NEUTRALISING ANTIBODIES TO THE MAJOR EXOTOXINS TCDA AND TCDB OF CLOSTRIDIUM DIFFICILE

Abstract

This present invention describes the derivation and selection of antibodies capable of neutralising the major exotoxins; TcdA and TcdB of Clostridium difficile. The invention also describes novel neutralisation and antigen binding properties of individual Mabs and mixtures thereof.

Description

[0001] The present invention relates to antibodies to exotoxins of Clostridium difficile, for example TcdA and TcdB, pharmaceutical compositions comprising the same, processes of producing said antibodies and compositions and use of the antibodies and compositions in treatment and/or prophylaxis, in particular treatment or prophylaxis of Clostridium difficile infection, pseudomembranous colitis, fulminant colitis and/or toxic mega colon.

[0002] The two major exotoxins TcdA and TcdB have been established as the major pathogenicity determinants of Clostridium difficile in a large number of in vitro and in vivo studies. Non-toxigenic strains are not pathogenic to animals and man (1, 2). To date a clear understanding of the role of binary toxin has yet to be established (3).

[0003] Both toxins are entero- and cyto-toxic, but the balance of evidence suggests that TcdA is a more powerful enterotoxin than TcdB, whilst TcdB is typically observed to be .about.1000.times. more cytotoxic than TcdA (4). Whilst both toxins are capable of inducing an inflammatory response, TcdA appears to aid the migration of the more inflammatory TcdB deeper into the gut mucosa (5).

[0004] In toto, a large collection of data generated for over 30 years support a model where both toxins are likely to be important in the human disease process. It is probable that TcdA initiates early (i.e. before TcdB) and rapid (i.e. 1-3 hours) gut damage through loss of tight junctions and destruction of villi tips and hence diarrhoea, probably through albumin driven fluid loss. This damage to the integrity of the gut lining enables TcdB to exert its superior molar potency (TcdB is typically cited as being 1000.times. more cytotoxic than TcdA) more rapidly and effectively (i.e. deeper into tissue, alternative cellular targets and damaging systemically accessed organs). Either toxin can be effective alone in vitro on human or animals cells and tissues. Either toxin can be effective alone in vivo in animals depending upon other eliciting factors such as mechanical damage, barrier overload and host specific sensitivities. It is now clear that in hamsters at least either TcdA or TcdB alone delivered by a Clostridium difficile gut infection can cause death (1). It is well established that A-B+ strains are capable of causing symptoms and death in humans (6,7). However, the majority (.about.95%) of clinical strains are A+B+ hence drugs aimed at treating Clostridium difficile infections (CDI) must be capable of neutralising the activities of and clearing both toxins effectively.

[0005] CDI is most typically a nosocomial infection of older patients or those with complicating co-morbidities. However, an increase in community acquired infections has been noted. Infection is almost always associated with or induced by use of broad spectrum antibiotics. Healthcare associated costs are estimated to be in excess of $1 bn per annum in the US alone. These costs are primarily due to patients having longer hospitals stays. Current therapies involve the use of antibiotics such as clindamycin, vancomycin or fidaxomicin which kill the Clostridium difficile cells within the gut. Current therapies address the bacterial infection but do not deal with or prevent directly the significant pathogenesis caused by TcdA and TcdB which are major contributors to CDI symptoms and mortality.

[0006] CDI symptoms in humans include mild to severe diarrhoea, pseudomembranous colitis (PMC) and fulminant colitis or so called toxic mega colon. Death results in 5-15% of patients receiving current best care. Thus at the present time there is no specific therapy available to patients to prevent the damage and injury caused by C. difficile toxins after infection.

[0007] Raising an antibody response through vaccination and parenteral administration of polyclonal and monoclonal antibodies have all been shown to be capable of protecting animals from symptoms of diarrhoea and death (8-15). Early studies in hamsters suggested that antibodies against TcdA alone were all that was necessary for protection. However, use of strains functionally deleted for TcdA or TcdB demonstrate that either toxin is capable of causing disease in hamsters, but that both toxins together are more effective (1).

[0008] For therapeutic applications, monoclonal antibodies (Mabs) can offer efficacy, safety, manufacturing and regulatory advantages over serum derived polyclonal antibodies or serum derived hyper-immune sera. For these reasons Mabs are usually the preferred option for therapeutic products.

[0009] There have been a number of attempts to generate protective Mabs against TcdA and TcdB. The most advanced of these in the clinic is a mixture of 2 IgG1 Mabs, one against each TcdA and TcdB originally called CDA1 and MDX1388 developed by MBL and Medarex. They were demonstrated to be unable to fully protect hamsters in models of acute or relapse infections (15). This Mab combination is now being developed as MK3415A by Merck Inc. In a human phase II trial MK3415A resulted in a statistically significant reduction in disease recurrence (p=0.006) (see also Lowy et al., NEJM (2010) 362: 197-205) but did not affect the duration/severity of diarrhoea or death rates (16). This may mean that these antibodies may only be useful for preventing recurrence of infection. Recurrence of infection results in approximately 25% of patients. Thus there likely to be a significant patient population in which these antibodies are not effective.

[0010] In order to be able to have a positive influence upon diarrhoea (for example as a result of acute damage to gut tight junctions due to TcdA) and death (for example resulting from prolonged poor nutritional status, dehydration stress and initiation of an inflammatory cascade, widespread anatomical damage to the gut lining and possibly damage to distant organs due to systemic toxin TcdB more so than TcdA) Mabs are required with superior affinity, toxin neutralisation, superior prevention of loss of TEER (trans-epithelial electrical resistance), antigen decoration and antigen immune clearance.

SUMMARY OF THE PRESENT INVENTION

[0011] The present invention provide a Mab(s) with a very high level of potency in vitro and in vivo which have the potential to have an impact upon duration and severity of diarrhoea and death rate in humans suffering from Clostridium difficile infection (CDI).

[0012] In one embodiment there is provided a monoclonal antibody specific to antigen TcdA or TcdB, wherein the antibody has high affinity for the target antigen and is suitable for reducing the duration and/or severity of diarrhoea and morbidity in a patient with Clostridium difficile infection or at risk of said infection.

[0013] In one embodiment there is provided a Mab specific to TcdA or TcdB, or a population of at least two Mabs at least one of which is specific to TcdA and at least one of which is specific to TcdB, wherein the EC.sub.50 of the or each antibody or the combination of antibodies is 200 ng/ml or less, for example 150 ng/ml or less such as 100 ng/ml.

[0014] The antibodies of the present disclosure are useful because they are likely to provide a means of treating the severity and duration of symptoms of a primary infection such as diarrhoea in a patient or preventing death and not just prevent the reoccurrence of disease symptoms.

[0015] In at least some embodiments the antibodies according to the present disclosure show no reduction in potency in the presence of high concentrations of toxin.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0016] Specific as employed herein is intended to refer to an antibody that only recognises the antigen to which it is specific or an antibody that has significantly higher binding affinity to the antigen to which is specific compared to binding to antigens to which it is non-specific, for example 5, 6, 7, 8, 9, 10 times higher binding affinity.

[0017] Binding affinity may be measured by standard assays such as surface plasmon resonance, such as BIAcore.

[0018] In one embodiment the EC.sub.50 is less than 75, 70, 60, 65, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.5 ng/ml Clostridium difficile infection in cell culture assays and the patient. This is significantly lower (more potent) than known antibodies and is thought to be a major factor as to why the antibodies of the present disclosure have a significant and positive impact on survival of subjects receiving treatment.

[0019] As employed herein potency is the ability of the antibody to elicit an appropriate biological response, for example neutralisation of the deleterious toxin effects, at a given dose or concentration. Examples of potency include the percent maximal neutralisation of toxin activity (extent of protection), the lowest relative concentration of Mab to antigen (e.g. EC.sub.50), the speed and durability of neutralisation activity.

[0020] In cell culture assays neutralisation might be observed as one or more of the following: prevention of binding of toxin to cells, immunoprecipitation of toxin from solution, prevention of loss of cell form and shape, prevention of loss of cytoskeletal structures, prevention of loss of cell monolayer tight junctions and trans-epithelial electrical resistance, prevention of cell death, apoptosis and production of pro-inflammatory cytokines such as TNF.alpha., IL-1.beta., IL-6 and MIP1.alpha..

[0021] In tissue section and explant assays neutralisation may, for example be observed as prevention of necrosis and/or oedematous fluid accumulation.

[0022] In in vivo assays neutralisation may be observed as one or more of the following: prevention of fluid accumulation in ligated ileal loops and prevention of gut tissue necrosis, diarrhoea, pseudo-membrane formation of death of animals,

[0023] Thus in one embodiment there is provided an antibody (for example an anti-toxin A antibody) comprising a CDR, such as 1, 2, 3, 4, 5 or 6 CDRs, selected from:

TABLE-US-00001 SEQ ID NO: 1 QASQSISNALA SEQ ID NO: 2 SASSLAS SEQ ID NO: 3 QYTHYSHTSKNP SEQ ID NO: 4 GFTISSYYMS SEQ ID NO: 5 IISSGGHFTWYANWAKG SEQ ID NO: 6 AYVSGSSFNGYAL

[0024] In one embodiment sequences 1 to 3 are in a light chain of the antibody.

[0025] In one embodiment sequences 4 to 6 are in a heavy chain of the antibody.

[0026] In one embodiment SEQ ID NO: 1 is CDR L1, SEQ ID NO: 2 is CDR L2 and SEQ ID NO; 3 is CDR L3.

[0027] In one embodiment SEQ ID NO: 4 is CDR H1, SEQ ID NO: 5 is CDR H2 and SEQ ID NO; 6 is CDR H3.

[0028] In one embodiment SEQ ID NO: 1 is CDR L1, SEQ ID NO: 2 is CDR L2, SEQ ID NO; 3 is CDR L3, SEQ ID NO: 4 is CDR H1, SEQ ID NO: 5 is CDR H2 and SEQ ID NO; 6 is CDR H3.

[0029] In one embodiment there is provided a variable region, such as a light chain variable region with the following sequence (Antibody 922 anti-toxin A antibody; Light chain Variable region sequence) SEQ ID NO: 7:

TABLE-US-00002 DPVMTQSPSTLSASVGDRVTITCQASQSISNALAWYQQKPGKAPKLLIYS ASSLASGVPSRFKGSGSGTEFTLTISSLQPDDFATYYCQYTHYSHTSKNP FGGGTKVEIK

wherein the CDRs are underlined and construct is referred to herein as 922.g1 VK (gL1).

[0030] The polynucleotide sequence encoding SEQ ID NO: 7 is shown in FIG. 1 and SEQ ID NO: 8 therein.

[0031] In one embodiment there is provided a variable region, such as a heavy chain variable region with the following sequence (Antibody 922 anti-toxin A antibody heavy chain variable region sequence) SEQ ID NO: 9:

TABLE-US-00003 EVQLVESGGGLVQPGGSLRLSCAASGFTISSYYMSWVRQAPGKGLEWIGI ISSGGHFTWYANWAKGRFTISSDSTTVYLQMNSLRDEDTATYFCARAYVS GSSFNGYALWGQGTLVTVS

wherein the CDRs are underlined and construct is referred to herein as 922.g1 VH (gH1)

[0032] The polynucleotide sequence encoding SEQ ID NO: 9 is shown in FIG. 1 and SEQ ID NO: 10 therein.

[0033] In one embodiment the antibody comprises the variable regions shown in SEQ ID NO: 7 and 9.

[0034] Thus in one embodiment there is provided an antibody (for example an anti-toxin A antibody) comprising a CDR, such as 1, 2, 3, 4, 5 or 6 CDRs, selected from:

TABLE-US-00004 SEQ ID NO: 11 QASQSISNYLA SEQ ID NO: 12 SASTLAS SEQ ID NO: 13 QYSHYGTGVFGA SEQ ID NO: 14 AFSLSNYYMS SEQ ID NO: 15 IISSGSNALKWYASWPKG SEQ ID NO: 16 NYVGSGSYYGMDL

[0035] In one embodiment sequences 11 to 13 are in a light chain of the antibody.

[0036] In one embodiment sequences 14 to 16 are in a heavy chain of the antibody.

[0037] In one embodiment SEQ ID NO: 11 is CDR L1, SEQ ID NO: 12 is CDR L2 and SEQ ID NO: 13 is CDR L3.

[0038] In one embodiment SEQ ID NO: 14 is CDR H1, SEQ ID NO: 15 is CDR H2 and SEQ ID NO; 16 is CDR H3.

[0039] In one embodiment SEQ ID NO: 11 is CDR L1, SEQ ID NO: 12 is CDR L2, SEQ ID NO: 13 is CDR L3, SEQ ID NO: 14 is CDR H1, SEQ ID NO: 15 is CDR H2 and SEQ ID NO; 16 is CDR H3.

[0040] In one embodiment there is provided a variable region, such as a light chain variable region with the following sequence (Antibody 923 anti-toxin A antibody; Light chain Variable region sequence) SEQ ID NO: 17:

TABLE-US-00005 DVVMTQSPSSLSASVGDRVTITCQASQSISNYLAWYQQKPGKVPKLLIYS ASTLASGVPSRFKGSGSGTQFTLTISSLQPEDVATYYCQYSHYGTGVFGA FGGGTKVEIK

wherein the CDRs are underlined and construct is referred to herein as CA923.g1 gL1

[0041] The polynucleotide sequence encoding SEQ ID NO: 17 is shown in FIG. 1 and SEQ ID NO: 18 therein.

[0042] In one embodiment there is provided a variable region, such as a heavy chain variable region with the following sequence (Antibody 923 anti-toxin A antibody heavy chain variable region sequence) SEQ ID NO: 19:

TABLE-US-00006 EVQLVESGGGLVQPGGSLRLSCAASAFSLSNYYMSWVRQAPGKGLEWIGI ISSGSNALKWYASWPKGRFTISKDSTTVYLQMNSLRAEDTATYFCARNYV GSGSYYGMDLWGQGTLVTVS

wherein the CDRs are underlined and construct is referred to herein as CA923.g1 gH1

[0043] The polynucleotide sequence encoding SEQ ID NO: 19 is shown in FIG. 2 and SEQ ID NO: 20 therein.

[0044] In one embodiment an antibody according to the invention comprises variable regions shown in SEQ ID NO 17: and SEQ ID NO: 19.

[0045] In one embodiment there is provided an antibody (for example an anti-toxin A antibody) comprising a CDR, such as 1, 2, 3, 4, 5 or 6 CDRs, selected from:

TABLE-US-00007 SEQ ID NO: 21 QASQSISSYFS SEQ ID NO: 22 GASTLAS SEQ ID NO: 23 QCTDYSGIYFGG SEQ ID NO: 24 GFSLSSYYMS SEQ ID NO: 25 IISSGSSTTFTWYASWAKG SEQ ID NO: 26 AYVGSSSYYGFDP

[0046] In one embodiment sequences 21 to 23 are in a light chain of the antibody.

[0047] In one embodiment sequences 24 to 26 are in a heavy chain of the antibody.

[0048] In one embodiment SEQ ID NO: 21 is CDR L1, SEQ ID NO: 22 is CDR L2 and SEQ ID NO; 23 is CDR L3.

[0049] In one embodiment SEQ ID NO: 24 is CDR H1, SEQ ID NO: 25 is CDR H2 and SEQ ID NO; 26 is CDR H3.

[0050] In one embodiment SEQ ID NO: 21 is CDR L1, SEQ ID NO: 22 is CDR L2, SEQ ID NO; 23 is CDR L3, SEQ ID NO: 24 is CDR H1, SEQ ID NO: 25 is CDR H2 and SEQ ID NO; 26 is CDR H3.

[0051] In one embodiment there is provided a variable region, such as a light chain variable region with the following sequence (Antibody 993 anti-toxin A antibody; Light chain Variable region sequence) SEQ ID NO: 27:

TABLE-US-00008 DVVMTQSPSTLSASVGDRVTITCQASQSISSYFSWYQQKPGKAPQLLIYG ASTLASGVPSRFKGSGSGTELTLTISSLQPDDFATYYCQCTDYSGIYFGG FGGGTKVEIK

wherein the CDRs are underlined and construct is referred to herein as CA993.g1 gL1

[0052] The polynucleotide sequence encoding SEQ ID NO: 27 is shown in FIG. 2 and SEQ ID NO: 28 therein.

[0053] In one embodiment there is provided a variable region, such as a heavy chain variable region with the following sequence (Antibody 993 anti-toxin A antibody heavy chain variable region sequence) SEQ ID NO: 29:

TABLE-US-00009 EVQLVESGGGLVQPGGSLKLSCTASGFSLSSYYMSWVRQAPGKGLEWIGI ISSGSSTTFTWYASWAKGRFTISKTSTTVYLQMNSLKTEDTATYFCARAY VGSSSYYGFDPWGQGTLVTVS

wherein the CDRs are underlined and construct is referred to herein as CA993.g1 gH1

[0054] The polynucleotide sequence encoding SEQ ID NO: 29 is shown in FIG. 2 and SEQ ID NO: 30 therein.

[0055] In one embodiment an antibody according to the invention comprises variable regions shown in SEQ ID NO: 27 and SEQ ID NO: 29.

[0056] In one embodiment there is provided an antibody (for example an anti-toxin A antibody) comprising a CDR, such as 1, 2, 3, 4, 5 or 6 CDRs, selected from:

TABLE-US-00010 SEQ ID NO: 31 QASQSINNYFS SEQ ID NO: 32 GAANLAS SEQ ID NO: 33 QNNYGVHIYGAA SEQ ID NO: 34 GFSLSNYDMI SEQ ID NO: 35 FINTGGITYYASWAKG SEQ ID NO: 36 VDDYIGAWGAGL

[0057] In one embodiment sequences 31 to 33 are in a light chain of the antibody.

[0058] In one embodiment sequences 34 to 36 are in a heavy chain of the antibody.

[0059] In one embodiment SEQ ID NO: 31 is CDR L1, SEQ ID NO: 32 is CDR L2 and SEQ ID NO; 33 is CDR L3.

[0060] In one embodiment SEQ ID NO: 34 is CDR H1, SEQ ID NO: 35 is CDR H2 and SEQ ID NO: 36 is CDR H3.

[0061] In one embodiment SEQ ID NO: 31 is CDR L1, SEQ ID NO: 32 is CDR L2, SEQ ID NO; 33 is CDR L3, SEQ ID NO: 34 is CDR H1, SEQ ID NO: 35 is CDR H2 and SEQ ID NO; 36 is CDR H3.

[0062] In one embodiment there is provided a variable region, such as a light chain variable region with the following sequence (Antibody 995 anti-toxin A antibody; Light chain Variable region sequence) SEQ ID NO: 37:

TABLE-US-00011 DVVMTQSPSTLSASVGDRVTITCQASQSINNYFSWYQQKPGKAPKLLIYG AANLASGVPSRFKGSGSGTEYTLTISSLQPDDFATYSCQNNYGVHIYGAA FGGGTKVEIK

wherein the CDRs are underlined

[0063] The polynucleotide sequence encoding SEQ ID NO: 37 is shown in FIG. 3 and SEQ ID NO: 38 therein.

[0064] In one embodiment there is provided a variable region, such as a heavy chain variable region with the following sequence (Antibody 995 anti-toxin A antibody heavy chain variable region sequence) SEQ ID NO: 39

TABLE-US-00012 EVQLVESGGGLVQPGGSLRLSCTASGFSLSNYDMIWVRQAPGKGLEYIGF INTGGITYYASWAKGRFTISRDSSTVYLQMNSLRAEDTATYFCARVDDYI GAWGAGLWGQGTLVTVS

wherein the CDRs are underlined

[0065] The polynucleotide sequence encoding SEQ ID NO: 39 is shown in FIG. 3 and SEQ ID NO: 40 therein.

[0066] In one embodiment an antibody according to the invention comprises variable regions shown in SEQ ID NO: 37 and SEQ ID NO: 39.

[0067] In one embodiment there is provided an antibody (for example an anti-toxin A antibody) comprising a CDR, such as 1, 2, 3, 4, 5 or 6 CDRs, selected from:

TABLE-US-00013 SEQ ID NO: 41 QASQSISSYLS SEQ ID NO: 42 RASTLAS SEQ ID NO: 43 LGVYGYSNDDGIA SEQ ID NO: 44 GIDLSSHHMC SEQ ID NO: 45 VIYHFGSTYYANWATG SEQ ID NO: 46 ASIAGYSAFDP

[0068] In one embodiment sequences 41 to 43 are in a light chain of the antibody.

