from an agarose gel followed by gel extraction. Purified or unpurified PCR product was directionally cloned into the expression plasmid pET24A+, using traditional restriction digest and ligation-dependent cloning strategy. Directionality and sequence were confirmed by traditional DNA sequencing, using the forward and reverse cloning primers. Due to the highly repetitive nature of the intervening sequences the sequence of the entire CTD was not confirmed. The translation start site is at the ATG in the forward primer sequence. Expression continues through the reverse primer sequence and the C-terminal tags encoded by the expression plasmid. Recombinant expression of these proteins yields a protein of the following sequence: Met-STSEENK-[toxinotype-specific B CTD aa's 1760-2352]-YYFDPDTA-LE-[6×His] (SEQ ID NO:240).
The cloned toxin A and B CTD proteins were expressed as soluble full-length His-tagged proteins by recombinant expression in the E. coli strain BL21 Star (DE3) using the IPTG-free Overnight Express Autoinduction System 1 as per the manufacturer's instructions (Novagen). Proteins were purified under native conditions by bind-and-elute affinity chromatography on Ni-NTA resin, followed by anion exchange in the negative purification mode. Purified CTD proteins were used in cell-based, in vitro neutralization assays to determine the toxinotype specificity of certain antibodies.
The A2 antibody was tested in the Vero cell competition assay to measure the impact of toxin A CTDs of toxinotype 0, III, V, XII, and XV on the neutralizing activity of A2. The Vero cell neutralization was carried out as described above with varying dilutions of the antibodies, plus the addition of 1 μg/ml of toxinotype 0, III, V, XII, or XV toxin A CTDs. A2 neutralizes toxin A induced cytotoxicity in Vero cells with high potency. Toxinotype 0, III, V, XII, and XV CTDs strongly inhibited the neutralizing activity of A2 at low concentrations of antibody (0.625 μg and below) but had minimal, if any effect, at A2 concentrations above 1.25 μg/ml.
The A2 antibody was also tested in a T-84 cell neutralization assay to measure the impact of toxin A CTDs of toxinotypes 0, III, V, XII, and XV on TEER in T-84 cells. The T-84 cell neutralization assay was carried out as described above with varying dilutions of A2, plus the addition of 0.4 μg/ml or 1 μg/ml of toxinotype 0, III, V, XII, and XV toxin A CTDs. A2 neutralizes toxin A induced loss of TEER in T-84 cells with high potency. CTDs from toxinotypes 0, III, V, XII, and XV strongly inhibited the neutralizing activity of A2 in the TEER assay.
The B6 antibody was similarly tested in the T-84 cell neutralization assay. Toxin B CTDs from toxinotypes 0, III, V, VIII, XII, and XV strongly inhibited the neutralizing activity of B6.
Epitope mapping of anti-toxin B antibodies was conducted by Western analysis using recombinant domains from C. difficile toxin A and toxin B. The recombinant domains from toxin A were used as negative controls. Segments of the genes for toxin A and toxin B were cloned by PCR from C. difficile DNA of strain VPI10463. The amino acid sequences of toxin A and toxin B from C. difficile strain VPI10463 are set forth in SEQ ID NO:230 and SEQ ID NO:230, respectively. The corresponding amino acid residues for the cloned gene segments of toxin A and B are set forth in the table below:
A methionine start codon was added to the N-terminus and a 6×His tag (SEQ ID NO:332) followed by a stop codon was added to the C-terminus. The resulting PCR products were ligated into the multiple cloning site of plasmid pET24+. The constructs were transformed into E. coli BL21(DE3) and induced by addition of IPTG.
Constructs A2 and A3 were expressed but were insoluble and were purified by denaturing chromatography, while constructs B1-B5 were at least partly soluble and purified by non-denaturing chromatography. Soluble constructs were grown to liter scale in LB medium at 37° C. Cells were pelleted by centrifugation and lysed by microfluidization (Microfluidics Corp, Newton Mass.) in 50 mM NaHPO4, 300 mM NaCl, 20 mM imidazole, pH 8.0. Insoluble material was removed by centrifugation and the cleared lysate was loaded onto a Ni-NTA column (Qiagen). The column was washed with 50 mM NaHPO4, 300 mM NaCl, 20 mM imidazole, pH 8.0 and eluted with 50 mM NaHPO4, 300 mM NaCl, 250 mM imidazole, pH 8.0. Insoluble constructs were grown and harvested as for soluble ones, but the cell pellet was resuspended in 8M Urea, 100 mM NaHPO4, 10 mM Tris-HCl, pH 8.0 before microfluidization. Insoluble material was removed by centrifugation and the cleared lysate was loaded onto a Ni-NTA column and washed with 8M Urea, 100 mM NaHPO4, 10 mM Tris-HCl, pH 6.3. The column was eluted with 8M Urea, 100 mM NaHPO4, 10 mM Tris-HCl, pH 4.5 and protein-containing fractions were dialysed with multiple changes against 50 mM NaHPO4, 300 mM NaCl, 250 mM imidazole, pH 8.0.