[0069] In one embodiment sequences 44 to 46 are in a heavy chain of the antibody.

[0070] In one embodiment SEQ ID NO: 41 is CDR L1, SEQ ID NO: 42 is CDR L2 and SEQ ID NO; 43 is CDR L3.

[0071] In one embodiment SEQ ID NO: 44 is CDR H1, SEQ ID NO: 45 is CDR H2 and SEQ ID NO: 46 is CDR H3.

[0072] In one embodiment SEQ ID NO: 41 is CDR L1, SEQ ID NO: 42 is CDR L2, SEQ ID NO; 43 is CDR L3, SEQ ID NO: 44 is CDR H1, SEQ ID NO: 45 is CDR H2 and SEQ ID NO; 46 is CDR H3.

[0073] In one embodiment there is provided a variable region, such as a light chain variable region with the following sequence (Antibody 997 anti-toxin A antibody; Light chain Variable region sequence) SEQ ID NO: 47:

TABLE-US-00014 ALVMTQSPSSFSASTGDRVTITCQASQSISSYLSWYQQKPGKAPKLLIYR ASTLASGVPSRFSGSGSGTEYTLTISCLQSEDFATYYCLGVYGYSNDDGI AFGGGTKVEIK

wherein the CDRs are underlined

[0074] The polynucleotide sequence encoding SEQ ID NO: 47 is shown in FIG. 3 and SEQ ID NO: 48 therein.

[0075] In one embodiment there is provided a variable region, such as a heavy chain variable region with the following sequence (Antibody 997 anti-toxin A antibody heavy chain variable region sequence) SEQ ID NO: 49:

TABLE-US-00015 EVQLVESGGGLVQPGGSLRLSCTVSGIDLSSHHMCWVRQAPGKGLEYIGV IYHFGSTYYANWATGRFTISKDSTTVYLQMNSLRAEDTATYFCARASIAG YSAFDPWGQGTLVTVS

wherein the CDRs are underlined

[0076] The polynucleotide sequence encoding SEQ ID NO: 49 is shown in FIG. 4 and SEQ ID NO: 50 therein.

[0077] In one embodiment an antibody according to the invention comprises variable regions shown in SEQ ID NO: 47 and SEQ ID NO: 49.

[0078] In one embodiment there is provided an antibody (for example an anti-toxin A antibody) comprising a CDR, such as 1, 2, 3, 4, 5 or 6 CDRs, selected from:

TABLE-US-00016 SEQ ID NO: 51 QASQSIYSYLA SEQ ID NO: 52 DASTLAS SEQ ID NO: 53 QGNAYTSNSHDNA SEQ ID NO: 54 GIDLSSDAVG SEQ ID NO: 55 IIATFDSTYYASWAKG SEQ ID NO: 56 TGSWYYISGWGSYYYGMDL

[0079] In one embodiment sequences 51 to 53 are in a light chain of the antibody.

[0080] In one embodiment sequences 54 to 56 are in a heavy chain of the antibody.

[0081] In one embodiment SEQ ID NO: 51 is CDR L1, SEQ ID NO: 52 is CDR L2 and SEQ ID NO: 53 is CDR L3.

[0082] In one embodiment SEQ ID NO: 54 is CDR H1, SEQ ID NO: 55 is CDR H2 and SEQ ID NO; 56 is CDR H3.

[0083] In one embodiment SEQ ID NO: 51 is CDR L1, SEQ ID NO: 52 is CDR L2, SEQ ID NO; 53 is CDR L3, SEQ ID NO: 54 is CDR H1, SEQ ID NO: 55 is CDR H2 and SEQ ID NO: 56 is CDR H3.

[0084] In one embodiment there is provided a variable region, such as a light chain variable region with the following sequence (Antibody 1000 anti-toxin A antibody; Light chain Variable region sequence) SEQ ID NO: 57:

TABLE-US-00017 EIVMTQSPSTLSASVGDRVTITCQASQSIYSYLAWYQQKPGKAPKLLIYD ASTLASGVPSRFKGSGSGTEFTLTISSLQPDDFATYYCQGNAYTSNSHDN AFGGGTKVEIK

wherein the CDRs are underlined.

[0085] The polynucleotide sequence encoding SEQ ID NO: 57 is shown in FIG. 4 and SEQ ID NO: 58 therein.

[0086] In one embodiment there is provided a variable region, such as a heavy chain variable region with the following sequence (Antibody 1000 anti-toxin A antibody heavy chain variable region sequence) SEQ ID NO: 59:

TABLE-US-00018 EVQLVESGGGLIQPGGSLRLSCTVSGIDLSSDAVGWVRQAPGKGLEYIGI IATFDSTYYASWAKGRFTISKASSTTVYLQMNSLRAEDTATYFCARTGSW YYISGWGSYYYGMDLWGQGTLVTVS

wherein the CDRs are underlined.

[0087] The polynucleotide sequence encoding SEQ ID NO: 59 is shown in FIG. 4 and SEQ ID NO: 60 therein.

[0088] In one embodiment an antibody according to the invention comprises variable regions shown in SEQ ID NO: 57 and SEQ ID NO: 59.

[0089] In one embodiment there is provided an antibody (for example an anti-toxin B antibody) comprising a CDR, such as 1, 2, 3, 4, 5 or 6 CDRs, selected from:

TABLE-US-00019 SEQ ID NO: 61 RASKSVSTLMH SEQ ID NO: 62 LASNLES SEQ ID NO: 63 QQTWNDPWT SEQ ID NO: 64 GFTFSNYGMA SEQ ID NO: 65 SISSSGGSTYYRDSVKG SEQ ID NO: 66 VIRGYVMDA

[0090] In one embodiment sequences 61 to 63 are in a light chain of the antibody.

[0091] In one embodiment sequences 64 to 66 are in a heavy chain of the antibody.

[0092] In one embodiment SEQ ID NO: 61 is CDR L1, SEQ ID NO: 62 is CDR L2 and SEQ ID NO: 63 is CDR L3.

[0093] In one embodiment SEQ ID NO: 64 is CDR H1, SEQ ID NO: 65 is CDR H2 and SEQ ID NO: 66 is CDR H3.

[0094] In one embodiment SEQ ID NO: 61 is CDR L1, SEQ ID NO: 62 is CDR L2, SEQ ID NO; 63 is CDR L3, SEQ ID NO: 64 is CDR H1, SEQ ID NO: 65 is CDR H2 and SEQ ID NO: 66 is CDR H3.

[0095] In one embodiment there is provided a variable region, such as a light chain variable region with the following sequence (Antibody 926 anti-toxin B antibody; Light chain Variable region sequence) SEQ ID NO: 67:

TABLE-US-00020 DTVLTQSPATLSLSPGERATLSCRASKSVSTLMHWFQQKPGQAPKLLIYL ASNLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQTWNDPWTFGG GTKVEIK

wherein the CDRs are underlined.

[0096] The polynucleotide sequence encoding SEQ ID NO: 67 is shown in FIG. 5 and SEQ ID NO: 68 therein.

[0097] In one embodiment there is provided a variable region, such as a heavy chain variable region with the following sequence (Antibody 926 anti-toxin B antibody heavy chain variable region sequence) SEQ ID NO: 69:

TABLE-US-00021 EVELLESGGGLVQPGGSLRLSCEASGFTFSNYGMAWVRQAPTKGLEWVTS ISSSGGSTYYRDSVKGRFTISRDNAKSSLYLQMNSLRAEDTATYYCTTVI RGYVMDAWGQGTLVTVS

wherein the CDRs are underlined.

[0098] The polynucleotide sequence encoding SEQ ID NO: 69 is shown in FIG. 5 and SEQ ID NO: 70 therein.

[0099] In one embodiment there is provided an antibody (for example an anti-toxin B antibody) comprising a CDR, such as 1, 2, 3, 4, 5 or 6 CDRs, selected from:

TABLE-US-00022 SEQ ID NO: 71 RASGSVSTLMH SEQ ID NO: 72 KASNLAS SEQ ID NO: 73 HQSWNSDT SEQ ID NO: 74 GFTFSNYGMA SEQ ID NO: 75 TINYDGRTTHYRDSVKG SEQ ID NO: 76 ISRSHYFDC

[0100] In one embodiment sequences 71 to 73 are in a light chain of the antibody.

[0101] In one embodiment sequences 74 to 76 are in a heavy chain of the antibody.

[0102] In one embodiment SEQ ID NO: 71 is CDR L1, SEQ ID NO: 72 is CDR L2 and SEQ ID NO: 73 is CDR L3.

[0103] In one embodiment SEQ ID NO: 74 is CDR H1, SEQ ID NO: 75 is CDR H2 and SEQ ID NO: 76 is CDR H3.

[0104] In one embodiment SEQ ID NO: 71 is CDR L1, SEQ ID NO: 72 is CDR L2, SEQ ID NO; 73 is CDR L3, SEQ ID NO: 74 is CDR H1, SEQ ID NO: 75 is CDR H2 and SEQ ID NO: 76 is CDR H3.

[0105] In one embodiment there is provided a variable region, such as a light chain variable region with the following sequence (Antibody 927 anti-toxin B antibody; Light chain Variable region sequence) SEQ ID NO: 77:

TABLE-US-00023 DTQMTQSPSTLSASVGDRVTITCRASGSVSTLMHWYQQKPGKAPKLLIYK ASNLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCHQSWNSDTFGQG TRLEIK

wherein the CDRs are underlined

[0106] The polynucleotide sequence encoding SEQ ID NO: 77 is shown in FIG. 5 and SEQ ID NO: 78 therein.

[0107] In one embodiment there is provided a variable region, such as a heavy chain variable region with the following sequence (Antibody 927 anti-toxin B antibody heavy chain variable region sequence) SEQ ID NO: 79:

TABLE-US-00024 EVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMAWVRQAPGKGLEWVAT INYDGRTTHYRDSVKGRFTISRDNSKSTLYLQMNSLRAEDTAVYYCTSIS RSHYFDCWGQGTLVTVS

wherein the CDRs are underlined.

[0108] The polynucleotide sequence encoding SEQ ID NO: 79 is shown in FIG. 5 and SEQ ID NO: 80 therein.

[0109] In one embodiment an antibody according to the invention comprises variable regions shown in SEQ ID NO: 77 and SEQ ID NO: 79.

[0110] In one embodiment there is provided an antibody (for example an anti-toxin B antibody) comprising a CDR, such as 1, 2, 3, 4, 5 or 6 CDRs, selected from:

TABLE-US-00025 SEQ ID NO: 81 KASKSISNHLA SEQ ID NO: 82 SGSTLQS SEQ ID NO: 83 QQYDEYPYT SEQ ID NO: 84 GFSLQSYTIS SEQ ID NO: 85 AISGGGSTYYNLPLKS SEQ ID NO: 86 PRWYPRSYFDY

[0111] In one embodiment sequences 81 to 83 are in a light chain of the antibody.

[0112] In one embodiment sequences 84 to 86 are in a heavy chain of the antibody.

[0113] In one embodiment SEQ ID NO: 81 is CDR L1, SEQ ID NO: 82 is CDR L2 and SEQ ID NO: 83 is CDR L3.

[0114] In one embodiment SEQ ID NO: 84 is CDR H1, SEQ ID NO: 85 is CDR H2 and SEQ ID NO: 86 is CDR H3.

[0115] In one embodiment SEQ ID NO: 81 is CDR L1, SEQ ID NO: 82 is CDR L2, SEQ ID NO; 83 is CDR L3, SEQ ID NO: 84 is CDR H1, SEQ ID NO: 85 is CDR H2 and SEQ ID NO: 86 is CDR H3.

[0116] In one embodiment there is provided a variable region, such as a light chain variable region with the following sequence (Antibody 1099 anti-toxin B antibody; Light chain Variable region sequence) SEQ ID NO: 87:

TABLE-US-00026 DVQLTQSPSFLSASVGDRVTITCKASKSISNHLAWYQEKPGKANKLLIHS GSTLQSGTPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYDEYPYTFGQ GTRLEIKRT

wherein the CDRs are underlined.

[0117] In one embodiment the last two amino acids (RT) of SEQ ID NO: 87 are omitted.

[0118] The polynucleotide sequence encoding SEQ ID NO: 87 is shown in FIG. 6 and SEQ ID NO: 88 therein. In one embodiment the codons encoding the last two amino acids (RT) are omitted.

[0119] In one embodiment there is provided a variable region, such as a heavy chain variable region with the following sequence (Antibody 1099 anti-toxin B antibody heavy chain variable region sequence) SEQ ID NO: 89:

TABLE-US-00027 EVQLQESGPGLVKPSETLSLTCTVSGFSLQSYTISWVRQPPGKGLEWIAA ISGGGSTYYNLPLKSRVTISRDTSKSQVSLKLSSVTAADTAVYYCTRPRW YPRSYFDYWGRGTLVTVS

wherein the CDRs are underlined

[0120] The polynucleotide sequence encoding SEQ ID NO: 89 is shown in FIG. 6 and SEQ ID NO: 90 therein.

[0121] In one embodiment an antibody according to the invention comprises variable regions shown in SEQ ID NO 87: and SEQ ID NO: 89.

[0122] In one embodiment there is provided an antibody (for example an anti-toxin B antibody) comprising a CDR, such as 1, 2, 3, 4, 5 or 6 CDRs, selected from:

TABLE-US-00028 SEQ ID NO: 91 RASQRISTSIH SEQ ID NO: 92 YASQSIS SEQ ID NO: 93 QQSYSSLYT SEQ ID NO: 94 GFTFSDSYMA SEQ ID NO: 95 SISYGGTIIQYGDSVKG SEQ ID NO: 96 RQGTYARYLDF

[0123] In one embodiment sequences 91 to 93 are in a light chain of the antibody.

[0124] In one embodiment sequences 94 to 96 are in a heavy chain of the antibody.

[0125] In one embodiment SEQ ID NO: 91 is CDR L1, SEQ ID NO: 92 is CDR L2 and SEQ ID NO; 93 is CDR L3.

[0126] In one embodiment SEQ ID NO: 94 is CDR H1, SEQ ID NO: 95 is CDR H2 and SEQ ID NO: 96 is CDR H3.

[0127] In one embodiment SEQ ID NO: 91 is CDR L1, SEQ ID NO: 92 is CDR L2, SEQ ID NO; 93 is CDR L3, SEQ ID NO: 94 is CDR H1, SEQ ID NO: 95 is CDR H2 and SEQ ID NO: 96 is CDR H3.

[0128] In one embodiment there is provided a variable region, such as a light chain variable region with the following sequence (Antibody 1102 anti-toxin B antibody; Light chain Variable region sequence) SEQ ID NO: 97:

TABLE-US-00029 NIVLTQSPATLSLSPGERATLSCRASQRISTSIHWYQQKPGQAPRLLIKY ASQSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSYSSLYTFGQ GTKLEIK

wherein the CDRs are underlined

[0129] The polynucleotide sequence encoding SEQ ID NO: 97 is shown in FIG. 6 and SEQ ID NO: 98 therein.

[0130] In one embodiment there is provided a variable region, such as a heavy chain variable region with the following sequence (Antibody 1102 anti-toxin B antibody heavy chain variable region sequence) SEQ ID NO: 99:

TABLE-US-00030 EVQLVESGGGLVQPGGSLRLSCAVSGFTFSDSYMAWVRQAPGKGLEWIAS ISYGGTIIQYGDSVKGRFTISRDNAKSSLYLQMNSLRAEDTAVYYCARRQ GTYARYLDFWGQGTLVTVS

wherein the CDRs are underlined.

[0131] The polynucleotide sequence encoding SEQ ID NO: 99 is shown in FIG. 7 and SEQ ID NO: 100 therein.

[0132] In one embodiment an antibody according to the invention comprises variable regions shown in SEQ ID NO 97: and SEQ ID NO: 99.

[0133] In one embodiment there is provided an antibody (for example an anti-toxin B antibody) comprising a CDR, such as 1, 2, 3, 4, 5 or 6 CDRs, selected from:

TABLE-US-00031 SEQ ID NO: 101 RASESVSTLLH SEQ ID NO: 102 KASNLAS SEQ ID NO: 103 HQSWNSPPT SEQ ID NO: 104 GFTFSNYGMA SEQ ID NO: 105 IINYDASTTHYRDSVKG SEQ ID NO: 106 YGRSHYFDY

[0134] In one embodiment sequences 101 to 103 are in a light chain of the antibody.

[0135] In one embodiment sequences 104 to 106 are in a heavy chain of the antibody.

[0136] In one embodiment SEQ ID NO: 101 is CDR L1, SEQ ID NO: 102 is CDR L2 and SEQ ID NO: 103 is CDR L3.

[0137] In one embodiment SEQ ID NO: 104 is CDR H1, SEQ ID NO: 105 is CDR H2 and SEQ ID NO: 106 is CDR H3.

[0138] In one embodiment SEQ ID NO: 101 is CDR L1, SEQ ID NO: 102 is CDR L2, SEQ ID NO; 103 is CDR L3, SEQ ID NO: 104 is CDR H1, SEQ ID NO: 105 is CDR H2 and SEQ ID NO; 106 is CDR H3.

[0139] In one embodiment there is provided a variable region, such as a light chain variable region with the following sequence (Antibody 1114 anti-toxin B antibody; Light chain Variable region sequence) SEQ ID NO: 107:

TABLE-US-00032 ATQMTQSPSSLSASVGDRVTITCRASESVSTLLHWYQQKPGKAPKLLIYK ASNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQSWNSPPTFGQ GTKLEIK

wherein the CDRs are underlined.

[0140] The polynucleotide sequence encoding SEQ ID NO: 107 is shown in FIG. 7 and SEQ ID NO: 108 therein.

[0141] In one embodiment there is provided a variable region, such as a heavy chain variable region with the following sequence (Antibody 1114 anti-toxin B antibody heavy chain variable region sequence) SEQ ID NO: 109:

TABLE-US-00033 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMAWVRQAPGKGLEWVAI INYDASTTHYRDSVKGRFTISRDNAKSSLYLQMNSLRAEDTAVYYCTRYG RSHYFDYWGQGTLVTVS

wherein the CDRs are underlined.

[0142] The polynucleotide sequence encoding SEQ ID NO: 109 is shown in FIG. 7 and SEQ ID NO: 110 therein.

[0143] In one embodiment an antibody according to the invention comprises variable regions shown in SEQ ID NO: 107 and SEQ ID NO: 109.

[0144] In one embodiment there is provided an antibody (for example an anti-toxin B antibody) comprising a CDR, such as 1, 2, 3, 4, 5 or 6 CDRs, selected from:

TABLE-US-00034 SEQ ID NO: 111 RASESVSTLLH SEQ ID NO: 112 KASNLAS SEQ ID NO: 113 HQSWNSPPT SEQ ID NO: 114 GFTFSNYGMA SEQ ID NO: 115 IINYDASTTHYRDSVK SEQ ID NO: 116 YGRSHYFDY

[0145] In one embodiment sequences 111 to 113 are in a light chain of the antibody.

[0146] In one embodiment sequences 114 to 116 are in a heavy chain of the antibody.

[0147] In one embodiment SEQ ID NO: 111 is CDR L1, SEQ ID NO: 112 is CDR L2 and SEQ ID NO: 113 is CDR L3.

[0148] In one embodiment SEQ ID NO: 114 is CDR H1, SEQ ID NO: 115 is CDR H2 and SEQ ID NO: 116 is CDR H3.

[0149] In one embodiment SEQ ID NO: 111 is CDR L1, SEQ ID NO: 112 is CDR L2, SEQ ID NO; 113 is CDR L3, SEQ ID NO: 114 is CDR H1, SEQ ID NO: 115 is CDR H2 and SEQ ID NO: 116 is CDR H3.

[0150] In one embodiment there is provided a variable region, such as a light chain variable region with the following sequence (Antibody 1114 graft 8 anti-toxin B antibody; Light chain Variable region sequence) SEQ ID NO: 117:

TABLE-US-00035 DTVLTQSPSSLSASVGDRVTITCRASESVSTLLHWYQQKPGKAPKLLIYK ASNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQSWNSPPTFGQ GTKLEIK

wherein the CDRs are underlined.

[0151] The polynucleotide sequence encoding SEQ ID NO: 117 is shown in FIG. 8 and SEQ ID NO: 118 therein.

[0152] In one embodiment there is provided a variable region, such as a heavy chain variable region with the following sequence (Antibody 1114 graft 8 anti-toxin B antibody heavy chain variable region sequence) SEQ ID NO: 119:

TABLE-US-00036 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMAWVRQAPGKGLEWVAI INYDASTTHYRDSVKGRFTISRDNAKSSLYLQMNSLRAEDTAVYYCTRYG RSHYFDYWGQGTLVTVS

wherein the CDRs are underlined.

[0153] The polynucleotide sequence encoding SEQ ID NO: 119 is shown in FIG. 8 and SEQ ID NO: 120 therein.

[0154] In one embodiment an antibody according to the invention comprises variable regions shown in SEQ ID NO: 117 and SEQ ID NO: 119.

[0155] In one embodiment there is provided an antibody (for example an anti-toxin B antibody) comprising a CDR, such as 1, 2, 3, 4, 5 or 6 CDRs, selected from:

TABLE-US-00037 SEQ ID NO: 121 KASQNIYMYLN SEQ ID NO: 122 NTNKLHT SEQ ID NO: 123 LQHKSFPYT SEQ ID NO: 124 GFTFRDSFMA SEQ ID NO: 125 SISYEGDKTYYGDSVKG SEQ ID NO: 126 LTITTSGDS

[0156] In one embodiment sequences 121 to 123 are in a light chain of the antibody.

[0157] In one embodiment sequences 124 to 126 are in a heavy chain of the antibody.

[0158] In one embodiment SEQ ID NO: 121 is CDR L1, SEQ ID NO: 122 is CDR L2 and SEQ ID NO: 123 is CDR L3.

[0159] In one embodiment SEQ ID NO: 124 is CDR H1, SEQ ID NO: 125 is CDR H2 and SEQ ID NO: 126 is CDR H3.

[0160] In one embodiment SEQ ID NO: 121 is CDR L1, SEQ ID NO: 122 is CDR L2, SEQ ID NO: 123 is CDR L3, SEQ ID NO: 124 is CDR H1, SEQ ID NO: 125 is CDR H2 and SEQ ID NO: 126 is CDR H3.

[0161] In one embodiment there is provided a variable region, such as a light chain variable region with the following sequence (Antibody 1125 anti-toxin B antibody; Light chain Variable region sequence) SEQ ID NO: 127:

TABLE-US-00038 DIQMTQSPSSLSASVGDRVTITCKASQNIYMYLNWYQQKPGKAPKRLIYN TNKLHTGVPSRFSGSGSGTEYTLTISSLQPEDFATYYCLQHKSFPYTFGQ GTKLEIK

wherein the CDRs are underlined.

[0162] The polynucleotide sequence encoding SEQ ID NO: 127 is shown in FIG. 8 and SEQ ID NO: 128 therein.

[0163] In one embodiment there is provided a variable region, such as a heavy chain variable region with the following sequence (Antibody 1125 anti-toxin B antibody heavy chain variable region sequence) SEQ ID NO: 129:

TABLE-US-00039 EVQLVESGGGLVQPGGSLRLSCAASGFTFRDSFMAWVRQAPGKGLEWVAS ISYEGDKTYYGDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARLT ITTSGDSWGQGTMVTVSS

wherein the CDRs are underlined.

[0164] In one embodiment the last amino acid (S) of SEQ ID NO: 129 is omitted.

[0165] The polynucleotide sequence encoding SEQ ID NO: 129 is shown in FIG. 9 and SEQ ID NO: 130 therein. In one embodiment the codon AGC encoding the last amino acid S is omitted.

[0166] In one embodiment an antibody according to the invention comprises variable regions shown in SEQ ID NO: 127 and SEQ ID NO: 129.

[0167] In one embodiment there is provided antibody (for example an anti-toxin B antibody) comprising a CDR, such as 1, 2, 3, 4, 5 or 6 CDRs, selected from:

TABLE-US-00040 SEQ ID NO: 131 KASQHVGTNVD SEQ ID NO: 132 GASIRYT SEQ ID NO: 133 LQYNYNPYT SEQ ID NO: 134 GFIFSNFGMS SEQ ID NO: 135 SISPSGGNAYYRDSVKG SEQ ID NO: 136 RAYSSPFAF

[0168] In one embodiment sequences 131 to 133 are in a light chain of the antibody.

[0169] In one embodiment sequences 134 to 136 are in a heavy chain of the antibody.

[0170] In one embodiment SEQ ID NO: 131 is CDR L1, SEQ ID NO: 132 is CDR L2 and SEQ ID NO: 133 is CDR L3.

[0171] In one embodiment SEQ ID NO: 134 is CDR H1, SEQ ID NO: 135 is CDR H2 and SEQ ID NO: 136 is CDR H3.

[0172] In one embodiment SEQ ID NO: 131 is CDR L1, SEQ ID NO: 132 is CDR L2, SEQ ID NO: 133 is CDR L3, SEQ ID NO: 134 is CDR H1, SEQ ID NO: 135 is CDR H2 and SEQ ID NO: 136 is CDR H3.

[0173] In one embodiment there is provided a variable region, such as a light chain variable region with the following sequence (Antibody 1129 anti-toxin B antibody; Light chain Variable region sequence) SEQ ID NO: 137:

TABLE-US-00041 DTQMTQSPSSLSASVGDRVTITCKASQHVGTNVDWYQQKPGKVPKLLIYG ASIRYTGVPDRFTGSGSGTDFTLTISSLQPEDVATYYCLQYNYNPYTFGQ GTKLEIK

wherein the CDRs are underlined.

[0174] The polynucleotide sequence encoding SEQ ID NO: 137 is shown in FIG. 8 and SEQ ID NO: 138 therein.

[0175] In one embodiment there is provided a variable region, such as a heavy chain variable region with the following sequence (Antibody 1129 anti-toxin B antibody heavy chain variable region sequence) SEQ ID NO: 139:

TABLE-US-00042 EVQLVESGGGVVQPGRSLRLSCATSGFIFSNFGMSWVRQAPGKGLEWVAS ISPSGGNAYYRDSVKGRFTISRDNSKTTLYLQMNSLRAEDTAVYYCTRRA YSSPFAFWGQGTLVTVSS

wherein the CDRs are underlined.

[0176] In one embodiment the last amino acid (S) of SEQ ID NO: 139 is omitted.

[0177] The polynucleotide sequence encoding SEQ ID NO: 139 is shown in FIG. 8 and SEQ ID NO: 140 therein. In one embodiment the codon AGC encoding the last amino acid S is omitted.

[0178] In one embodiment an antibody according to the invention comprises variable regions shown in SEQ ID NO: 137 and SEQ ID NO: 139.

[0179] In one embodiment there is provided an antibody (for example an anti-toxin B antibody) comprising a CDR, such as 1, 2, 3, 4, 5 or 6 CDRs, selected from:

TABLE-US-00043 SEQ ID NO: 141 KASKSISNHLA SEQ ID NO: 142 SGSTLQP SEQ ID NO: 143 QQYDEYPYT SEQ ID NO: 144 GFSLNSYTIT SEQ ID NO: 145 AISGGGSTYFNSALKS SEQ ID NO: 146 PRWYPRSYFDY

[0180] In one embodiment sequences 141 to 143 are in a light chain of the antibody.

[0181] In one embodiment sequences 144 to 146 are in a heavy chain of the antibody.

[0182] In one embodiment SEQ ID NO: 141 is CDR L1, SEQ ID NO: 142 is CDR L2 and SEQ ID NO: 143 is CDR L3.

[0183] In one embodiment SEQ ID NO: 144 is CDR H1, SEQ ID NO: 145 is CDR H2 and SEQ ID NO: 146 is CDR H3.

[0184] In one embodiment SEQ ID NO: 141 is CDR L1, SEQ ID NO: 142 is CDR L2, SEQ ID NO: 143 is CDR L3, SEQ ID NO: 144 is CDR H1, SEQ ID NO: 145 is CDR H2 and SEQ ID NO: 146 is CDR H3.

[0185] In one embodiment there is provided a variable region, such as a light chain variable region with the following sequence (Antibody 1134 anti-toxin B antibody; Light chain Variable region sequence):

TABLE-US-00044 SEQ ID NO: 147 DVQLTQSPSFLSASVGDRVTITCKASKSISNHLAWYQEKPGKANKLLIH SGSTLQPGTPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYDEYPYTF GQGTRLEIK

wherein the CDRs are underlined.

[0186] The polynucleotide sequence encoding SEQ ID NO: 147 is shown in FIG. 9 and SEQ ID NO: 148 therein.

[0187] In one embodiment there is provided a variable region, such as a heavy chain variable region with the following sequence (Antibody 1134 anti-toxin B antibody heavy chain variable region sequence) SEQ ID NO: 149:

TABLE-US-00045 EVQLQESGPGLVKPSETLSLTCTVSGFSLNSYTITWVRQPPGKGLEWIA AISGGGSTYFNSALKSRVTISRDTSKSQVSLKLSSVTAADTAVYYCTRP RWYPRSYFDYWGRGTLVTVS

wherein the CDRs are underlined

[0188] The polynucleotide sequence encoding SEQ ID NO: 149 is shown in FIG. 9 and SEQ ID NO: 150 therein.

[0189] In one embodiment an antibody according to the invention comprises variable regions shown in SEQ ID NO 147: and SEQ ID NO: 149.

[0190] In one embodiment there is provided antibody (for example an anti-toxin B antibody) comprising a CDR, such as 1, 2, 3, 4, 5 or 6 CDRs, selected from:

TABLE-US-00046 SEQ ID NO: 151 KASQNVGNNVA SEQ ID NO: 152 YASNRFT SEQ ID NO: 153 QRVYQSTWT SEQ ID NO: 154 GFSLTSYYVH SEQ ID NO: 155 CIRTGGNTEYQSEFKS SEQ ID NO: 156 GNYGFAY

[0191] In one embodiment sequences 151 to 153 are in a light chain of the antibody.

[0192] In one embodiment sequences 154 to 156 are in a heavy chain of the antibody.

[0193] In one embodiment SEQ ID NO: 151 is CDR L1, SEQ ID NO: 152 is CDR L2 and SEQ ID NO: 153 is CDR L3.

[0194] In one embodiment SEQ ID NO: 154 is CDR H1, SEQ ID NO: 155 is CDR H2 and SEQ ID NO: 156 is CDR H3.

[0195] In one embodiment SEQ ID NO: 151 is CDR L1, SEQ ID NO: 152 is CDR L2, SEQ ID NO; 153 is CDR L3, SEQ ID NO: 154 is CDR H1, SEQ ID NO: 155 is CDR H2 and SEQ ID NO; 156 is CDR H3.

[0196] In one embodiment there is provided a variable region, such as a light chain variable region with the following sequence (Antibody 1151 anti-toxin B antibody; Light chain Variable region sequence) SEQ ID NO: 157:

TABLE-US-00047 AIQMTQSPSSLSASVGDRVTITCKASQNVGNNVAWYQHKPGKAPKLLIY YASNRFTGVPSRFTGGGYGTDFTLTISSLQPEDFATYYCQRVYQSTWTF GQGTKVEIK

wherein the CDRs are underlined.

[0197] The polynucleotide sequence encoding SEQ ID NO: 157 is shown in FIG. 9 and SEQ ID NO: 158 therein.

[0198] In one embodiment there is provided a variable region, such as a heavy chain variable region with the following sequence (Antibody 1151 anti-toxin B antibody heavy chain variable region sequence) SEQ ID NO: 159:

TABLE-US-00048 EVQLQESGPGLVKPSETLSLTCTVSGFSLTSYYVHWVRQPPGKGLEWMG CIRTGGNTEYQSEFKSRVTISRDTSKNQVSLKLSSVTAADTAVYYCARG NYGFAYWGQGTLVTVS

wherein the CDRs are underlined.

[0199] The polynucleotide sequence encoding SEQ ID NO: 159 is shown in FIG. 9 and SEQ ID NO: 160 therein.

[0200] In one embodiment an antibody according to the invention comprises variable regions shown in SEQ ID NO: 157 and SEQ ID NO: 159.

[0201] In one embodiment there is provided an antibody (for example an anti-toxin B antibody) comprising a CDR, such as 1, 2, 3, 4, 5 or 6 CDRs, selected from:

TABLE-US-00049 SEQ ID NO: 161 KASQNINKYLD SEQ ID NO: 162 NIQSLHT SEQ ID NO: 163 FQHNSGW SEQ ID NO: 164 GFTFTQAAMF SEQ ID NO: 165 RISTKSNNFATYYPDSVKG SEQ ID NO: 166 PAYYYDGTVPFAY

[0202] In one embodiment sequences 161 to 163 are in a light chain of the antibody.

[0203] In one embodiment sequences 164 to 166 are in a heavy chain of the antibody.

[0204] In one embodiment SEQ ID NO: 161 is CDR L1, SEQ ID NO: 162 is CDR L2 and SEQ ID NO: 163 is CDR L3.

[0205] In one embodiment SEQ ID NO: 164 is CDR H1, SEQ ID NO: 165 is CDR H2 and SEQ ID NO: 166 is CDR H3.

[0206] In one embodiment SEQ ID NO: 161 is CDR L1, SEQ ID NO: 162 is CDR L2, SEQ ID NO: 163 is CDR L3, SEQ ID NO: 164 is CDR H1, SEQ ID NO: 165 is CDR H2 and SEQ ID NO: 166 is CDR H3.

[0207] In one embodiment there is provided a variable region, such as a light chain variable region with the following sequence (Antibody 1153 anti-toxin B antibody; Light chain Variable region sequence) SEQ ID NO: 167:

TABLE-US-00050 DIQMTQSPSSLSASVGDRVTITCKASQNINKYLDWYQQKPGKVPKLLIY NIQSLHTGIPSRFSGSGSGTDFTLTISSLQPEDVATYYCFQHNSGWTFG QGTRLEIK

wherein the CDRs are underlined.

[0208] The polynucleotide sequence encoding SEQ ID NO: 167 is shown in FIG. 10 and SEQ ID NO: 168 therein.

[0209] In one embodiment there is provided a variable region, such as a heavy chain variable region with the following sequence (Antibody 1153 anti-toxin B antibody heavy chain variable region sequence) SEQ ID NO: 169:

TABLE-US-00051 EVQLVESGGGLVQPGGSLKLSCAASGFTFTQAAMFWVRQASGKGLEGIA RISTKSNNFATYYPDSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYC TAPAYYYDGTVPFAYWGQGTLVTVS

wherein the CDRs are underlined.

[0210] The polynucleotide sequence encoding SEQ ID NO: 169 is shown in FIG. 10 and SEQ ID NO: 170 therein.

[0211] In one embodiment an antibody according to the invention comprises variable regions shown in SEQ ID NO: 167 and SEQ ID NO: 169.

[0212] In one embodiment there is provided antibody comprising 6 CDRs independently selected from SEQ ID NOs 1, 2, 3, 4, 5, 6, 11, 12, 13, 14, 15, 16, 21, 22, 23, 24, 25, 26, 31, 32, 33, 34, 35, 36, 41, 42, 43, 44, 45, 46, 51, 52, 53, 54, 55, 56, 61, 62, 63, 64, 65, 66, 71, 72, 73, 74, 75, 76, 81, 82, 83, 84, 85, 86, 91, 92, 93, 94, 95, 96, 101, 102, 103, 104, 105, 106, 111, 112, 113, 114, 115, 116, 121, 122, 123, 124, 125, 126, 131, 132, 133, 134, 135, 136, 141, 142, 143, 144, 145, 146, 151, 152, 153, 154, 155, 156, 161, 162, 163, 164, 165 and 166.

[0213] In one embodiment there is provided an anti-TcdA antibody comprising 6 CDRs independently selected from SEQ ID NOs 1, 2, 3, 4, 5, 6, 11, 12, 13, 14, 15, 16, 21, 22, 23, 24, 25, 26, 31, 32, 33, 34, 35, 36, 41, 42, 43, 44, 45, 46, 51, 52, 53, 54, 55 and 56.

[0214] In one embodiment there is provided an anti-TcdB antibody comprising 6 CDRs independently selected from SEQ ID NOs 61, 62, 63, 64, 65, 66, 71, 72, 73, 74, 75, 76, 81, 82, 83, 84, 85, 86, 91, 92, 93, 94, 95, 96, 101, 102, 103, 104, 105, 106, 111, 112, 113, 114, 115, 116, 121, 122, 123, 124, 125, 126, 131, 132, 133, 134, 135, 136, 141, 142, 143, 144, 145, 146, 151, 152, 153, 154, 155, 156, 161, 162, 163, 164, 165 and 166.

[0215] In one embodiment there is provided an antibody which comprises two variable regions independently selected from SEQ ID NOs: 7, 9, 17, 19, 27, 29, 37, 39, 47, 49, 57, 59, 67, 69, 77, 79, 87, 89, 97, 99, 107, 109, 117, 119, 127, 129, 137, 139, 147, 149, 157 and 159.

[0216] In one embodiment there is provided an antibody which comprises two variable regions independently selected from SEQ ID NOs: 7, 9, 17, 19, 27, 29, 37, 39, 47, 49, 57 and 59.

[0217] In one embodiment there is provided an antibody which comprises two variable regions independently selected from SEQ ID NOs: 67, 69, 77, 79, 87, 89, 97, 99, 107, 109, 117, 119, 127, 129, 137, 139, 147, 149, 157 and 159.

[0218] In one embodiment the antibodies according to the invention are humanized.

[0219] In one embodiment the antibody or antibodies are directed to the C terminal "cell binding" portion of the TcdA and/or TcdB toxin.

[0220] In one embodiment an antibody according to the invention is suitable for neutralising toxin A or toxin B.

[0221] Neutralising as employed herein is intended to refer to the elimination or reduction of harmful/deleterious effects of the target toxin, for example at least a 50% reduction in the relevant harmful effect.

[0222] The inventors have established by using internal comparisons between antibodies discovered in this application and by comparison against antibodies well described in the art (Babcock et al. 2006; Lowy et al., 2010) that some antibodies have the desirable characteristic of maintaining effective neutralization (for example low EC.sub.50 and high % protection) even at high toxin concentrations. Other antibodies including those described in the art do not maintain effective toxin neutralization at high toxin concentrations.

[0223] Effective toxin concentrations can be defined as a `lethal dose` (LD) in titration studies in the absence of neutralizing antibodies. Neutralisation assays are typically conducted at an LD of 50% of complete cell killing (i.e. an LD.sub.50) but may be more rigorously conducted at an LD.sub.50.

[0224] Assays may also be performed under considerably more challenging conditions such as LD.sub.90, LD.sub.95 and/or LD.sub.max (LD.sub.max is the maximal toxin quantity which can be included in an assay as constrained by assay volume and maximum toxin concentration/solubility). Such assays aim to mimic the early stages of infection of humans when C. difficile growth in the bowel is rampant and diarrhea and other symptoms lead one to hypothesise that toxin concentrations are at their highest. Antibodies which effectively neutralize damaging toxin activities under high toxin concentration conditions are thought by the present inventors to have special clinical value for the control of symptoms in human infections. In one embodiment the antibody or antibodies of the present disclosure have useful, for example low EC.sub.50 values and/or high % protection from cell death for one or more the LD.sub.80, LD.sub.90, LD.sub.95 and/or LD.sub.max. In one embodiment the EC.sub.50 in the one or more of the latter situations is 15 ng/ml or less, for example 10 ng/ml or less, such as 5 ng/ml or less, in particular 1 ng/ml or less. In one embodiment the % protection from cell death is >90%, or >75% or >50%.

[0225] Thus in one embodiment the present disclosure provides an antibody or a combination of antibodies which maintain toxin neutralization even in the presence of high levels of toxin, for example as measured in an assay provided herein.

[0226] The harmful effect of toxin may, for example be measured in a suitable in vitro assay. In one embodiment the neutralization is measured in an assay given in Example 1 below. Also provided is an antibody or antibodies identified in a neutralization assay, for example wherein the potency of the antibody is maintained in the presence of high levels of toxin.

[0227] Toxin A is used interchangeably with TcdA.

[0228] Toxin B is used interchangeably with TcdB.

[0229] In one embodiment an antibody according to the invention is a monoclonal antibody or binding fragment thereof.

[0230] In one embodiment a monoclonal antibody according to the invention is capable of neutralising TcdA with very high potency and affinity.

[0231] In one embodiment a monoclonal antibody according to the invention is capable of neutralising TcdA with very high potency and affinity and high avidity.

[0232] Avidity as employed herein refers to the combined strength of multiple binding affinities.

[0233] In one embodiment a monoclonal antibody according to the invention is capable of neutralising TcdA with very high potency and affinity and high avidity and high valency of binding.