The binding of the B1, B2, B4, and B6 antibodies to the recombinant domains was assessed by Western analysis. Purified recombinant domains (about 400 ng) were analyzed using SDS-PAGE on a NuPAGE® (Life Technologies) 4-12% polyacrylamide gel run at 200V using SeeBlue2® standards (Invitrogen). The proteins in the gel were transferred to a nitrocellulose membrane in 6 min, using the iBlot® (Invitrogen) gel blotting system. The blot was blocked with PBST (10 mM sodium phosphate, 2 mM potassium phosphate, 2.7 mM potassium chloride, 137 mM sodium chloride, 0.1% Tween 20) containing 5% nonfat dry milk (NFDM) for 1 h at room temperature. The blot was probed with the antibody diluted 1:5000 in 2.5% NFDM/PBST for 1 h at RT, then washed 3×5 min with PBST. The blot was incubated with goat anti-human Alkaline Phosphatase conjugate (Sigma, A1543) [1:6600 in 2.5% NFDM/PBST,] for 1 h at RT. The blot was washed 3×5 min with PBST and developed with 1 BCIP/NBT tablet (Sigma, B5655) in 10 ml water. Development was stopped by putting the blot into deionized water.
Western analysis revealed that B1 and B2 bound to an epitope in the glucosyl transferase domain (amino acids 10-520 of SEQ ID NO:231) of toxin B, while B4 bound to an epitope in the N-terminal translocation domain (amino acids 1110-1530 of SEQ ID NO:231) of toxin B. The B6 antibody bound to an epitope in the receptor binding domain (amino acids 1750-2360 of SEQ ID NO:231) of toxin B.
The PepSet ELISA was used to identify linear epitopes of toxin A recognized by the A2 antibody. For the CTD, the following non-overlapping peptides of varying length were designed to cover the repetitive oligopeptide units:
15 amino acid sequences with overlapping 5 amino acids domains and a moving window of 10 amino acids were also designed to cover the gaps between those repetitive units. All peptides were synthesized and probed for binding to A2.
Peptide binding was measured by ELISA. Briefly, 100 μl of 5 μg/ml streptavidin (Southern Biotech) in a sodium carbonate/sodium bicarbonate coating buffer solution (pH 9.6) was added to each well of NUNC Maxisorp4 (eBiosciences) 96 well plates and incubated at 4° C. overnight. The plates were washed 4 times with PBS Tween 20 (PBST) using a volume of 300 μl/well before blocking with 3% BSA solution for 60 minutes. Biotinylated C. difficile toxin A CTD peptides were diluted to a concentration of 100 ng/ml, in diluent (3% BSA in PBST), added to each well (100 μl/well), and incubated at 25° C. for 60 minutes. The plates were then washed 4 times with PBST using a volume of 300 μl/well. The antibody solution was diluted to the appropriate dilution in diluent buffer (3% BSA in PBST), added to the plates (100 μl/well), and incubated at 25° C. for 60 minutes. Following the incubation with antibody, the plates were again washed 4 times with PBST using a volume of 300 μl/well.
For the secondary antibody reaction, horseradish peroxidase (HRP)-goat anti human IgG (Jackson ImmunoResearch) was diluted to 1:2000 in diluent buffer, added to the plates (100 μl/well), and incubated at 25° C. for 60 minutes. Following the incubation with secondary antibody, the plates were again washed 4 times with PBST using a volume of 300 μl/well. SureBlue Reserve™ TMB peroxidase substrate (KPL Inc.) was then added to each well (100 μl/well) and incubated at 25° C. for 10 minutes. The reaction was stopped by adding 100 μl/well of TMB stop solution (KPL Inc.). The plates were read at a wavelength of 450 nm at 25° C. using a Molecular Devices, Model Spectra Max MS.