[0234] Valency of binding as employed herein refers to the ability for a monoclonal antibody to bind to an antigen multiple times. High valency of binding hence results in high levels of decoration of antigen with antibodies and/or high levels of cross-linking of toxin molecules, which is thought to be advantageous.

[0235] Anti-TcdA Mabs according to the present disclosure may be suitable for neutralising the early effects of TcdA, for example on cells such as loss of tight junctions.

[0236] Tight junction as employed herein is intended to refer to impermeable zone of connection between cells within a monolayer or anatomical tissue structure. Fluid loss does not occur when tight junctions retain their structural and functional integrity. Loss of tight junctions is an indication that the cell has been compromised by toxin and is well documented as being an early step in the toxic effects of TcdA and TcdB (25) and results in loss of fluid containing serum, immunoglobulin and ions (26, 3). Loss of tight junctions is thought to be a first step on the onset of diarrhoea in humans.

[0237] The TEER assay system, can be used to measure the loss of tight junction in vitro. TEER is an acronym for trans epithelial electric resistance assay and it is generally employed to measure the permeability of a differentiated cell layer representative of a gut endothelial lining. However, in the context of screening for antibodies TEER loss can be employed to identify antibodies that slow or prevent damage to the tight junctions and hence is a surrogate for protection against tissue damage leading to diarrhoea.

[0238] Often Caco-2 cells are employed since they are derived from human colon cells and are known to form differentiated monolayers with cells connected by tight junctions. A kit is commercially available from Becton-Dickinson named the Caco-2 BioCoat HTS plate system (BD Biosciences/354802). The instructions in the kit are suitable for testing in the present context. The resistance of the membrane changes when the membrane has been compromised.

[0239] Generally the antibody will be pre-incubated with the toxin before addition to the TEER system to establish if the antibody can prevent or slow the damage to the membrane caused by the toxin. The assay may be performed over a suitable period, for example 24 hours taking measurements at certain time-points. The present inventors have established that the 4 hour time point is particularly discriminating for therapeutically useful antibodies. The concentration of toxin employed in the TEER assay is generally in the range 100-200 ng/ml, most preferably 125 ng/ml

[0240] The concentration of antibody (for example IgG1) employed in the TEER assay is generally in the range of 4 to 2000 ng/ml, for example 50 to 1000 ng/ml, such as 100 to 500 ng/ml.

[0241] In one embodiment the EC.sub.50 of the antibody in the TEER assay employed in said condition is at least 200 ng/ml, for example less than 100 ng/ml, such as about 60-80 ng/ml.

[0242] In one embodiment there is provided an anti-TcdA antibody or an anti-TcdB antibody suitable for use as a therapeutic agent in the treatment or prevention of C. difficile infection, wherein said antibody was screened and selected employing a TEER assay.

[0243] In one aspect there is provided a method of screening an antibody in a TEER assay for the ability to slow or prevent loss of tight junctions. In one embodiment the antibody or antibodies screened are anti-TcdA antibodies. In one embodiment the antibody or antibodies screened are anti-TcdB antibodies. In one embodiment the antibody or antibodies screened are a combination of anti-TcdA and anti-TcdB antibodies. In one embodiment the method comprises the step of identifying an appropriate antibody or antibodies and expressing suitable quantities of same. In one embodiment the method comprises the further step of formulating said antibody or antibodies in a pharmaceutical formulation. In one embodiment the method comprises the further step of administering said antibody or antibodies or said formulation to a patient in need thereof.

[0244] In one embodiment multiple antibodies of the disclosure may be capable of binding to the target toxin (TcdA or TcdB), which may aid immune clearance of the toxin.

[0245] Multiple antibodies as employed herein is intended to refer to multiple copies of an antibody with the same sequence or an antibody with the same amino acid sequence or an antibody specific to the same target antigen but with a different amino acid sequence.

[0246] In one embodiment the antibodies according to the invention are specific to the target antigen, for example specific to an epitope in the target antigen.

[0247] In one embodiment the antibodies of the invention are able to bind to the target antigen in two or more locations, for example two or three locations, such as four, five, six, seven, eight, nine, ten or more locations, for example the toxin may comprise repeating domains and thus an antibody may be specific to an epitope and in fact that epitope may be present in the antigen several times i.e. in more than one location. Thus given antibodies may bind the same epitope multiple times in different locations in the antigen.

[0248] In one embodiment the antibody binds to the given antigen multiple times, for example 2 to 20 times such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 times. In one embodiment the antibody binds the given antigen at least 3 times. This multiple binding is thought to be important in neutralisation and/or clearance of the toxin. Whilst not wishing to be bound by theory it is thought that multiple binding, for example 3 more times, i.e. by decoration with 3 or more Fc fragments is important in triggering rapid clearance of the toxin (24) primarily via the liver and spleen (27, 28).

[0249] In one embodiment the anti-TcdA antibody binds 3 or more times, for example 3 to 16 times.

[0250] In one embodiment the anti-TcdA antibody binds 12 times.

[0251] In one embodiment the anti-TcdA antibody binds 2 times.

[0252] In one embodiment an anti-TcdA antibody binds in the catalytic-terminal cell binding domain of TcdA.

[0253] In one embodiment the anti-TcdB antibody binds 2 or more times, for example 2 times.

[0254] In one embodiment an anti-TcdB antibody binds in the catalytic-terminal cell binding domain of TcdB.

[0255] In one embodiment the antibody or antibodies according to disclosure are capable of cross-linking toxin molecules, for example one arm of the antibody molecule binds one toxin molecule and another of the antibody binds a epitope in a different toxin molecule, thereby forming a sort of immune complex. The formation of the latter may also facilitate activation of the immune system to clear the relate toxin and thereby minimise the deleterious in vivo effects of the same.

[0256] In one embodiment an innate immune response, such as complement is activated.

[0257] In one embodiment the antibody or antibodies of the disclosure have high potency against toxins derived from strains of different ribotypes, for example 003, 027, 078.

[0258] In one embodiment antibodies against TcdA may have an EC.sub.50 in the range of 0.1-100 ng/ml, such as 1 to 10 ng/ml and a maximal inhibition in the range of 50-100% at toxin concentrations of LD.sub.80-95, for example against toxins from strains of ribotypes 003, 027 and 078.

[0259] In one embodiment antibodies against TcdA may have an EC.sub.50 in the range of 0.1 100 ng/ml, such as 1 to 10 ng/ml and a maximal inhibition in the range of 60-100%, 70-100%, 80-100% or 90-100% at toxin concentrations of LD.sub.80-95, for example against toxins from strains of ribotypes 003, 027 and 078.

[0260] In one embodiment antibodies against TcdB may have EC.sub.50 in the range of 0.1-100 ng/ml, such as 1 to 10 ng/ml and a maximal inhibition in the range of 50-100% at toxin concentrations of LD.sub.80-95, for example against toxins from strains of ribotype 003.

[0261] In one embodiment antibodies against TcdB may have EC.sub.50 in the range of 0.1-100 ng/ml, such as 1 to 10 ng/ml and a maximal inhibition in the range of 60-100%, 70-100%, 80-100% or 90-100% at toxin concentrations of LD.sub.80-95, for example against toxins from strains of ribotype 003.

[0262] In one embodiment there are provided combinations of antibodies according to the invention, for example combinations of antibodies specific to TcdA, combinations of antibodies specific to TcdB or combinations of antibodies to specific to TcdA and antibodies specific to TcdB.

[0263] Combinations of antibodies specific to TcdA will generally refer to combinations of antibodies directed to different epitopes on the target antigen TcdA, or at least with different binding properties.

[0264] Combinations of antibodies specific to TcdB will generally refer to combinations of antibodies directed to different epitopes on the target antigen TcdB, or at least with different binding properties.

[0265] The combinations may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 distinct antibodies, for example 2, 3, 4 or 5 antibodies.

[0266] In one embodiment there is provided a combination of one anti-TcdA antibody and two anti-TcdB, for example wherein the anti-TcdA antibody is 997 and where the anti-TcdB antibodies are 1125 and 1151

[0267] In particular there is provided a combination of one anti-TcdA antibody comprising a heavy variable region with a sequence as shown in SEQ ID NO:49 and a light variable region with a sequence shown in SEQ ID NO: 47 and two anti-TcdB antibodies the first with a heavy variable region shown in SEQ ID NO: 129 and a light variable region shown in SEQ ID NO: 127, and the second with a heavy variable region shown in SEQ ID NO: 159 and light variable region shown in SEQ ID NO: 157.

[0268] Distinct antibodies as employed herein is intended to refer to antibodies with different amino acid sequences, which may bind the same epitope or different epitopes on the target antigen.

[0269] Also provided by the present invention is a specific region or epitope of TcdA which is bound by an antibody provided by the present invention, in particular an antibody comprising the heavy chain sequence given in SEQ ID NO:49 and the light chain sequence given in SEQ ID NO:47.

[0270] Also provided by the present invention is a specific region or epitope of TcdB which is bound by an antibody provided by the present invention, in particular an antibody comprising the heavy chain sequence given in SEQ ID NO:129 and the light chain sequence given in SEQ ID NO:127 or an antibody comprising the heavy chain sequence given in SEQ ID NO:159 and the light chain sequence given in SEQ ID NO:157.

[0271] This specific region or epitope of the TcdA or TcdB toxins can be identified by any suitable epitope mapping method known in the art in combination with any one of the antibodies provided by the present invention. Examples of such methods include screening peptides of varying lengths derived from the toxins for binding to the antibody of the present invention with the smallest fragment that can specifically bind to the antibody containing the sequence of the epitope recognised by the antibody. The peptides may be produced synthetically or by proteolytic digestion of the toxin polypeptide. Peptides that bind the antibody can be identified by, for example, mass spectrometric analysis. In another example, NMR spectroscopy or X-ray crystallography can be used to identify the epitope bound by an antibody of the present invention. Once identified, the epitopic fragment which binds an antibody of the present invention can be used, if required, as an immunogen to obtain additional antagonistic antibodies which bind the same epitope.

[0272] Antibodies which cross-block the binding of an antibody according to the present invention may be similarly useful in neutralizing toxin activity. Accordingly, the present invention also provides a neutralizing antibody having specificity for TcdA or TcdB, which cross-blocks the binding of any one of the antibodies described above to TcdA or TcdB and/or is cross-blocked from binding these toxins by any one of those antibodies. In one embodiment, such an antibody binds to the same epitope as an antibody described herein above. In another embodiment the cross-blocking neutralising antibody binds to an epitope which borders and/or overlaps with the epitope bound by an antibody described herein above. In another embodiment the cross-blocking neutralising antibody of this aspect of the invention does not bind to the same epitope as an antibody of the present invention or an epitope that borders and/or overlaps with said epitope.

[0273] Cross-blocking antibodies can be identified using any suitable method in the art, for example by using competition ELISA or BIAcore assays where binding of the cross blocking antibody to TcdA or TcdB prevents the binding of an antibody of the present invention or vice versa.

[0274] In one embodiment there is provided a method of generating an anti-TcdA or anti-TcdB antibody, in particular a neutralizing antibody and/or an antibody which cross-blocks the binding of an antibody described herein, said method comprising the steps of immunizing a host with a suitable antigen, for example an antigen shown in any one of SEQ ID Nos 173 to 194 or a combination thereof. The said method may also comprise one or more the following steps, for example identifying an antibody of interest (in particular using a functional assay such as TEER assay), expressing the antibody of interest, and optionally formulating the antibody as a pharmaceutically acceptable composition.

[0275] Thus in one aspect the present disclosure provides a method of immunizing a host with an amino acid sequence shown in SEQ ID Nos 173 to 194 or a combination thereof.

[0276] In one embodiment the antibodies according to the invention have an affinity to the target antigen of 10 nM or less, for example 1 nM or less such as 900 pM, in particular 800 pM, 700 pM, 600 pM or 500 pM, such as 60 pM.

[0277] In one embodiment the affinity is for TcdA or TcdB or a fragment thereof. In one example the fragment is TcdA123 corresponding to residues S1827-D2249 of TcdA. In one example the fragment is TcdA456 corresponding to residues G2205-R2608. In one embodiment the fragment is TcdB1234 corresponding to residues S1833-E2366 of TcdB.

[0278] In one embodiment antibodies according to the invention or a combination thereof have an EC.sub.50 of 200 ng/ml or less, for example 150 ng/ml or less such as 100 ng/ml or less, such as in the range 0.1 to 10 ng/ml.

[0279] The individual component antibodies of mixtures are not required to have an EC.sub.50 in said range provided that when they are used in combination with one or more antibodies the combination has an EC.sub.50 in said range.

[0280] Advantageously, the antibodies of the invention are stable, for example are thermally stable at temperatures above 50.degree. C. such as 60 or 70.degree. C.

[0281] The antibodies and combinations according to the present invention also have one or more of the following advantageous properties: slow off rate, high affinity, high potency, the ability to bind multiple times to the target antigen, to neutralise the toxin by a mechanism which reduces the loss of measurable TEER activity, to stimulate or assist the hosts natural immune response, to catalyse or assist in immune clearance of the pathogen (or toxin) and/or to educate the immune system to respond appropriately to the pathogen (or toxin).

[0282] The residues in antibody variable domains are conventionally numbered according to a system devised by Kabat et al. This system is set forth in Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereafter "Kabat et al. (supra)"). This numbering system is used in the present specification except where otherwise indicated.

[0283] The Kabat residue designations do not always correspond directly with the linear numbering of the amino acid residues. The actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or complementarity determining region (CDR), of the basic variable domain structure. The correct Kabat numbering of residues may be determined for a given antibody by alignment of residues of homology in the sequence of the antibody with a "standard" Kabat numbered sequence.

[0284] The CDRs of the heavy chain variable domain are located at residues 31-35 (CDR-H1), residues 50-65 (CDR-H2) and residues 95-102 (CDR-H3) according to the Kabat numbering system. However, according to Chothia (Chothia, C. and Lesk, A. M. J. Mol. Biol., 196, 901-917 (1987)), the loop equivalent to CDR-H1 extends from residue 26 to residue 32. Thus unless indicated otherwise `CDR-H1` as employed herein is intended to refer to residues 26 to 35, as described by a combination of the Kabat numbering system and Chothia's topological loop definition.

[0285] The CDRs of the light chain variable domain are located at residues 24-34 (CDR-L1), residues 50-56 (CDR-L2) and residues 89-97 (CDR-L3) according to the Kabat numbering system.

[0286] Antibodies for use in the present invention may be obtained using any suitable method known in the art. The toxin A and/or toxin B polypeptide/protein including fusion proteins, for example toxin-Fc fusions proteins or cells (recombinantly or naturally) expressing the polypeptide (such as activated T cells) can be used to produce antibodies which specifically recognise the target toxins. The toxin polypeptide may be the full length polypeptide or a biologically active fragment or derivative thereof.

[0287] Polypeptides may be prepared by processes well known in the art from genetically engineered host cells comprising expression systems or they may be recovered from natural biological sources. In the present application, the term "polypeptides" includes peptides, polypeptides and proteins. These are used interchangeably unless otherwise specified. The sequence for TcdA from ribotype 027 is given in SEQ ID NO: 171 (Uniprot accession number C9YJ37) and the sequence for TcdB from ribotype 027 is given is SEQ ID NO: 172 (Uniprot accession number C9YJ35).

[0288] The antigen polypeptide may in some instances be part of a larger protein such as a fusion protein for example fused to an affinity tag.

[0289] Antibodies generated against the antigen polypeptide may be obtained, where immunisation of an animal is necessary, by administering the polypeptides to an animal, preferably a non-human animal, using well-known and routine protocols, see for example Handbook of Experimental Immunology, D. M. Weir (ed.), Vol 4, Blackwell Scientific Publishers, Oxford, England, 1986). Many warm-blooded animals, such as rabbits, mice, rats, sheep, cows, camels or pigs may be immunized. However, mice, rabbits, pigs and rats are generally most suitable.

[0290] Monoclonal antibodies may be prepared by any method known in the art such as the hybridoma technique (Kohler & Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today, 4:72) and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp 77-96, Alan R Liss, Inc., 1985).

[0291] Antibodies for use in the invention may also be generated using single lymphocyte antibody methods by cloning and expressing immunoglobulin variable region cDNAs generated from single lymphocytes selected for the production of specific antibodies by, for example, the methods described by Babcook, J. et al., 1996, Proc. Natl. Acad. Sci. USA 93(15):7843-78481; WO92/02551; WO2004/051268 and International Patent Application number WO2004/106377.

[0292] Humanised antibodies (which include CDR-grafted antibodies) are antibody molecules having one or more complementarity determining regions (CDRs) from a non-human species and a framework region from a human immunoglobulin molecule (see, e.g. U.S. Pat. No. 5,585,089; WO91/09967). It will be appreciated that it may only be necessary to transfer the specificity determining residues of the CDRs rather than the entire CDR (see for example, Kashmiri et al., 2005, Methods, 36, 25-34). Humanised antibodies may optionally further comprise one or more framework residues derived from the non-human species from which the CDRs were derived.

[0293] As used herein, the term `humanised antibody molecule` refers to an antibody molecule wherein the heavy and/or light chain contains one or more CDRs (including, if desired, one or more modified CDRs) from a donor antibody (e.g. a murine monoclonal antibody) grafted into a heavy and/or light chain variable region framework of an acceptor antibody (e.g. a human antibody). For a review, see Vaughan et al, Nature Biotechnology, 16, 535-539, 1998. In one embodiment rather than the entire CDR being transferred, only one or more of the specificity determining residues from any one of the CDRs described herein above are transferred to the human antibody framework (see for example, Kashmiri et al., 2005, Methods, 36, 25-34). In one embodiment only the specificity determining residues from one or more of the CDRs described herein above are transferred to the human antibody framework. In another embodiment only the specificity determining residues from each of the CDRs described herein above are transferred to the human antibody framework.

[0294] When the CDRs or specificity determining residues are grafted, any appropriate acceptor variable region framework sequence may be used having regard to the class/type of the donor antibody from which the CDRs are derived, including mouse, primate and human framework regions. Suitably, the humanised antibody according to the present invention has a variable domain comprising human acceptor framework regions as well as one or more of the CDRs provided herein.

[0295] Thus, provided in one embodiment is a humanised antibody which binds toxin A or toxin B wherein the variable domain comprises human acceptor framework regions and non-human donor CDRs.

[0296] Examples of human frameworks which can be used in the present invention are KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (Kabat et al., supra). For example, KOL and NEWM can be used for the heavy chain, REI can be used for the light chain and EU, LAY and POM can be used for both the heavy chain and the light chain. Alternatively, human germline sequences may be used; these are available at: http://vbase.mrc-cpe.cam.ac.uk/

[0297] In a humanised antibody of the present invention, the acceptor heavy and light chains do not necessarily need to be derived from the same antibody and may, if desired, comprise composite chains having framework regions derived from different chains.

[0298] Also, in a humanised antibody of the present invention, the framework regions need not have exactly the same sequence as those of the acceptor antibody. For instance, unusual residues may be changed to more frequently-occurring residues for that acceptor chain class or type. Alternatively, selected residues in the acceptor framework regions may be changed so that they correspond to the residue found at the same position in the donor antibody (see Reichmann et al., 1998, Nature, 332, 323-324). Such changes should be kept to the minimum necessary to recover the affinity of the donor antibody. A protocol for selecting residues in the acceptor framework regions which may need to be changed is set forth in WO 91/09967.

[0299] Generally the antibody sequences disclosed in the present specification are humanised.

[0300] The invention also provides sequences which are 80%, 90%, 91%, 92%, 93% 94%, 95% 96%, 97%, 98% or 99% similar to a sequence or antibody disclosed herein.

[0301] "Identity", as used herein, indicates that at any particular position in the aligned sequences, the amino acid residue is identical between the sequences. "Similarity", as used herein, indicates that, at any particular position in the aligned sequences, the amino acid residue is of a similar type between the sequences. For example, leucine may be substituted for isoleucine or valine. Other amino acids which can often be substituted for one another include but are not limited to:

[0302] phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains);

[0303] lysine, arginine and histidine (amino acids having basic side chains);

[0304] aspartate and glutamate (amino acids having acidic side chains);

[0305] asparagine and glutamine (amino acids having amide side chains); and

[0306] cysteine and methionine (amino acids having sulphur-containing side chains).