No binding was observed between A2 and the longer, overlapping peptide sequences. The A2 antibody bound to non-overlapping peptides 2, 18, 19, 20, 24 and 30:
Thus, the A2 antibody recognizes a minimal linear epitope in the C-terminal domain of toxin A comprising the amino acid sequence X1TGWQTI (SEQ ID NO:232), where X1 is A or V. The A2 antibody also recognizes a longer consensus sequence comprising the amino acid sequence of: X2TGWQTIX3GKX4YYF (SEQ ID NO:233), where X2 is A or V, X3 is N or D and X4 is K or V.
As discussed above, it was determined by Western analysis that the B1 and B2 antibodies bound to an epitope in the glucosyl transferase domain (GTD) of toxin B. In addition, sequence analysis showed that B1 and B3 light and heavy chains differed by only 3 CDR and 2 FR mutations in the light chain and 2 FR mutations in the heavy chain and predicted that the B1 and B3 antibodies would bind to the same epitope. Thus, the PepSet peptide binding assay was also used to identify linear epitopes in the GTD of toxin B recognized by B1, B2, and B3, using the general protocol described above for the A2 antibody. For the GTD, the following peptides with 15 amino acid sequences and 5 amino acid overlap were designed to cover the N terminal domain of the molecule:
All peptides were synthesized and probed for binding to B1, B2, and B3.
The B1 and B3 antibodies both bound to peptides 190, 191, and 192 from the toxin B GTD:
Thus, the B1 and B3 antibodies both recognize a minimal linear epitope in the GTD of toxin B comprising the amino acid sequence SGRNK (SEQ ID NO:234). This epitope maps to amino acids 56-80 of SEQ ID NO:231. The B2 antibody binds very weakly to the P190, P191, and P192 peptides but did not bind strongly to any of the GTD short repeat sequences. The B1 and B3 antibodies were also shown to bind to the same epitope by Octet analysis (data not shown).
The N-terminal 91 amino acids of the GTD shares homology with a domain found in cholera toxin and other pathogens. In cholera, this domain, referred to as the 4-helix bundle (4HB) or membrane localization domain (MLD), has been shown to be involved in direct binding of the toxin to the cell membrane and mutagenesis of several amino acids in the MLD abolishes this function (Geissler et al, PNAS, 2010). The SGRNK (SEQ ID NO:234) sequence identified through peptide binding analysis is located in a loop between alpha helices 3 and 4 of the MLD.
Amino acids 1-91 of toxin B were cloned into a pET28a expression construct (GeneArt) with an LPETG (SEQ ID NO:336) motif (which allows for sortase-catalyzed conjugation of labels, such as biotin) and a C-terminal 6×His tag both with the wild-type SGRNK (SEQ ID NO:234) sequence and a mutated version: AGANK (SEQ ID NO:337). These constructs had the following amino acid sequences:
The GTD enzymatic domain (amino acids 95-586; “ASE”) was also cloned into a pET28a expression construct (GeneArt) with an LPETG motif and a C-terminal 6×His tag. This construct has the following amino acid sequence:
Using both Western and dot blot analysis, B2 was found to bind strongly to the wild type MLD sequence and to the mutant MLD sequence to a much lesser extent. The B1 and B3 antibodies did not bind to either the wild type or mutant MLD sequence by Western or dot blot analysis. No binding to the ASE domain was observed with any of the antibodies.
Binding of the B1, B2, and B3 antibodies to the wild type and mutant toxin B MLD sequences and the wild type toxin B ASE sequence was also assessed using Bio-Layer Interferometry on a Octet® RED96 (FortéBio) at 30° C., as described above in Example 2. As expected, all three antibodies bind to the full length toxin B and toxin B GTD by Octet analysis. B2 bound to the wild type and mutant MLD sequences, while neither B1 nor B3 bound to either MLD sequence. Unexpectedly, all three antibodies were found to bind the toxin B ASE domain by Octet analysis, suggesting that the non-denatured ASE domain may possess some non-specific binding activity due to misfolding or a hydrophobic surface generated by separating the ASE domain from the MLD. In the Western analysis, under denaturing conditions, only the positive control (6×His) antibody bound the ASE domain.
The Octet analysis was also conducted for the B1 and B2 antibodies using the cloned GTD (aa 1-586) with the wild-type SGRNK (SEQ ID NO:234) sequence and a mutated version: AGANK (SEQ ID NO:337). Both antibodies bind strongly to the wild type GTD. B2 binding was reduced by the mutations to the SGRNK motif (SEQ ID NO:234, leading to an approximately 100-fold difference in Kd. B1 binding was unaffected by the mutations.