[0307] Degrees of identity and similarity can be readily calculated (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 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987, Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991, the BLAST.TM. software available from NCBI (Altschul, S. F. et al., 1990, J. Mol. Biol. 215:403-410; Gish, W. & States, D. J. 1993, Nature Genet. 3:266-272. Madden, T. L. et al., 1996, Meth. Enzymol. 266:131-141; Altschul, S. F. et al., 1997, Nucleic Acids Res. 25:3389-3402; Zhang, J. & Madden, T. L. 1997, Genome Res. 7:649-656,).

[0308] The antibody molecules of the present invention include a complete antibody molecule having full length heavy and light chains or a fragment thereof and may be, but are not limited to Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single domain antibodies (e.g. VH or VL or VHH), scFv, bi, tri or tetra-valent antibodies, Bis-scFv, diabodies, triabodies, tetrabodies and epitope-binding fragments of any of the above (see for example Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews--Online 2(3), 209-217). The methods for creating and manufacturing these antibody fragments are well known in the art (see for example Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). Other antibody fragments for use in the present invention include the Fab and Fab' fragments described in International patent applications WO2005/003169, WO2005/003170 and WO2005/003171. Multi-valent antibodies may comprise multiple specificities e.g bispecific or may be monospecific (see for example WO 92/22853 and WO05/113605). Bispecific and multispecific antibody variants are especially considered in this example since the aim is to neutralise two independent target proteins: TcdA and TcdB. Variable regions from antibodies disclosed herein may be configured in such a way as to produce a single antibody variant which is capable of binding to and neutralising TcdA and TcdB.

[0309] In one embodiment the antibody according to the present disclosure is provided as TcdA or TcdB binding antibody fusion protein which comprises an immunoglobulin moiety, for example a Fab or Fab' fragment, and one or two single domain antibodies (dAb) linked directly or indirectly thereto, for example as described in WO2009/040562.

[0310] In one embodiment the fusion protein comprises two domain antibodies, for example as a variable heavy (VH) and variable light (VL) pairing, optionally linked by a disulphide bond, for example as described in WO2010/035012.

[0311] In one embodiment the Fab or Fab' element of the fusion protein has the same or similar specificity to the single domain antibody or antibodies. In one embodiment the Fab or Fab' has a different specificity to the single domain antibody or antibodies, that is to say the fusion protein is multivalent. In one embodiment a multivalent fusion protein according to the present invention has an albumin binding site, for example a VH/VL pair therein provides an albumin binding site.

[0312] In one embodiment the multivalent fusion protein according to the invention binds TcdA and TcdB.

[0313] In one embodiment the multivalent fusion protein according to the invention binds TcdB in multiple positions, for example has distinct binding regions specific for two different epitopes.

[0314] The constant region domains of the antibody molecule of the present invention, if present, may be selected having regard to the proposed function of the antibody molecule, and in particular the effector functions which may be required. For example, the constant region domains may be human IgA, IgD, IgE, IgG or IgM domains. In particular, human IgG constant region domains may be used, especially of the IgG1 and IgG3 isotypes when the antibody molecule is intended for therapeutic uses and antibody effector functions are required. Alternatively, IgG2 and IgG4 isotypes may be used when the antibody molecule is intended for therapeutic purposes and antibody effector functions are not required, e.g. for simply neutralising or agonising an antigen. It will be appreciated that sequence variants of these constant region domains may also be used. For example IgG4 molecules in which the serine at position 241 has been changed to proline as described in Angal et al., Molecular Immunology, 1993, 30 (1), 105-108 may be used. It will also be understood by one skilled in the art that antibodies may undergo a variety of posttranslational modifications. The type and extent of these modifications often depends on the host cell line used to express the antibody as well as the culture conditions. Such modifications may include variations in glycosylation, methionine oxidation, diketopiperazine formation, aspartate isomerization and asparagine deamidation. A frequent modification is the loss of a carboxy-terminal basic residue (such as lysine or arginine) due to the action of carboxypeptidases (as described in Harris, R J. Journal of Chromatography 705:129-134, 1995).

[0315] In one embodiment the antibody heavy chain comprises a CH1 domain and the antibody light chain comprises a CL domain, either kappa or lambda.

[0316] Biological molecules, such as antibodies or fragments, contain acidic and/or basic functional groups, thereby giving the molecule a net positive or negative charge. The amount of overall "observed" charge will depend on the absolute amino acid sequence of the entity, the local environment of the charged groups in the 3D structure and the environmental conditions of the molecule. The isoelectric point (pI) is the pH at which a particular molecule or solvent accessible surface thereof carries no net electrical charge. In one example, the antibody and fragments of the invention may be engineered to have an appropriate isoelectric point. This may lead to antibodies and/or fragments with more robust properties, in particular suitable solubility and/or stability profiles and/or improved purification characteristics.

[0317] Thus in one aspect the invention provides a humanised antibody engineered to have an isoelectric point different to that of the originally identified antibody from which it is derived. The antibody may, for example be engineered by replacing an amino acid residue such as replacing an acidic amino acid residue with one or more basic amino acid residues. Alternatively, basic amino acid residues may be introduced or acidic amino acid residues can be removed. Alternatively, if the molecule has an unacceptably high pI value acidic residues may be introduced to lower the pI, as required. It is important that when manipulating the pI care must be taken to retain the desirable activity of the antibody or fragment. Thus in one embodiment the engineered antibody or fragment has the same or substantially the same activity as the "unmodified" antibody or fragment.

[0318] Programs such as ** ExPASY http://www.expasy.ch/tools/pi_tool.html, and http://www.iut-arles.up.univ-mrs.fr/w3bb/d_abim/compo-p.html, may be used to predict the isoelectric point of the antibody or fragment.

[0319] It will be appreciated that the affinity of antibodies provided by the present invention may be altered using any suitable method known in the art. The affinity of the antibodies or variants thereof may be measured using any suitable method known in the art, including BIAcore, using an appropriate isolated natural or recombinant protein or a suitable fusion protein/polypeptide.

[0320] The present invention therefore also relates to variants of the antibody molecules of the present invention, which have an improved affinity for TcdA or TcdB, as appropriate. Such variants can be obtained by a number of affinity maturation protocols including mutating the CDRs (Yang et al., J. Mol. Biol., 254, 392-403, 1995), chain shuffling (Marks et al., Bio/Technology, 10, 779-783, 1992), use of mutator strains of E. coli (Low et al., J. Mol. Biol., 250, 359-368, 1996), DNA shuffling (Patten et al., Curr. Opin. Biotechnol., 8, 724-733, 1997), phage display (Thompson et al., J. Mol. Biol., 256, 77-88, 1996) and sexual PCR (Crameri et al., Nature, 391, 288-291, 1998). Vaughan et al. (supra) discusses these methods of affinity maturation.

[0321] Improved affinity as employed herein in this context refers to an improvement refers to an improvement over the starting molecule.

[0322] If desired an antibody for use in the present invention may be conjugated to one or more effector molecule(s). It will be appreciated that the effector molecule may comprise a single effector molecule or two or more such molecules so linked as to form a single moiety that can be attached to the antibodies of the present invention. Where it is desired to obtain an antibody fragment linked to an effector molecule, this may be prepared by standard chemical or recombinant DNA procedures in which the antibody fragment is linked either directly or via a coupling agent to the effector molecule. Techniques for conjugating such effector molecules to antibodies are well known in the art (see, Hellstrom et al., Controlled Drug Delivery, 2nd Ed., Robinson et al., eds., 1987, pp. 623-53; Thorpe et al., 1982, Immunol. Rev., 62:119-58 and Dubowchik et al., 1999, Pharmacology and Therapeutics, 83, 67-123). Particular chemical procedures include, for example, those described in WO 93/06231, WO 92/22583, WO 89/00195, WO 89/01476 and WO03031581. Alternatively, where the effector molecule is a protein or polypeptide the linkage may be achieved using recombinant DNA procedures, for example as described in WO 86/01533 and EP0392745.

[0323] The term effector molecule as used herein includes, for example, biologically active proteins, for example enzymes, other antibody or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof e.g. DNA, RNA and fragments thereof, radionuclides, particularly radioiodide, radioisotopes, chelated metals, nanoparticles and reporter groups such as fluorescent compounds or compounds which may be detected by NMR or ESR spectroscopy.

[0324] Other effector molecules may include chelated radionuclides such as 111In and 90Y, Lu177, Bismuth213, Californium252, Iridium192 and Tungsten188/Rhenium188; or drugs such as but not limited to, alkylphosphocholines, topoisomerase I inhibitors, taxoids and suramin.

[0325] Other effector molecules include proteins, peptides and enzymes. Enzymes of interest include, but are not limited to, proteolytic enzymes, hydrolases, lyases, isomerases, transferases. Proteins, polypeptides and peptides of interest include, but are not limited to, immunoglobulins, toxins such as abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin, a protein such as insulin, tumour necrosis factor, .alpha.-interferon, .beta.-interferon, nerve growth factor, platelet derived growth factor or tissue plasminogen activator, a thrombotic agent or an anti-angiogenic agent, e.g. angiostatin or endostatin, or, a biological response modifier such as a lymphokine, interleukin-1 (IL-1), interleukin-2 (IL-2), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), nerve growth factor (NGF) or other growth factor and immunoglobulins.

[0326] Other effector molecules may include detectable substances useful for example in diagnosis. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive nuclides, positron emitting metals (for use in positron emission tomography), and nonradioactive paramagnetic metal ions. See generally U.S. Pat. No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics. Suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; suitable prosthetic groups include streptavidin, avidin and biotin; suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin; suitable luminescent materials include luminol; suitable bioluminescent materials include luciferase, luciferin, and aequorin; and suitable radioactive nuclides include 125I, 131I, 111In and 99Tc.

[0327] In another example the effector molecule may increase the half-life of the antibody in vivo, and/or reduce immunogenicity of the antibody and/or enhance the delivery of an antibody across an epithelial barrier to the immune system. Examples of suitable effector molecules of this type include polymers, albumin, albumin binding proteins or albumin binding compounds such as those described in WO05/117984.

[0328] Where the effector molecule is a polymer it may, in general, be a synthetic or a naturally occurring polymer, for example an optionally substituted straight or branched chain polyalkylene, polyalkenylene or polyoxyalkylene polymer or a branched or unbranched polysaccharide, e.g. a homo- or hetero-polysaccharide.

[0329] Specific optional substituents which may be present on the above-mentioned synthetic polymers include one or more hydroxy, methyl or methoxy groups.

[0330] Specific examples of synthetic polymers include optionally substituted straight or branched chain poly(ethyleneglycol), poly(propyleneglycol) poly(vinylalcohol) or derivatives thereof, especially optionally substituted poly(ethyleneglycol) such as methoxypoly(ethyleneglycol) or derivatives thereof.

[0331] Specific naturally occurring polymers include lactose, amylose, dextran, glycogen or derivatives thereof.

[0332] "Derivatives" as used herein is intended to include reactive derivatives, for example thiol-selective reactive groups such as maleimides and the like. The reactive group may be linked directly or through a linker segment to the polymer. It will be appreciated that the residue of such a group will in some instances form part of the product as the linking group between the antibody fragment and the polymer.

[0333] The size of the polymer may be varied as desired, but will generally be in an average molecular weight range from 500 Da to 50000 Da, for example from 5000 to 40000 Da such as from 20000 to 40000 Da. The polymer size may in particular be selected on the basis of the intended use of the product for example ability to localize to certain tissues such as tumors or extend circulating half-life (for review see Chapman, 2002, Advanced Drug Delivery Reviews, 54, 531-545). Thus, for example, where the product is intended to leave the circulation and penetrate tissue, for example for use in the treatment of a tumour, it may be advantageous to use a small molecular weight polymer, for example with a molecular weight of around 5000 Da. For applications where the product remains in the circulation, it may be advantageous to use a higher molecular weight polymer, for example having a molecular weight in the range from 20000 Da to 40000 Da.

[0334] Suitable polymers include a polyalkylene polymer, such as a poly(ethyleneglycol) or, especially, a methoxypoly(ethyleneglycol) or a derivative thereof, and especially with a molecular weight in the range from about 15000 Da to about 40000 Da.

[0335] In one example antibodies for use in the present invention are attached to poly(ethyleneglycol) (PEG) moieties. In one particular example the antibody is an antibody fragment and the PEG molecules may be attached through any available amino acid side-chain or terminal amino acid functional group located in the antibody fragment, for example any free amino, imino, thiol, hydroxyl or carboxyl group. Such amino acids may occur naturally in the antibody fragment or may be engineered into the fragment using recombinant DNA methods (see for example U.S. Pat. Nos. 5,219,996; 5,667,425; WO98/25971, WO2008/038024). In one example the antibody molecule of the present invention is a modified Fab fragment wherein the modification is the addition to the C-terminal end of its heavy chain one or more amino acids to allow the attachment of an effector molecule. Suitably, the additional amino acids form a modified hinge region containing one or more cysteine residues to which the effector molecule may be attached. Multiple sites can be used to attach two or more PEG molecules.

[0336] Suitably PEG molecules are covalently linked through a thiol group of at least one cysteine residue located in the antibody fragment. Each polymer molecule attached to the modified antibody fragment may be covalently linked to the sulphur atom of a cysteine residue located in the fragment. The covalent linkage will generally be a disulphide bond or, in particular, a sulphur-carbon bond. Where a thiol group is used as the point of attachment appropriately activated effector molecules, for example thiol selective derivatives such as maleimides and cysteine derivatives may be used. An activated polymer may be used as the starting material in the preparation of polymer-modified antibody fragments as described above. The activated polymer may be any polymer containing a thiol reactive group such as an .alpha.-halocarboxylic acid or ester, e.g. iodoacetamide, an imide, e.g. maleimide, a vinyl sulphone or a disulphide. Such starting materials may be obtained commercially (for example from Nektar, formerly Shearwater Polymers Inc., Huntsville, Ala., USA) or may be prepared from commercially available starting materials using conventional chemical procedures. Particular PEG molecules include 20K methoxy-PEG-amine (obtainable from Nektar, formerly Shearwater; Rapp Polymere; and SunBio) and M-PEG-SPA (obtainable from Nektar, formerly Shearwater).

[0337] In one embodiment, the antibody is a modified Fab fragment or diFab which is PEGylated, i.e. has PEG (poly(ethyleneglycol)) covalently attached thereto, e.g. according to the method disclosed in EP 0948544 or EP1090037 [see also "Poly(ethyleneglycol) Chemistry, Biotechnical and Biomedical Applications", 1992, J. Milton Harris (ed), Plenum Press, New York, "Poly(ethyleneglycol) Chemistry and Biological Applications", 1997, J. Milton Harris and S. Zalipsky (eds), American Chemical Society, Washington D.C. and "Bioconjugation Protein Coupling Techniques for the Biomedical Sciences", 1998, M. Aslam and A. Dent, Grove Publishers, New York; Chapman, A. 2002, Advanced Drug Delivery Reviews 2002, 54:531-545]. In one example PEG is attached to a cysteine in the hinge region. In one example, a PEG modified Fab fragment has a maleimide group covalently linked to a single thiol group in a modified hinge region. A lysine residue may be covalently linked to the maleimide group and to each of the amine groups on the lysine residue may be attached a methoxypoly(ethyleneglycol) polymer having a molecular weight of approximately 20,000 Da. The total molecular weight of the PEG attached to the Fab fragment may therefore be approximately 40,000 Da.

[0338] Particular PEG molecules include 2-[3-(N-maleimido)propionamido]ethyl amide of N,N'-bis(methoxypoly(ethylene glycol) MW 20,000) modified lysine, also known as PEG2MAL40K (obtainable from Nektar, formerly Shearwater).

[0339] Alternative sources of PEG linkers include NOF who supply GL2-400MA2 (wherein m in the structure below is 5) and GL2-400MA (where m is 2) and n is approximately 450:

##STR00001##

[0340] That is to say each PEG is about 20,000 Da.

[0341] Further alternative PEG effector molecules of the following type:

##STR00002##

are available from Dr Reddy, NOF and Jenkem.

[0342] In one embodiment there is provided an antibody which is PEGylated (for example with a PEG described herein), attached through a cysteine amino acid residue at or about amino acid 226 in the chain, for example amino acid 226 of the heavy chain (by sequential numbering).

[0343] In one embodiment one certain antibodies according to the present disclosure have the following properties:

TABLE-US-00052 Affinity (pM) Valency of binding Antibody TcdA.sub.123 TcdA.sub.456 TcdA, est. EC.sub.50 (ng/ml) CA922 4.06 2.59 16 1.21 CA923 64.7 312 12 160.42 CA995 nil 119 1 37.64 CA997 132 66.8 12 6.25 CA1000 73.3 84.1 2 19.73

[0344] The present invention also provides compositions such as a pharmaceutical composition of antibody or combination of antibodies defined herein.

[0345] The present invention also provides a composition that comprises at least two antibodies according to the invention, for example wherein at least one antibody therein is specific to TcdA and at least one antibody therein is specific to TcdB or alternatively at least two antibodies specific to TcdA or at least two antibodies specific to TcdB.

[0346] In one embodiment there is provided a composition that comprises multiple antibodies specific to TcdA and optionally one or more antibodies specific to TcdB.

[0347] In one embodiment there is provided a composition that comprises multiple antibodies specific to TcdB and optionally one or more antibodies specific to TcdA.

[0348] Thus in one embodiment there is provided a composition comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 antibodies according to the invention i.e. distinct antibodies.

[0349] The invention describes one particular mixture comprising 3 Mabs, one Mab of which is specific for TcdA and two Mabs of which are specific for TcdB. This mixture demonstrated very high levels of protection from death and gut inflammation from a lethal infective oral dose of Clostridium difficile in hamsters.

[0350] In particular there is provided a composition comprising a combination of one anti-TcdA antibody comprising a heavy variable region with a sequence as shown in SEQ ID NO:49 and a light variable region with a sequence shown in SEQ ID NO: 47 and two anti-TcdB the first with a heavy variable region shown in SEQ ID NO: 129 and a light variable region shown in SEQ ID NO: 127, and the second with a heavy variable region shown in SEQ ID NO: 159 and light variable region shown in SEQ ID NO: 157.

[0351] In one embodiment wherein the composition comprises 3 antibodies, such as one anti-TcdA and two anti-TcdB antibodies the antibodies are in the ratio of 50%, 25% and 25% respectively of the total antibody content thereof.

[0352] In one embodiment there is provided a composition comprising 2, 3, 4 or 5 antibodies specific to TcdA and optionally 1, 2, 3, 4 or 5 antibodies specific to TcdB.

[0353] In one embodiment the compositions provided according to the invention are well defined, for example are mixtures of monoclonal antibodies rather than simply polyclonal compositions derived from an immunised or immune competent host.

[0354] In one embodiment the composition of antibodies has an EC.sub.50 of 200 ng/ml or less, for example 150 ng/ml or less, such as 100 ng/ml or less, such as 0.1 to 10 ng/ml.

[0355] Advantageously the antibodies described herein have very high levels of biophysical stability and so are suitable for inclusion in mixtures of antibodies.

[0356] In one aspect a pharmaceutical formulation or composition according to the invention further comprises a pharmaceutically acceptable excipient.

[0357] Pharmaceutically acceptable carriers in therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents or pH buffering substances, may be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions, for ingestion by the patient.

[0358] Suitable forms for administration include forms suitable for parenteral administration, e.g. by injection or infusion, for example by bolus injection or continuous infusion. Where the product is for injection or infusion, it may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and it may contain formulatory agents, such as suspending, preservative, stabilising and/or dispersing agents. Alternatively, the antibody molecule may be in dry form, for reconstitution before use with an appropriate sterile liquid.

[0359] Once formulated, the compositions of the invention can be administered directly to the subject. The subjects to be treated can be animals. However, in one or more embodiments the compositions are adapted for administration to human subjects.

[0360] Suitably in formulations according to the present disclosure, the pH of the final formulation is not similar to the value of the isoelectric point of the antibody or fragment, for example if the pH of the formulation is 7 then a pI of from 8-9 or above may be appropriate. Whilst not wishing to be bound by theory it is thought that this may ultimately provide a final formulation with improved stability, for example the antibody or fragment remains in solution.

[0361] In one embodiment the composition or formulation of the present disclosure comprises 1-200 mg/mL of antibodies, that this to say the combined antibody content, for example 150 mg/mL or less, such as 100 mg/mL or less, in particular 90, 80, 70, 60, 50, 40, 30, 20, 10 mg/mL or less.

[0362] In one embodiment a composition or formulation according to the present disclosure comprises 20 mg/mL of each antibody therein.

[0363] In one embodiment there is provided a formulation comprising:

[0364] 33 mg/mL or less of one anti-TcdA antibody comprising a heavy variable region with a sequence as shown in SEQ ID NO: 49 and a light variable region with a sequence shown in SEQ ID NO: 47, and

[0365] 28 mg/mL or less of a first anti-TcdB with a heavy variable region shown in SEQ ID NO: 129 and a light variable region shown in SEQ ID NO: 127, and 25 mg/mL of a second anti-TcdB with a heavy variable region shown in SEQ ID NO: 159 and light variable region shown in SEQ ID NO: 157.

[0366] In one embodiment the pharmaceutical formulation at a pH in the range of 4.0 to 7.0 comprises: 1 to 200 mg/mL of an antibody according to the present disclosure, 1 to 100 mM of a buffer, 0.001 to 1% of a surfactant,

[0367] a) 10 to 500 mM of a stabiliser,

[0368] b) 5 to 500 mM of a tonicity agent, or

[0369] c) 10 to 500 mM of a stabiliser and 5 to 500 mM of a tonicity agent.

[0370] In one embodiment the composition or formulation according to the present disclosure comprises the buffer phosphate buffered saline.

[0371] For example the formulation at approximately pH6 may comprise 1 to 50 mg/mL of antibody, 20 mM L-histidine HCl, 240 mM trehalose and 0.02% polysorbate 20. Alternatively a formulation at approximately pH 5.5 may comprise 1 to 50 mg/mL of antibody, 20 mM citrate buffer, 240 mM sucrose, 20 mM arginine, and 0.02% polysorbate 20.

[0372] The pharmaceutical compositions of this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, transcutaneous (for example, see WO98/20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal routes. Hyposprays may also be used to administer the pharmaceutical compositions of the invention. Typically, the therapeutic compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.

[0373] Direct delivery of the compositions will generally be accomplished by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue.

[0374] The compositions can also be administered into a lesion or directly into the gastrointestinal tract by for examples, encapsulated oral dosage for swallowing, through a nasogastric tube to the stomach or ileum, through a rectal tube or enema solutions or by rectal capsule. Dosage treatment may be a single dose schedule or a multiple dose schedule.

[0375] It will be appreciated that the active ingredient in the composition will be an antibody molecule. As such, it will be susceptible to degradation in the gastrointestinal tract. Thus, if the composition is to be administered by a route using the gastrointestinal tract, the composition will need to contain agents which protect the antibody from degradation but which release the antibody once it has been absorbed from the gastrointestinal tract.

[0376] A thorough discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Publishing Company, N.J. 1991).

[0377] The present invention also provides an antibody or antibody combination or a composition comprising the same, as described herein, for treatment, for example for the treatment or prophylaxis of C. difficile infection or complications associated with the same such as diarrhoea, colitis in particular pseudomembranous colitis, bloating, abdominal pain and toxic megacolon.

[0378] Prophylaxis can also be achieved by the administration of pre-formed complexes of inactivated toxin antigen (toxoid) and antibody in order to create a vaccine.

[0379] In one embodiment the antibodies, combinations thereof and compositions comprising the same according to the invention are suitable for treating infection with so-called super strains of C. difficile, i.e. hypervirulent strains such as ribotype 027.

[0380] The antibodies and compositions according to the present invention are suitable for use in the treatment or prophylaxis of acute and/or chronic effects of the relevant C. difficile toxins during primary infection.

[0381] The antibodies and compositions according to the present invention are suitable for use in the treatment or prophylaxis of effects of the relevant C. difficile toxins during secondary infection or re-infection. International guidelines enshrine time intervals after a primary infection which hence defines a secondary (or recurrent) infection as being distinct from a continuation of existing symptoms sometimes described as a relapse (29). Research has shown that secondary infections can be the result of the same strain or ribotype as the primary infection. In such cases recurrence rather than relapse relies on agreed temporal constraints. However, research also clearly shows that secondary infection can also be the result of infection of a strain or ribotype distinct from that of the primary infection. In one study, 48% of disease recurrences were the result of a second strain distinct from that having caused the first infection (30). In another study, more than 56% of disease recurrences were the result of a second strain distinct from that having caused the first infection (31).

[0382] In one embodiment the antibodies, combinations thereof and compositions comprising the same according to the invention are suitable for use in the prevention of damage, for example long term structural damage to the epithelium of the colon.

[0383] In one embodiment the antibodies, combinations and composition are suitable for preventing C. difficile infection including recurrence of infection, in particular nosocomial infection.

[0384] In one embodiment the antibodies, combinations thereof and compositions comprising the same according to the invention are suitable for reducing the risk of recurrence of C. difficile infection.

[0385] Advantageously, the antibodies of the present disclosure can be administered prophylactically to prevent infection or re-infection because in the absence of toxin to which the antibody is specific the antibody is simply to be cleared from the body without causing adverse interactions with the subjects body tissues.

[0386] Advantageously the antibodies of the present disclosure seem to elicit a rapid response after administration, for example within one or two days of administration rapid clearance of the target toxin is invoked, this may prevent vital organs such as the lungs, heart and kidneys being damaged. This is the first time that agents have been made available with can be employed to prevent damage or injury to a patient by toxins A and/or B in the acute C. difficile infection stage.

[0387] Thus in one embodiment the antibodies, combinations thereof and compositions comprising the same according to the invention are suitable for preventing damage to vital organs.

[0388] In one embodiment the antibody, combinations or formulations described herein are suitable for preventing death of an infected patient, if administered within an appropriate time frame before irreparable damage has been done by the toxins.

[0389] The antibodies of the present disclosure have fast on-rates, which facilitates the rapid effect in vivo.

[0390] In one embodiment the patient population is over 60, such as over 65 years of age.

[0391] In one embodiment the patient population is 5 years old or less.

[0392] The antibodies according the invention may be employed in combination with antibiotic treatment for example metronidazole, vancomycin or fidaxomicin.

[0393] A range of in vitro data exemplify the properties of the Mabs and Mab mixtures. We show that one mixture of 3 Mabs (50% molar quantities of anti-TcdA and 50% molar quantities of anti-TcdB components) was able to protect hamsters from a lethal CDI.

[0394] In one embodiment there is provided a method of treating a patient in need thereof by administering a therapeutically effective amount of an antibody as described herein or antibody combination or a composition comprising the same, for example in the treatment or prophylaxis of C. difficile infection or complications associated with the same such as diarrhoea, colitis in particular pseudomembranous colitis, bloating, abdominal pain and toxic megacolon.

[0395] In one embodiment the antibody, combination or formulation is administered by a parenteral route, for example subcutaneously, intraperitoneally, intravenously or intramuscularly. The data in the Examples generated in hamsters indicates that the doses administered by this route reach the gut and thus are able to generate a therapeutic effect.

[0396] In one embodiment the antibody, combination or formulation is administered orally, for example an enterically coated formulation.

[0397] In one embodiment there is provided use of an antibody, combination or formulation as described herein for the manufacture of a medicament for the treatment or prophylaxis of C. difficile infection.

[0398] In one embodiment the dose administered is in the range 1 to 1000 mg/Kg, for example 10 to 75 mg/Kg, such 20 to 50 mg/Kg.

[0399] In one embodiment the half-life of the antibody or antibodies in mice and hamsters in vivo is in the range 6 to 8 days in healthy (uninfected) animals and hence are expected to have half-lives in humans in the range of 14-28 days.

[0400] In one embodiment the antibody or antibodies are given as one dose only.

[0401] In one embodiment the antibody or antibodies are given as a weekly or biweekly dose.

[0402] In one embodiment the antibody or antibodies are given as once daily doses.

[0403] In one embodiment there is provided complex comprising TcdA or an immunogenic fragment thereof, complexed with one or more anti-TcdA antibodies defined herein. The complex may be employed as the antigen in a vaccine formulation, for example suitable for generating protective antibodies to toxin A in vivo after administration to a human.

[0404] In one embodiment there is provided complex comprising TcdB or an immunogenic fragment thereof, complexed with one or more anti-TcdB antibodies defined herein. The complex may be employed as the antigen in a vaccine formulation, for example suitable for generating protective antibodies to toxin B in vivo after administration to a human.

[0405] Th1-type immunostimulants which may be formulated to produce adjuvants suitable for use in the present invention include and are not restricted to the following.

[0406] In one embodiment there is provided a complex comprising TcdA or an immunogenic fragment thereof and TcdB or an immunogenic fragment thereof, wherein each toxin or fragment is complexed with one or more antibodies specific thereto, wherein the complex is suitable for administration as a vaccine formulation.

[0407] Antibody:antigen complexes are known to be taken up by the immune system in an Fc receptor mediated process (27, 28) and pre-formed complexes of antibody:antigen complexes have been successfully use as vaccines in human clinical trials (22).

[0408] In one or more embodiments the vaccine formulation further comprises an adjuvant as an immunostimulant.

[0409] Monophosphoryl lipid A, in particular 3-de-O-acylated monophosphoryl lipid A (3D-MPL), is a preferred Th1-type immunostimulant for use in the invention. 3D-MPL is a well known adjuvant manufactured by Ribi Immunochem, Montana. Chemically it is often supplied as a mixture of 3-de-O-acylated monophosphoryl lipid A with either 4, 5, or 6 acylated chains. It can be purified and prepared by the methods taught in GB 2122204B, which reference also discloses the preparation of diphosphoryl lipid A, and 3-O-deacylated variants thereof. Other purified and synthetic lipopolysaccharides have been described (U.S. Pat. No. 6,005,099 and EP 0 729 473 B1; Hilgers et al., 1986, Int. Arch. Allergy. Immunol., 79(4):392-6; Hilgers et al., 1987, Immunology, 60(1):141-6; and EP 0 549 074 B1). A preferred form of 3D-MPL is in the form of a particulate formulation having a small particle size less than 0.2 mm in diameter, and its method of manufacture is disclosed in EP 0 689 454.

[0410] Saponins are also preferred Th1 immunostimulants in accordance with the invention. Saponins are well known adjuvants and are taught in: Lacaille-Dubois, M and Wagner H. (1996. A review of the biological and pharmacological activities of saponins. Phytomedicine vol 2 pp 363-386). For example, Quil A (derived from the bark of the South American tree Quillaja Saponaria Molina), and fractions thereof, are described in U.S. Pat. No. 5,057,540 and "Saponins as vaccine adjuvants", Kensil, C. R., Crit Rev Ther Drug Carrier Syst, 1996, 12 (1-2):1-55; and EP 0 362 279 B1. The haemolytic saponins QS21 and QS17 (HPLC purified fractions of Quil A) have been described as potent systemic adjuvants, and the method of their production is disclosed in U.S. Pat. No. 5,057,540 and EP 0 362 279 B1. Also described in these references is the use of QS7 (a non-haemolytic fraction of Quil-A) which acts as a potent adjuvant for systemic vaccines. Use of QS21 is further described in Kensil et al. (1991. J. Immunology vol 146, 431-437). Combinations of QS21 and polysorbate or cyclodextrin are also known (WO 99/10008). Particulate adjuvant systems comprising fractions of QuilA, such as QS21 and QS7 are described in WO 96/33739 and WO 96/11711. One such system is known as an Iscorn and may contain one or more saponins.

[0411] Another preferred immunostimulant is an immunostimulatory oligonucleotide containing unmethylated CpG dinucleotides ("CpG"). CpG is an abbreviation for cytosine-guanosine dinucleotide motifs present in DNA. CpG is known in the art as being an adjuvant when administered by both systemic and mucosal routes (WO 96/02555, EP 468520, Davis et al., J. Immunol, 1998, 160(2):870-876; McCluskie and Davis, J. Immunol., 1998, 161(9):4463-6). Historically, it was observed that the DNA fraction of BCG could exert an anti-tumour effect. In further studies, synthetic oligonucleotides derived from BCG gene sequences were shown to be capable of inducing immunostimulatory effects (both in vitro and in vivo). The authors of these studies concluded that certain palindromic sequences, including a central CG motif, carried this activity. The central role of the CG motif in immunostimulation was later elucidated in a publication by Krieg, Nature 374, p 546 1995. Detailed analysis has shown that the CG motif has to be in a certain sequence context, and that such sequences are common in bacterial DNA but are rare in vertebrate DNA. The immunostimulatory sequence is often: Purine, Purine, C, G, pyrimidine, pyrimidine; wherein the CG motif is not methylated, but other unmethylated CpG sequences are known to be immunostimulatory and may be used in the present invention.

[0412] In certain combinations of the six nucleotides a palindromic sequence is present. Several of these motifs, either as repeats of one motif or a combination of different motifs, can be present in the same oligonucleotide. The presence of one or more of these immunostimulatory sequences containing oligonucleotides can activate various immune subsets, including natural killer cells (which produce interferon g and have cytolytic activity) and macrophages (Wooldrige et al Vol 89 (no. 8), 1977). Other unmethylated CpG containing sequences not having this consensus sequence have also now been shown to be immunomodulatory.

[0413] CpG when formulated into vaccines, is generally administered in free solution together with free antigen (WO 96/02555; McCluskie and Davis, supra) or covalently conjugated to an antigen (WO 98/16247), or formulated with a carrier such as aluminium hydroxide ((Hepatitis surface antigen) Davis et al. supra; Brazolot-Millan et al., Proc. Natl. Acad. Sci., USA, 1998, 95(26), 15553-8).

[0414] Such immunostimulants as described above may be formulated together with carriers, such as for example liposomes, oil in water emulsions, and or metallic salts, including aluminium salts (such as aluminium hydroxide). For example, 3D-MPL may be formulated with aluminium hydroxide (EP 0 689 454) or oil in water emulsions (WO 95/17210); QS21 may be advantageously formulated with cholesterol containing liposomes (WO 96/33739), oil in water emulsions (WO 95/17210) or alum (WO 98/15287); CpG may be formulated with alum (Davis et al. supra; Brazolot-Millan supra) or with other cationic carriers.

[0415] Combinations of immunostimulants are also preferred, in particular a combination of a monophosphoryl lipid A and a saponin derivative (WO 94/00153; WO 95/17210; WO 96/33739; WO 98/56414; WO 99/12565; WO 99/11241), more particularly the combination of QS21 and 3D-MPL as disclosed in WO 94/00153. Alternatively, a combination of CpG plus a saponin such as QS21 also forms a potent adjuvant for use in the present invention.

[0416] Alternatively the saponin may be formulated in a liposome or in an Iscorn and combined with an immunostimulatory oligonucleotide.

[0417] Thus, suitable adjuvant systems include, for example, a combination of monophosphoryl lipid A, preferably 3D-MPL, together with an aluminium salt.

[0418] Thus is one embodiment the adjuvant is a combination of QS21 and 3D-MPL in an oil in water or liposomal formulation.

[0419] An enhanced system involves the combination of a monophosphoryl lipid A and a saponin derivative particularly the combination of QS21 and 3D-MPL as disclosed in WO 94/00153, or a less reactogenic composition where the QS21 is quenched in cholesterol containing liposomes (DQ) as disclosed in WO 96/33739. This combination may additionally comprise an immunostimulatory oligonucleotide.

[0420] A particularly potent adjuvant formulation involving QS21, 3D-MPL & tocopherol in an oil in water emulsion is described in WO 95/17210 and is another preferred formulation for use in the invention.

[0421] Another preferred formulation comprises a CpG oligonucleotide alone or together with an aluminium salt.

[0422] In a further aspect of the present invention there is provided a method of manufacture of a vaccine formulation as herein described, wherein the method comprises admixing a polypeptide according to the invention with a suitable adjuvant.

[0423] Particularly suitable adjuvant combinations for use in the formulations according to the invention are as follows:

i) 3D-MPL+QS21 in a liposome

ii) Alum+3D-MPL

[0424] iii) Alum+QS21 in a liposome+3D-MPL

iv) Alum+CpG

[0425] v) 3D-MPL+QS21+oil in water emulsion

vi) CpG

[0426] As used herein, the term "comprising" in context of the present specification should be interpreted as "including".

[0427] Embodiments and preferences may be combined as technically appropriate.

[0428] The disclosure herein describes embodiments comprising certain integers. The disclosure also extends to the same embodiments consisting or consisting essentially of said integers.

FIGURES

[0429] FIG. 1-10 shows various antibody and fragment sequences

[0430] FIG. 11 shows sera titres for TcdA and TcdB

[0431] FIG. 12 shows anti TcdA (Ribotype 003) in-vitro neutralization data for single Mabs

[0432] FIG. 13 shows anti TcdA (Ribotype 003) in-vitro neutralization data for single and paired Mabs

[0433] FIG. 14-15 shows anti TcdA (Ribotype 003) in-vitro neutralization data for paired Mabs

[0434] FIG. 16-18 shows anti TcdA (Ribotype 003) in-vitro neutralization data for three Mab mixtures

[0435] FIG. 19-20 shows anti TcdA (Ribotype 003) in-vitro neutralization data for four and five Mab mixtures

[0436] FIG. 21-22 shows anti TcdA (Ribotype 003) in-vitro neutralization data for single and paired Mabs at different TcdA concentrations

[0437] FIG. 23-24 shows anti TcdA (Ribotype 003) in-vitro neutralization data for single and to five Mab mixtures at different TcdA concentrations

[0438] FIG. 25-26 shows anti TcdB (Ribotype 003) in-vitro neutralization data for single Mabs

[0439] FIG. 27-30 shows anti TcdB (Ribotype 003) in-vitro neutralization data for paired Mabs

[0440] FIG. 31-33 shows anti TcdB (Ribotype 003) in-vitro neutralization data for three Mab mixtures

[0441] FIG. 34-40 shows anti TcdB (Ribotype 003) in-vitro neutralization data for two Mab mixtures at different toxin concentrations

[0442] FIG. 41-45 shows anti TcdB (Ribotype 003) in-vitro neutralization data for two Mab mixtures at different relative Mab ratios and different toxin concentrations

[0443] FIG. 46-59 shows TcdB neutralisation data for single antibodies and pairs of antibodies

[0444] FIG. 60 shows the amino acid sequence for TcdA

[0445] FIG. 61 shows the amino acid sequence for TcdB

[0446] FIG. 62 shows TEER assay data for TcdA in a histogram format

[0447] FIG. 62A shows TEER assay data for TcdA in line graph format

[0448] FIG. 63 shows a meier-kaplan curve for the combination of antibodies 997, 1125 and 1151, high concentration is 50 mg/Kg and low concentration is 5 mg/Kg 50 mg/kg' dose gave 100% protection to day 11, .about.82% protection to day 28. 5 mg/kg' dose resulted in non-durable and incomplete protection.

[0449] FIG. 64 shows bodyweight changes for vancomycin and vehicle treated hamsters

[0450] FIG. 65 shows the bodyweight for low dose antibodies 5 mg/Kg and high dose antibodies 50 mg/Kg

[0451] FIG. 66 shows photographs of a colon where the animal received treatment with antibodies according to the present disclosure vs a control

[0452] FIG. 67-68 show effects of vortexing on antibody stability

[0453] FIG. 69 shows a comparison of aggregation stability for various antibodies

[0454] FIG. 70-73 show neutralisation of TcdA for various ribotypes

EXAMPLES

Antibody Generation

[0455] A range of different immunogens and screening reagents were either purchased or produced by conventional E. coli expression techniques in order to provide a diverse and broad immune response and to facilitate identification and characterisation of monoclonal antibodies (listed in Table 1). In cases where recombinant proteins or peptides were generated, sequences were based on ribotype 027. The sequence for TcdA from ribotype 027 is given in SEQ ID NO: 171 (Uniprot accession number C9YJ37) and the sequence for TcdB from ribotype 027 is given is SEQ ID NO: 172 (Uniprot accession number C9YJ35).