Hydrogen-Deuterium Exchange Mass Spectroscopy shows that the binding of the B2 antibody strongly reduces solvent exchange of the N-terminal helix of the GTD, while the SGRNK (SEQ ID NO:234) sequence is barely protected by B2 binding (data not shown). Saturation binding on the Octet shows that B1 and B2 do not interfere with each other's ability to bind toxin B, thus suggesting that the two antibodies recognize different epitopes.
In summary, while PepSet ELISA showed that the B1 and B3 antibodies bind strongly to a linear epitope comprising the SRGNK (SEQ ID NO:234) motif of the MLD, neither antibody bound to the toxin B MLD by Western, dot blot, or Octet analysis. However, the B1 and B3 antibodies do bind to the toxin B GTD by Western and Octet analysis, suggesting that the conformational epitope recognized by the B1 and B3 antibodies may not be exposed or present when the MLD is expressed separately from the ASE domain. On the other hand, the B2 antibody, which binds very weakly to linear epitopes comprising the SRGNK (SEQ ID NO:234) motif, was found to bind the toxin B MLD by Western, dot blot, and Octet analysis. Mutating the SRGNK (SEQ ID NO:234) sequence in the toxin B MLD and GTD reduces the binding of the B2 antibody. Together, these observations suggest that the B2 antibody binds to a conformational epitope within the MLD. The SRGNK (SEQ ID NO:234) motif within the MLD may play a role in contributing to the tertiary structure of the epitope recognized by B2 or may interact non-specifically with the B2 antibody.
The hamster model is widely recognized as the optimal choice for the evaluation of novel treatment strategies against C. difficile (Best et al. Gut, 2012, 3(2):145-167; Babcock et al. Infection & Immunity, 2006, 74(11):6339-6347). Once the normal intestinal flora of these animals is compromised by antibiotic treatment, challenge with live toxigenic C. difficile bacteria or viable spores from a toxigenic strain leads to colonization followed by lethal cecitis. Diarrhea, histological damage and cecitis result from the action of C. difficile toxin A and B and the stimulation of local inflammation. These symptoms are very similar to the symptoms observed in human suffering from C. difficile-associated diarrhea (CDAD). Thus, the in vivo efficacy of the human anti-toxin A and anti-toxin B antibodies was evaluated in a hamster CDAD model (also known as the hamster C. difficile infection (CDI) model).
Female Golden Syrian hamsters (Mesocricetus auratus), obtained from Charles River Laboratories, were individually caged and allowed to acclimate to the animal facility for at least 48 hours prior to any treatment, challenge, or other manipulation. All procedures involving animals were conducted under protocols approved by the Institutional Animal Care and Use Committee (IACUC).
For the primary challenge, animals were intraperitoneally (IP) injected with anti-toxin A and B antibodies at doses ranging from 6-50 mg/kg, every day for 4 consecutive days on days −3, −2, −1, and 0 relative to bacterial challenge on day 0. The test antibodies were injected as a combination of one human anti-toxin A antibody and one human anti-toxin B antibody. Combinations tested included 1) A2 (anti toxin A) and B6 (anti toxin B), 2) A2 (anti toxin A) and B4 (anti toxin B), 3) A2 (anti toxin A) and B1 (anti toxin B), and 4) A2 (anti toxin A) and B2 (anti toxin B). Control animals were also injected on the same 4-day injection schedule with 2 ml of PBS. In addition, 24 hours prior to bacterial challenge all animals were weighed and IP injected with 1 ml of a 1 mg/ml clindamycin solution. This antibiotic pretreatment disrupts the normal intestinal flora and facilitates gut colonization with C. difficile.
On the day of challenge, animals received their final IP injection of antibodies or PBS prior to intragastric (IG) challenge with a LD100 dose of C. difficile spores (toxinotype 0 strain 630). To prepare C. difficile spores, the bacteria were grown for 24 hours in thioglycollate medium. This culture was used to inoculate anaerobic blood agar plates which were incubated at 37° C. until the bacterial were confluent (3-4 days). After reaching confluence, plates were incubated for an additional 3 days to induce spore formation. Spores were harvested into PBS without Ca or Mg, washed once and then heat shocked at 56° C. for 10 minutes to kill the vegetative cells. The spore suspension was centrifuged at 500 g for 30 minutes and re-suspended in 20% glycerol in PBS. Spore preparations were frozen at −65° C. or less for long term storage. Viable spore counts (CFU ml−1) were assessed by thawing the spore stock at 37° C. and performing serial 10-fold dilutions in water. Dilutions were plated in triplicate onto pre-reduced CDSA agar plates. Plates were incubated under anaerobic conditions at 37° C. for no less than 48 hours. The colonies were counted and CFU ml−1 was calculated. After completion of IP injection and IG challenge, animals were housed individually in sterile caging that consists of autoclaved sterilized bedding, autoclaved sterilized water, and irradiated food.