[0456] Sprague Dawley rats and half-lop rabbits were immunised with either synthetic peptides mapping to regions common to both TcdA and TdcdB full-length toxin, formaldehyde-inactivated toxoid A, binding domain fragments of Toxin A (CROPs1,2,3 or CROPs4,5,6) or binding domain fragment of Toxin B (CROPs1,2,3,4), or in some cases, a combination of the above. Following 2 to 6 immunisations, animals were sacrificed and PBMC, spleen and bone marrow harvested. Sera were monitored for binding to Toxin A domains, toxin B domains, toxin or toxoid by ELISA. Sera titres of 2 such immunisations are shown in FIG. 11. UCB SLAM was used as a means to generate monoclonal antibodies. B cells were cultured directly from immunised animals (Zubler et al., 1985). This step enabled sampling of a large percentage of the B cell repertoire. By incorporating the selected lymphocyte antibody method (SLAM) (Babcook et al., 1996) it was possible to deconvolute positive culture wells and identify antigen-specific antibody-secreting cells. Here we used a modified version of SLAM (UCB SLAM (Tickle et al. 2009)) that utilises a fluorescence-based method to identify antigen-specific B cells from culture wells. B cell cultures were set up and supernatants were first screened for their ability to bind a relevant purified toxin domain (binding, translocation or catalytic) in a bead-based assay using an Applied Biosystem 8200 cellular detection system. This was a homogeneous assay using B cell culture supernatant containing IgG, biotinylated toxin domains coated onto streptavidin beads and a goat anti-rat/rabbit Fc-Cy5 conjugate. Cell cultures positive for binding to TcdA or TcdB components from this assay were selected for use in cell-based functional assays to identify neutralisers of toxin-induced cytotoxicity. Approximately 12,000 toxin-specific positives were identified in the primary binding screen from a total of 40.times.50-plate experiments. This equated to the screening of approximately 0.5 billion B cells. Heavy and light variable region gene pairs were isolated from single cells harvested by micromanipulation from approximately 100 toxin-neutralising wells following reverse transcription (RT)-PCR. These V-region genes were then cloned as mouse IgG1/kappa full-length antibodies for rat variable regions and rabbit IgG/kappa full-length antibodies for rabbit variable regions. Antibodies were re-expressed in a HEK-293 transient expression system. These recombinant antibodies were then retested for their ability to neutralise toxin in cell based assays. Recombinant antibodies were also screened by BIAcore to determine affinity for a given toxin domain and to also determine the specificity and approximate the number of binding events of antibody to toxin. Based on in vitro activity in cell based assays and affinity measurements, lead candidates were selected for humanisation. Unless otherwise stated, all the data herein was generated using the humanised antibodies.

[0457] A panel of recombinant, E. coli-produced toxin fragments (TcdA), C. difficile-derived toxin or toxoid (A) and synthetic peptides (B) were generated or purchased from commercial sources.

TABLE-US-00053 TABLE 1 Toxin A (TcdA) sequence related reagents for screening and immunizations. Fragment Residue number Source TcdA catalytic M1-E659 UCB E. coli expression TcdA translocation K577-D1350 UCB E. coli expression TcdA CROPS.sub.123 (TcdA123) S1827-D2249 UCB E. coli expression TcdA CROPS.sub.456 (TcdA456) G2205-R2608 UCB E. coli expression TcdA CROP.sub.1 S1827-N1978 UCB E. coli expression TcdA CROP.sub.2 G1966-N2133 UCB E. coli expression TcdA CROP.sub.3 G2100-D2249 UCB E. coli expression TcdA CROP.sub.4 G2213-N2381 UCB E. coli expression TcdA CROP.sub.5 G2328-N2494 UCB E. coli expression TcdA CROP.sub.6 G2462-N2609 UCB E. coli expression TcdA CROP.sub.7 R2554-D2701 UCB E. coli expression TcdB catalytic M1-A593 UCB E. coli expression TcdB translocation R576-D1349 UCB E. coli expression TcdB binding (TcdB1234) S1833-E2366 UCB E. coli expression TcdB CROP.sub.1 S1833-S1981 UCB E. coli expression TcdB CROP.sub.2 G1968-D2113 UCB E. coli expression TcdB CROP.sub.3 G2100-E2247 UCB E. coli expression TcdB CROP.sub.4 G2234-E2366 UCB E. coli expression Toxin A Full length purchased Toxin B Full length purchased Toxoid A Full length purchased

TABLE-US-00054 TABLE 2 Toxin B (TcdB) sequence related reagents for screening and immunizations. Toxin Domain Amino acid Sequence Catalytic SPVEKNLHFVWIGGEVSD SEQ ID NO: 173 Catalytic NLAAASDIVRL SEQ ID NO: 174 Catalytic CGGVYLDVDMLPGIH SEQ ID NO: 175 Catalytic CGGVYLDVDMLPGIHSDLFK SEQ ID NO: 176 Catalytic CWEMIKLEAIMKYK SEQ ID NO: 177 Catalytic CTNLVIEQVKNR SEQ ID NO: 178 Catalytic PEARSTISLSGP SEQ ID NO: 179 Catalytic CSNLIVKQIENR SEQ ID NO: 180 Catalytic TEQEINSLWSFDQA SEQ ID NO: 181 Catalytic TEQEINSLWSFDPEARSTISLSGPC SEQ ID NO: 182 Translocation NVEETYPGKLLLC SEQ ID NO: 183 Translocation Acetyl-CANQYEVRINSEGR SEQ ID NO: 184 Translocation VNTLNAAFFIQSLIC SEQ ID NO: 185 Translocation YAQLFSTGLNTIC SEQ ID NO: 186 Translocation CAGISAGIPSLVNNEL SEQ ID NO: 187 Translocation DDLVISEIDFNNNSIC SEQ ID NO: 188 Translocation MEGGSGHTVT SEQ ID NO: 189 Translocation AVNDTINVLPTITEGIPIVSTILDGINLGAAIK EL SEQ ID NO: 190 Binding CGFEYFAPANTDANNIEGQA SEQ ID NO: 191 Binding CGYKYFAPANTVNDNIYGQA SEQ ID NO: 192 Binding CKYYFNTNTAEA SEQ ID NO: 193 Binding CKYYFDEDTAEA SEQ ID NO: 194

Expression and Purification of C. difficile Anti-Toxin Mabs

[0458] Separate light chain and heavy chain mammalian expression plasmids were combined in equimolar ratios and used to transfect HEK-293 or CHO-S cells. For small scale expression studies lipofectamine and HEK-293 cells were used whereas for production of larger batches of IgG electroporation into CHO-S was preferred.

[0459] Culture supernatants were loaded onto a Mab Select SuRe column (in PBS pH 7.4). Antibody was eluted with 100% 0.1M Sodium Citrate pH 3.4 buffer. Samples were neutralized to pH7.4 with Tris.Cl pH8.0. Aggregate was removed by Superdex 200 Gel Filtration column in PBS pH 7.4.

TABLE-US-00055 TABLE 3 Cell Volume of Expression Amount Antibody type SN (L) type purified (mg) CA164_00997.g1_P3 CHO 10 Transient 755.93 CA164_00922.g1_P3 CHO 0.5 Transient 129.36 CA164_01125.g2_P3 CHO 10 Transient 498.96 CA164_01151.g4_P3 CHO 5 Transient 262.43

Example 1 In-Vitro Neutralization of TcdA Activity by Purified Mabs

[0460] All neutralisation screening assays were run in 96 well polystyrene plates. The assay uses CACO-2 cells grown, and screened in MEM+20% FCS, 2 mM Q, and NEAA. Any antibody combinations are at equal molar ratios unless stated otherwise. Day 1: Cells were plated @ 3000 per well in 50 .mu.l media, and incubated for 24 hrs; Day 2: Purified samples of humanised Mab were added to 96 well round bottomed polypropylene sterile plates; Spike PP plates with toxin A at a concentration sufficient to generated the appropriate lethal dose i.e. LD.sub.80 or above and incubate for 1 hr, at 37.degree. C.; Add 50 .mu.l of this mixture to cell plates and incubate for 96 hrs; Day 5: Add Methylene blue (0.5% Methylene Blue 50% ethanol); Incubate for 1 hr at room temperature; Lyse the cells with 1% N-Lauryl Sarcosine, and Read on the BIOTEK Synergy2 plate reader at 405 nm. The results are shown in FIGS. 12 to 24. EC.sub.50 and % maximum neutralization of TcdA activity shown confirm that the selected antibodies have very high potencies as single agents. Combinations of 2 to 5 of these did not improve upon the best EC.sub.50 or % maximum neutralization. Lack of any synergy when combining Mabs CA922, 923, 995, 997 and 1000 is an important observation and may be due to the fact the each antibody alone has exceptionally high levels of affinity and potency. Supporting data in Example 5 also show that some of the Mabs (e.g. CA997) are capable of binding to TcdA subdomains many times. Hence it seems probable that these 5 Mabs represent that the maximum affinity, potency and valency that is achievable when targeting the C-terminal cell binding domain of TcdA. The antibodies were also effective at neutralising very high toxin concentrations ranging from LD80 to greater than LD.sub.95 (LD.sub.max) but some modest increases in EC.sub.50 (i.e. decreases in potency) were observed with very high levels of [TcdA]. These data are also surprising since others have shown substantial reductions in potency when testing elevated TcdA concentrations (20).

[0461] The high potency and affinity of the Mabs described here, e.g. for CA997; is not due solely to their high valency of binding. Others (20 and WO06/071877) describe anti-TcdA Mabs capable of binding up to 14 times. These Mabs only had affinities in the range 0.3 to 100 nM and showed incomplete protection against TcdA mediated cell killing, alone (26-63% protection) or in pairs (31-73% protection). Hence it has been demon-strated that high valency of binding to TcdA does not necessarily invoke either high affinity of binding to or neutralisation of TcdA. Neither the affinities nor valency of binding to TcdA were described for Mab CDA-1 (18 and U.S. Pat. No. 7,625,559). Thus Mabs described herein to have surprising affinity, potency and valency.

TABLE-US-00056 TABLE 4 Anti TcdA 1, 2 & 3 Mab combinations at a single TcdA conc. (LD.sub.80) Final (highest) EC.sub.50 Antibody Mab conc.ng/ml (ng/ml) 922 500 1.21 923 500 160.42 995 500 37.64 997 500 6.25 1000 500 19.73 922 + 923 500 3.58 922 + 925 500 3.326 922 + 997 500 2.88 922 + 1000 500 2.64 923 + 995 500 60.23 923 + 997 500 7.54 923 + 1000 500 9.24 995 + 997 500 7.29 995 + 1000 500 19.63 997 + 1000 500 4.46 922 + 923 + 995 500 4.72 922 + 923 + 997 500 3.23 922 + 923 + 1000 500 3.21 922 + 995 + 997 500 2.22 922 + 995 + 1000 500 2.85 922 + 997 + 1000 500 2.22 923 + 995 + 997 500 5.04 923 + 995 + 1000 500 9.49 995 + 997 + 1000 500 5.84 922 + 923 + 995 + 997 500 2.75 922 + 923 + 995 + 1000 500 3.75 922 + 995 + 997 + 1000 500 3.46 923 + 995 + 997 + 1000 500 4.81 922 + 923 + 997 + 1000 500 3.06 922 + 923 + 995 + 997 + 1000 500 4.72

TABLE-US-00057 TABLE 5 Anti TcdA single, paired, and triplet Mab combinations at various TcdA concentrations, where TcdA is at its LD.sub.80, LD.sub.90, LD.sub.95 and LD.sub.max. Final Mab EC.sub.50 Toxin TcdA Sample conc.ng/ml (ng/ml) @ 3000 pg/ml 922 500 4.89 (LD.sub.MAX) 997 500 10.99 1000 500 50.17 922 + 997 500 7.18 922 + 1000 500 6.99 997 + 1000 500 9.437 922 + 997 + 1000 500 10.80 922 + 997 + 1000 + 995 500 15.03 922 + 997 + 1000 + 995 + 923 500 7.16 @ 1000 pg/ml 922 500 1.24 (LD.sub.95) 997 500 3.42 1000 500 9.60 922 + 997 500 1.85 922 + 1000 500 2.51 997 + 1000 500 3.61 922 + 997 + 1000 500 2.40 922 + 997 + 1000 + 995 500 2.74 922 + 997 + 1000 + 995 + 923 500 2.38 @ 700 pg/ml 922 500 0.84 (LD.sub.90) 997 500 2.40 1000 500 6.23 922 + 997 500 1.19 922 + 1000 500 1.33 997 + 1000 500 2.68 922 + 997 + 1000 500 1.84 922 + 997 + 1000 + 995 500 2.17 922 + 997 + 1000 + 995 + 923 500 2.06 @ 350 pg/ml 922 500 0.39 (LD.sub.80) 997 500 1.18 1000 500 2.76 922 + 997 500 0.67 922 + 1000 500 0.85 997 + 1000 500 2.06 922 + 997 + 1000 500 0.83 922 + 997 + 1000 + 995 500 0.97 922 + 997 + 1000 + 995 + 923 500 0.98

Example 2 Anti TcdB In-Vitro Neutralization by Purified Mab

Assay Methods Description:

[0462] All neutralisation screening assays were run in 96 well polystyrene plates.

[0463] The assay uses CACO-2 cells grown, and screened in MEM+20% FCS, 2 mM Q, and NEAA. Unless stated all Ab combinations are in equal ratios.

[0464] Day 1: Cells are plated @ 3000 per well in 50 .mu.l media, and incubated for 24 hrs

[0465] Day 2: Purified samples of humanised Mab were added to 96 well round bottomed polypropylene sterile plates

[0466] Spike PP plates with toxin B lot #031 and incubate for 1 hr, at 37.degree. C.

[0467] Add 50 .mu.l of this mixture to cell plates

[0468] Incubate for 96 hrs

[0469] Day 5: Add Methylene blue (0.5% Methylene Blue 50% ethanol)

[0470] Incubate for 1 hr at room temperature

[0471] Lyse the cells with 1% N-Lauryl Sarcosine

[0472] Read on the BIOTEK Synergy2 plate reader at 405 nm

[0473] The data in FIGS. 25 to 33 show that single Mabs alone were relatively ineffective at neutralizing TcdB, both in terms of % maximum neutralization and activity (EC.sub.50). However, when the antibodies were combined in two's and three's considerable improvements in both % maximum neutralization and activity (EC.sub.50) were observed. 1125 and 1151 were selected as a best pairing, although other good pairings were observed: 1125+1153, 1125+1134.

[0474] The most effective pairs of Mabs were selected empirically and were found retrospectively to make unexpected and surprising combinations when regarding the individual potencies of each Mab. For example, in Table 6 only CA927 had a TcdB neutralisation potential which could result in a defined EC.sub.50 whilst the TcdB neutralisation potential of both CA1125 and CA1151 were insufficient under these assay conditions to result in a defined EC.sub.50. However, CA927 was not found to be the most effective Mab to use within a combination. The best CA927 containing combination had an EC.sub.50 of 13.5 ng/ml whereas other two Mab combinations had EC.sub.50's as low as 2.59 and 4.71 ng/ml. In another example, in Table 8 CA1099 had the lowest TcdB neutralisation EC.sub.50 under the assay conditions used. However, CA1099 was not found to be the most effective Mab to use within a combination. The best CA1099 containing combination had an EC.sub.50 of Eng/ml whereas other two Mab combinations had EC.sub.50's as low as 2 and 1 ng/ml. We might speculate that the most effective pairings of Mabs are defined by their cooperative binding modalities especially as defined by having non-overlapping epitopes.

TABLE-US-00058 TABLE 6 Anti-TcdB Mab combinations and relative Mab ratios at constant toxin concentration. Final Mab Sample conc.ng/ml EC.sub.50(ng/ml) 1125.g2 1000 >1000 1134.g5 1000 >1000 927.g2 1000 12.89 1153.g8 1000 >1000 1102.g4 1000 >1000 927 + 1099 1000 >1000 927 + 1102 1000 >1000 927 + 1114 1000 >111.111 927 + 1125 1000 13.55 927 + 1134 1000 51.58 1099 + 1114 1000 >1000 1102 + 1114 1000 >333.333 1102 + 1125 1000 15.51 1114 + 1134 1000 19.70 1114 + 1151 1000 25.69 1114 + 1153 1000 27.48 1125 + 1134 1000 2.59 1125 + 1151 1000 4.71 1125 + 1153 1000 21.23 1125 + 1134 + 1114 1000 3.77 1125 + 1134 + 927 1000 2.63 1125 + 1151 + 1114 1000 4.90 1125 + 1151 + 927 1000 5.69 1125.g2 + 1134.g5 + 927.g2 1000 5.83 1125.g2 + 1134.g5 + 1153.g8 1000 9.89 1125.g2 + 1134.g5 + 1102.g4 1000 2.72

Example 3 Neutralisation of TcdB by Combinations of Purified Mab

[0475] All neutralisation screening assays were run in 96 well polystyrene plates.

[0476] The assay uses CACO-2 cells grown, and screened in MEM+20% FCS, 2 mM Q, and NEAA.

[0477] Day 1: Cells are plated @ 3000 per well in 50 .mu.l media, and incubated for 24 hrs

[0478] Day 2: Purified samples of humanised Mab were added to 96 well round bottomed polypropylene sterile plates

[0479] Spike PP plates with toxin B (VPI 10463) and incubate for 1 hr, at 37.degree. C.

[0480] Add 50 .mu.l of this mixture to cell plates

[0481] Incubate for 72 hrs

[0482] Day 5: Add Methylene blue (0.5% Methylene Blue 50% ETOH)

[0483] Incubate for 1 hr at room temperature

[0484] Lyse the cells with 1% N-Lauryl Sarcosine

[0485] Read on the BIOTEK Synergy2 plate reader at 405 nm

[0486] The results are shown in FIGS. 34 to 45.

[0487] These data show that the best pair of Mabs for neutralizing TcdB at a range of toxin concentrations was CA1125 and CA1151. Moreover, the 1125+1151 combination was largely unaffected by changes in the relative molar ratios which is in contrast to 1125+1153.

TABLE-US-00059 TABLE 7 Anti-TcdB Mab combinations and relative Mab ratios at 3 different toxin cones. EC50 values (ng/ml) Antibody combination TcdB LD60 TcdB LD77 TcdB LD85 1125.g2 + 927.g2 (50:50) 2.8 6 11.3 1125.g2 + 1102.g4 (50:50) 4 13 44 1125.g2 + 1114.g8 (50:50) 3.5 7.1 25.4 1125.g2 + 1134.g5 (50:50) 0.48 1.4 4 1125.g2 + 1151.g4 (50:50) 0.85 0.85 1.5 1125.g2 + 1153.g8 (50:50) 2.7 5.2 25.2 1125.g2 + 1134.g5 (25:75) <0.15 0.84 7.2 1125.g2 + 1151.g4 (25:75) 0.73 1 2.1 1125.g2 + 1153.g8 (25:75) 7 10 27 1125.g2 + 1134.g5 (75:25) 0.66 1.2 2.5 1125.g2 + 1151.g4 (75:25) 1.4 1.2 8.3 1125.g2 + 1153.g8 (75:25) 2.9 7.5 30

[0488] The data show that even the most active specific paired combinations have surprisingly and non-predictably different properties relative to each other. The EC.sub.50 of the preferred combination of CA1125 and CA1151 in equimolar ratios is largely unaffected by an increasing [TcdB]. The three relative molar ratios of Mabs tested (i.e. 25:75 vs 50:50 vs 75:25) have very similar EC.sub.50's to each other, suggesting that CA1125 and CA1151 have especially complementary modes of action. This is in contrast to the combination of CA1125 with CA1134 where the increase in EC.sub.50 (i.e. reduction of potency) with higher [TcdB] is more substantial and where the three Mab molar ratios are not equally effective: The CA1125:CA1134 ratio of 25:75 is notably less potent than 50:50 and 75:25. This suggests that the combined potency of CA1125+CA1134 is more dependent upon the CA1125 component. The EC.sub.50 of all three molar combinations of CA1125 and CA1153 is substantially affected by increasing [TcdB] suggesting that CA1153 is a less suitable partner for combination with CA1125. In toto, these data show that CA1125 and CA1151 are a particularly favourable combination since the highest potency is maintained across a range of Mab and TcdB molar ratios.