After challenge, animals were observed at least twice a day for morbidity and mortality and were weighed as per approved protocol. Both diarrheal disease and animal behavior were assessed. Diarrheal disease was scored numerically on a scale of 0-3: 0—no disease, 1—loose feces, 2—wet tail and perianal region, 3—wet perianal region, belly and hind paws. Behavior is evaluated categorically using the following criteria: N—animal appears normal; QAR—animal appears slightly lethargic, but alert and arousable; I—animal appears severely dehydrated, immobile, and exhibits hunched posture and/or ruffled fur. If an animal received a behavior score of I, the animal was immediately euthanized via CO2 overdose. Percent weight loss was also calculated and if the animal lost >30% of its pre-challenge body weight it was considered moribund and was immediately euthanized via CO2 overdose. All animals in a study were observed until all animals had either died or been euthanized or there was a period of at least 48 hours with no animals displaying any diarrheal symptoms or behaviors of illness.
In initial tests, the A2 antibody was paired with either the B4 or B6 antibody. Control hamsters that did not receive an antibody usually died by day 4 of the study. With these antibody combinations, a dose of 50 mg/kg provided optimal results (data not shown). At a dose of 50 mg/kg, A2+B4 conferred survival on all animals tested through the end of the study (15 days post challenge), whereas only 60% of animals treated with A2 and B6 survived.
Subsequent testing in the CDAD model compared A2+B4 to A2+B1 and A2+B2. At the 50 mg/kg dose, both A2+B1 and A2+B2 conferred survival on all animals tested through the end of the study (12 days post challenge).
Fecal pellets were collected from challenged animals throughout the study, usually but not always on days 4, 7 and 12 post-challenge. In order to ensure collection of fresh fecal pellets, fecal matter was collected 1 day after animals were transferred into clean cages. Fecal matter was cultured to determine the C. difficile colonization status of animals. To culture fecal matter, fecal pellets were weighed and at least 40 mg of feces were homogenized with 5×volumes of DPBS and 5×volumes ethanol per mg of sample. Material was serially diluted and 100 μl of diluted homogenized fecal matter was cultured on reduced C. difficile selective Agar plates (CDSA plates) at 37° C. in an anaerobic jar. After 56-72 hours of growth, colonies, which should appear as flat to low umbonate yellow colonies with ground glass-like appearance and a slightly filamentous edge, were counted.
Treatment with 6 mg/kg A2+B1 or A2+B2 did not affect the initial colonization and C. difficile burden, as measured by a C. difficile fecal culture tested on Day 4 post challenge.
Based on the results from Example 9, the A2+B2 antibody combination was tested against highly virulent strains of C. difficile: the toxinotype 0 strain VPI10463 and toxinotype III (ribotype 027) strain 13695#7. The hamster CDAD model was used as described in Example 9. At the 6 mg/kg dose, A2+B2 prolonged life as compared to the PBS control but resulted in 0% survival at days 6 and 4, respectively for the VPI10463 and 13695#7 strains.
Adding a third antibody to the A2+B2 combination significantly enhances survival in the hamster CDAD model. The following three antibody combinations were tested at low (6 mg/kg) and high doses (50 mg/kg) using epidemic highly virulent strain 13695#7: A2 (6 mg/kg or 50 mg/kg)+B1 (3 mg/kg or 25 mg/kg)+B2 (3 mg/kg or 25 mg/kg), A2 (6 mg/kg or 50 mg/kg)+B2 (3 mg/kg or 25 mg/kg)+B4 (3 mg/kg or 25 mg/kg), and A2 (6 mg/kg or 50 mg/kg)+B2 (3 mg/kg or 25 mg/kg)+B6 (3 mg/kg or 25 mg/kg). All three combinations conferred survival on all animals tested through the end of the study (10 days post challenge) except for the low dose of A2+B2+B4, which conferred 80% survival through the end of the study.
While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
This application is a U.S. National Stage application of PCT/US2014/028637 filed 14 Mar. 2014, which claims the benefit of, and relies on the filing date of, U.S. provisional patent application number 61/794,071, filed 15 Mar. 2013, the entire disclosure of which is incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
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| PCT/US2014/028637 | 3/14/2014 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2014/144292 | 9/18/2014 | WO | A |
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| 20160068591 A1 | Mar 2016 | US |
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| 61794071 | Mar 2013 | US |