TABLE-US-00060 TABLE 8 TcdB neutralisation - 1 or 2 anti-TcdB Mabs at constant toxin dose (LD.sub.80). Antibody IC50 (ng/ml) 1099 2 1102 N/A 1114 103 1125 N/A 1134 8 1151 182 1153 260 926 N/A 927 N/A 1099 + 1125 6 1114 + 1125 7 1151 + 1125 2 1134 + 1125 1 1102 + 1125 6 1125 + 1153 12 926 + 1125 42 927 + 1125 4

TABLE-US-00061 TABLE 9 TcdB neutralisation - 1 or 2 anti-TcdB Mabs at various TcdB doses. EC50 values (ng/ml) Maximum neutralisation Antibody combination TcdB LD75 TcdB LD86 TcdB LD90 TcdB LD75 TcdB LD86 TcdB LD90 1125.g2 n/a n/a n/a 40% 25% 15% 1114.g8 n/a n/a n/a 45% 25% 15% 1134.g5 n/a n/a n/a 45% 25% 15% 1151.g4 n/a n/a n/a 45% 25% 20% 1153.g8 28.3 n/a n/a 65% 35% 28% 1125.g2 + 1114.g8 (50:50) 10.1 243.8 n/a 85% 65% 40% 1125.g2 + 1134.g5 (50:50) 1.7 22.6 n/a 87% 60% 40% 1125.g2 + 1153.g8 (50:50) 6.1 32.2 n/a 95% 75% 48% 1125.g2 + 1151.g4 (50:50) 0.8 2.8 19.1 85% 80% 55% 1125.g2 + 1151.g4 (25:75) 1.2 2.8 47.2 85% 75% 60% 1125.g2 + 1151.g4 (75:25) 2.9 3.8 2.6 75% 70% 60%

[0489] These data show that combination of Mabs, especially CA1125 and CA1151 improve both the potency as measured by EC.sub.50 but also as measured by % maximum protection. The % maximum protection is of particular relevance in this assay method since the Mab:TcdB mixture is incubated with cells for a long time (72 h). Since TcdB is toxic to Caco-2 cells in the range of pg/ml in 2-4 h this measure may be considered to be a very difficult test of Mab neutralisation ability and may reflect the ability of Mab mixture with regard to their binding kinetics or modalities. In turn this may reflect the ability of Mab mixtures to protect against the effects of TcdB during an established infection when there may be substantial quantities of TcdB within tissues for many hours. Selected data from Tables 6-9 are further illustrated in FIGS. 46-59.

Example 4 Valency of Binding of Mabs to TcdB Sub-Domains

[0490] The number of moles of binding events of anti-C. difficile TcdB antibodies to TcdB.sub.1234 was determined by Surface Plasmon Resonance (SPR) on a Biacore 3000 (GE Healthcare). Streptavidin was immobilized on a CM5 sensor chip (GE Healthcare) to a level of .about.4000RU via amine coupling and biotinylated TcdB.sub.1234 was bound at 500-600RU. Two 20 .mu.l injections of the same anti-TcdB antibody mixtures (final concentration of each antibody was 500 nM) were injected over this surface at 10 .mu.l/min and the saturating binding response recorded. The surface was regenerated after every cycle using HCl. All the data was corrected for background binding using the response to the streptavidin only reference flowcell.

TABLE-US-00062 TABLE 10 Surface plasmon resonance analysis of the number of IgG binding sites on TcdB.sub.1234 No. of Binding Binding relative Antibody binding Response to CA927 combination cycle repeats (RU) average response CA1125.g2 10 750 0.9 CA1151.g4 10 1232 1.6 CA1125_CA1151 4 1941 2.5 CA1125_CA927 3 1570 2.0 CA1151_CA927 3 1959 2.5 CA927 8 791 1.0

[0491] All responses have been expressed relative to a multiple of CA927 average response (final column table 10) since CA927 appears to be representative of a Mab which binds to TcdB.sub.1234 once only.

[0492] Immobilized CA1125, when bound to TcdB.sub.1234, does not allow CA1125 to bind further supporting the idea that CA1125 has one binding site on TcdB.sub.1234 and that after this has been saturated that no other binding site for CA1125 can be found. However, when TcdB.sub.1234 has been saturated by CA1125, CA1151 can still bind. This demonstrates that CA1151 binds at alternative sites to that occupied by CA1125. Together these data show that CA1125 is a single binder of TcdB.sub.1234 whereas 1151 IgG binds to TcdB.sub.1234 more than once, most likely twice. Hence a mixture of CA1125 and CA1151 can bind to TcdB.sub.1234 approximately 3 times.

[0493] All antibodies combinations have an additive binding response showing that there are 2 or more non-competitive sites on TcdB.sub.1234 bound by these combinations.

Example 5 Valency of Binding of Mabs to TcdA Sub-Domains

[0494] The number of moles of binding events of anti-C. difficile TcdA antibodies to TcdA.sub.123 and A.sub.456 were determined by Surface Plasmon Resonance (SPR) on a Biacore 3000 (GE Healthcare). Streptavidin was immobilized on a CM5 sensor chip (GE Healthcare) via amine coupling to a level of .about.4000RU and biotinylated TcdA.sub.123 was bound to one flowcell and TcdA.sub.456 was bound to a different flowcell to a response of .about.500RU. Two 30 .mu.l injections of the same anti-TcdA antibody at 1 .mu.M were injected over both flowcells at 10 .mu.l/min and the saturating binding response recorded. The surface was regenerated after every cycle using HCl. All the data was corrected for background binding using the response to the streptavidin only reference flowcell.

TABLE-US-00063 TABLE 11 SPR analysis of the binding responses of IgGs to immobilised TcdA.sub.123 and TcdA.sub.456 CA997 CA1000 CA997/CA1000 ratio TcdA123 1069 166 6 TcdA456 1285 407 3

[0495] Antibodies CA997 and CA1000 bind to TcdA.sub.123 in a ratio of six CA997's to one CA1000 whereas they bind to TcdA.sub.456 in a ratio of three CA997's to one CA1000 (Table 2).

[0496] The maximum antibody response for CA997, corrected for molecular weight and immobilized toxin level is similar for TcdA.sub.123 and TcdA.sub.456. This suggests that CA997 binds TcdA.sub.456 six times and CA1000 binds twice to TcdA.sub.456. Hence antibody CA997 likely binds to TcdA whole toxin (TcdA) approximately 12 times.

[0497] Overall CA997 binds six times or more to A.sub.123 and six times or more to A.sub.456, whereas CA1000 binds at least once to A.sub.123 and twice to A.sub.456.

[0498] Increased valency of binding to TcdA and TcdB may have two important effects in vivo. The first is that any Mab or Mab mixture which is capable of binding TcdB more than once will have increased potential to form inter-toxin binding events and hence immunoprecipitation. Immunoprecipitation can contribute to potency by reducing the solubility of toxin and forming very large macromolecular complexes which hence reduce the effective working concentration of toxin. Such large protein complexes may be taken up by macrophages and monocytes resident in the tissue and may contribute to an augmented host immune response. Antigen:antibody complexes bearing Fc fragments have been specifically shown to be capable of priming a host immune response against a gut pathogen (21). Also, soluble antigen:antibody complexes have been successfully used as a vaccine directed against the antigen in human clinical trials (22). In addition, immune decoration of toxin with Fc bearing IgG may contribute to immune clearance using normal mechanisms through the liver and spleen. In general, higher levels of Fc decoration of antigen lead to faster and more complete levels of clearance (23) Critically, it may be that presence of 2 or more Mab Fc domains per toxin, especially 3 Fc domains per toxin may represent a critical number of Fcs required for very rapid and substantial clearance of toxin (24). The anti-TcdA Mab CA997 is likely capable of binding to TcdA up to 12 times and the combination of CA1125 and CA1151 is likely capable of binding to TcdB 3 times. Hence the 3 Mab mixture is very likely to be capable of providing for these kinds of additional potency mechanisms in vivo.

Example 6 Mab Neutralisation of Loss of TEER Caused by TcdA

[0499] C. difficile monolayer integrity assay is performed using the Becton-Dickinson (BD) Caco-2 BioCoat HTS plate system.

[0500] Day 1--Caco-2 cells seeded @ 2.times.10.sup.5/ml per well of the plate insert in 500 .mu.l Basal seeding medium (provided by BD). 35 ml of Basal seeding medium added to the feeder tray. Cells incubated for 24 hours at 37.degree. C. Day 2--Basal seeding medium removed from inserts and feeder tray, and replaced with Entero-STIM differentiation medium (supplied by BD). 500 .mu.l added per well insert and 35 ml to the feeder tray. Cells incubate for a further 72 hrs at 37.degree. C. Day 5--Antibodies prepared at 2.times. concentration relative to that to be used in the assay well in a polypropylene plate and toxin A. Toxin A added to antibodies at a concentration of 125 ng/ml and plate incubated for 1 hr at 37.degree. C. 1 ml of Caco-2 growth medium (MEM+20% FCS, 2 mM Q, NEAA) added to each well of a standard 24-well TC plate. BioCoat insert plate transferred to the 24-well TC plate. Entero-STIM medium removed from inserts and replaced with 400 .mu.l of toxin:Ab mixture. Loss of tight junctions between gut cells is the key early effect of TcdA on cell monolayers and gut tissue sections and is the primary cause of diarrhoea. Albumin and other serum proteins are lost into the gut lumen along with accompanying serum fluid. The loss of trans-epithelial electrical resistance in differentiated cultured cells which have formed a monolayer is a useful surrogate for the protection against the acute effects of TcdA. Three antibodies shown have good levels of protection against TEER loss, FIG. 62. It is notable and surprising that the abilities of these Mabs in TEER assays do not reflect those seen in toxin neutralization as measured in a cell proliferation assay. CA922 has the best performance in a cell proliferation assay (EC.sub.50=1.21 ng/ml) and yet this is considerably out-performed in the TEER assay by an antibody (CA1000) which has >10.times. lower potency in a cell proliferation assay (EC.sub.50=19.73 ng/ml). CA997 had the best performance in the TEER assay since it had both high levels of protection and maintained this at the lower Mab concs. CA997 had a substantial potential to neutralize TEER loss with maximal inhibition approaching 80% and an EC.sub.50 of approximately 80 ng/ml at 4 h. These observations are unexpected since the Mabs in question all had high affinities for TcdA domains (CA922 .about.4 pM, CA997 .about.132 pM, CA1000 .about.73 pM). These data suggest that CA997 and CA1000 recognise epitopes important in TEER loss or neutralize TcdA by different mechanism to other Mabs. Furthermore, since CA1000 is estimated to bind to holotoxin twice (once in TcdA.sub.123 and once in TcdA.sub.456) CA1000 may define `TEER critical` epitopes within the TcdA cell binding regions which might have particular value for defining vaccine immunogens. Results are shown in FIG. 62.

Example 7 Affinity of Anti-C. difficile Toxin Antibodies for Sub-Domains of TcdA and TcdB: TcdA.sub.123, TcdA.sub.456 and TcdB.sub.1234

[0501] Kinetic constants for the interactions of anti-C. difficile TcdA and TcdB antibodies were determined by surface plasmon resonance conducted on a BIAcore 3000 using CM5 sensor chips. All experiments were performed at 25.degree. C. Affinipure F(ab').sub.2 fragment goat anti-human IgG, Fc fragment specific (Jackson ImmunoResearch) was immobilised on a CM5 Sensor Chip (GE) via amine coupling chemistry to a capture level of .apprxeq.7000 response units (RUs). HBS-EP buffer (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, Biacore AB) was used as the running buffer with a flow rate of 10 .mu.L/min. A 10 .mu.L injection of each antibody at 1 ug/ml or lower was used for capture by the immobilised anti-human IgG, Fc. TcdA123, TcdA456 or TcdB1234 were titrated over captured purified antibodies at doubling dilutions from 12.5 nM at a flow rate of 30 .mu.L/min. For antibodies present in culture supernatants, a single concentration of 12.5 nM of TcdA123 or TcdA456 and 50 nM of TcdB1234 was passed over the antibodies at 30 ul/min. Kinetics were calculated on n=2 The surface was regenerated at a flowrate of 10 uL/min by two 10 .mu.L injections of 40 mM HCl, and a 5 .mu.L injection of 5 mM NaOH. Double referenced background subtracted binding curves were analysed using the BIAevaluation software (version 3.2) following standard procedures. Kinetic parameters were determined from the fitting algorithm.

TABLE-US-00064 TABLE 12 Anti-TcdA Mab affinities and binding kinetics Antibody ID ka (1/Ms) kd (1/s) KD (M) KD (pM) Material/Assay TcdA123 CA164_00922.g1 1.09E+06 4.43E-06 4.06E-12 4.06 Purified Mab 5 point CA164_00923.g1 5.36E+05 3.47E-05 6.47E-11 64.7 titration CA164_00995.g1 No binding No binding CA164_00997.g1 7.84E+05 1.03E-04 1.32E-10 132 CA164_01000.g1 1.33E+05 9.78E-06 7.33E-11 73.3 CA164_00993.g1 9.00E+05 5.00E-06 5.56E-12 5.56 Supernatant 2x 1 point titration TcdA456 CA164_00922.g1 1.29E+06 3.33E-06 2.59E-12 2.59 CA164_00923.g1 6.16E+05 1.92E-04 3.12E-10 312 Purified Mab 5 point CA164_00995.g1 2.87E+05 3.42E-05 1.19E-10 119 titration CA164_00997.g1 9.21E+05 6.15E-05 6.68E-11 66.8 CA164_01000.g1 3.55E+05 2.98E-05 8.41E-11 84.1 CA164_00993.g1 1.25E+06 5.00E-06 4.00E-12 4.00 Supernatant 2x 1 point titration

TABLE-US-00065 TABLE 13 Anti-TcdB Mab affinities and binding kinetics Antibody ID ka (1/Ms) kd (1/s) KD (M) KD (pM) Material/Assay TcdB1234 CA164_1125.g2 2.64E+05 3.23E-05 1.22E-10 122 Purified Mab 3 point titration CA164_1151.g4 7.49E+05 4.13E-04 5.51E-10 551 Purified Mab 3 point titration CA164_926.g1 1.38E+05 7.12E-05 5.16E-10 516 Supernatant 2x 1 point titration CA164_927.g2 3.97E+05 3.61E-05 9.11E-11 91 Purified Mab 3 point titration CA164_1099.g2 5.24E+05 1.63E-05 3.10E-11 31 Purified Mab 3 point titration CA164_1102.g4 1.17E+05 3.78E-04 3.25E-09 3250 Supernatant 2x 1 point titration CA164_1114.g2 2.87E+05 1.97E-03 6.87E-09 6870 Supernatant 2x 1 point titration CA164_1114.g8 2.55E+05 1.85E-03 7.25E-09 7250 Supernatant 2x 1 point titration CA164_1129.g1 1.89E+05 2.30E-04 1.22E-09 1220 Supernatant 2x 1 point titration CA164_1134.g5 5.09E+05 2.45E-05 4.81E-11 48 Purified Mab 3 point titration CA164_1153.g8 1.43E+05 4.48E-05 3.14E-10 314 Purified Mab 3 point titration

[0502] The anti-TcdA affinities are particularly high compared to the published affinities of other Mabs. We demonstrate that affinities as low as 4 pM are achievable. The preferred CA997 has an affinity of 132 pM, CA1125 122 pM and CA115 551 pM. CA995 clearly shows that it does not bind to CROPs A.sub.123 and hence that demonstrates that the Mab shown here have properties which are different from each other in surprising and unexpected ways. CA922, 923, 997 and 1000 do bind at least once to CROPs A123 and A456. Hence these 4 Mabs confirming that each must bind to holotoxin at least twice. We have been unable to derive affinities for the binding of these Mabs to holotoxin due to technical constraints. However, given the high affinities and valencies demonstrated for the anti-TcdA Mabs it is possible to speculate that the functional affinities against holotoxin may be even stronger than those illustrated for binding to toxin sub-domains. The anti-TcdB Mabs also demonstrated strong affinities reaching as low as 31 pM. In particular CA1125, 1151, 927, 1099, 1134 and 1153 show affinities which surpass those demonstrated by others.

Example 8 Biophysical Characterisation of C. difficile Anti-Toxin Humanised IgG1 Molecules

Molecules Analysed

Anti-TcdA IgG1:

CA164_00922.g1

CA164_0923.g1

CA164_0995.g1

CA164_0997.g1

CA164_01000.g1

Anti-TcdB IgG1

CA164_01125.g1

CA164_01125.g2

CA164_01134.g4

CA164_01134.g5

CA164_01134.g6

CA164_01102.g1

CA164_01102.g4

CA164_01151.g4

[0503] Antibody combinations need to be made up of Mabs having high levels of stability in order to mitigate potential risks of aggregation during long term storage. Thermal stability (Tm) is used as one measure. Of special value to Mab mixtures is measuring their propensity to aggregate due to physical stress such as agitation or shaking. Aggregates are undesirable components of drug compositions since they may reduce storage life time and may pose a safety risk to patients at certain levels. The Tm data show that all 5 anti-TcdA Mabs have high Tm stability, whilst three (CA922, 923 and 997) have very high Tm's in the range of 79-81.degree. C. Of the anti-TcdB Mabs tested all but two have very high Tm's. Of note is that CA997, CA1125 and CA1151 which were tested in the hamster infection study (Example 9) had very high Tm's (79.2.degree. C., 79.3.degree. C. and 80.8.degree. C. respectively) which makes them suitable for use in a Mab mixture.

[0504] In the shaking aggregation assay, CA997 and 922 had the lowest propensity to aggregate of the 5 anti-TcdA Mabs. Similarly, CA115 and 1151 had the lowest aggregation propensities of the anti-TcdB Mabs. Hence the use of CA997, 1125 and 1151 as a Mab mixture may have special value since they are more likely to survive co-formulation and storage at high protein concentrations.

Estimation of Isoelectric Point (pI) by Capillary IEF

[0505] Samples were prepared by mixing the following: 30 ul Protein sample at 2 mg/ml, 0.35% Methylcellulose, 4% pH3-10 ampholytes (Pharmalyte), synthetic pI markers (4.65 and 9.77), 1 ul of each stock solution, and HPLC grade water to make up the final volume to 200 ul. The mixture was then analysed using iCE280 IEF analyzer (pre-focusing at 1500V for 1 min followed by focusing at 3000V for 6 mins). The calibrated electropherograms were then integrated using Empower software (from Waters)

Thermal Stability (Tm) Measured Via Thermofluor Assay.

[0506] This method uses Sypro orange fluorescent dye to monitor the unfolding process of protein domains. The dye binds to exposed hydrophobic regions that become exposed as a consequence of unfolding which results in a change to the emission spectrum.

[0507] The sample (5 ul at 1 mg/ml) is mixed with a 5 ul of a stock solution of Sypro orange (30.times.) and the volume made up to 50 ul with PBS, pH 7.40.

[0508] 10 ul aliquots of this solution is applied towells in a 384 well plate (n=4).

[0509] The plate is placed in a 7900HT fast real-time PCR system containing a heating device for accurate temperature control. The temperature is increased from 20.degree. C. to 99.degree. C. (Ramp rate of 1.1.degree. C./min). A CCD device simultaneously monitors the fluorescence changes in the wells. An algorithm is used to process intensity data and take into account multiple transitions.

Stressing of Samples by Agitation.

[0510] During manufacture antibody samples are subjected to mechanical stress generated by processes such as pumping and filtration. This may cause denaturation and consequently aggregation due to exposure of the protein to air-liquid interfaces and shear forces resulting in the ultimate loss of bioactivity. Stress by vortexing is a method to screen the robustness of the antibody samples for prediction of aggregation stability.

[0511] Both anti-TcdA and anti-TcdB IgG1 molecules were subjected to stress by agitation, by vortexing using an Eppendorf Thermomixer Comfort at 25.degree. C., 1400 rpm. Sample size was 250 uL, (.times.3 per sample) in a 1.5 mL conical Eppendorf-style capped tube (plastic), in PBS pH 7.4. Each sample was brought to a concentration of 1 mg/ml (using extinction coefficient calculated from sequence) and aggregation was monitored by absorbance at 340 nm and/or 595 nm, by use of a Varian Cary 50-Bio spectrophotometer, measured at intervals for up to 24 hours.

[0512] Results Table 14 provides a summary of the measured pI and Tm data for both anti-TcdA and anti-TcdB IgG1 molecules.

TABLE-US-00066 TABLE 14 Compilation of pI and Tm Data measured Tm(Fab) Anti-TcdA IgG1 pI in PBS Tm(CH2) CA164_00922.g1 8.8 81 69.2 CA164_0923.g1 9.2 79 69.3 CA164_0995.g1 8.5 71 no data* CA164_0997.g1 8.3 79.2 68.4 CA164_01000.g1 7.74 70.5 no data* Anti-TcdB IgG1 CA164_01125.g1 9.2 79.3 69.4 CA164_01125.g2 9.2 79.5 69.3 CA164_01134.g4 9.3 78.4 69.4 CA164_01134.g5 9.2 76.4 69.2 CA164_01134.g6 9.2 76.6 69.6 CA164_01102.g1 9.1 69 no data* CA164_01102.g4 9.1 69.1 no data* CA164_01151.g4 9.2 80.8 69.8 *denotes that it was not possible to discern the Fab and CH2 domains.

Measured pI

[0513] The measured pI of the molecules were high (except for CA164_01000.g1_P3) and away from the pH of formulation buffers such as PBS, pH 7.4 and 50 m sodium acetate/125 mM sodium chloride, pH 5. This may mean that buffers with pH's suitable for co-formulation of two or more Mabs can be selected.

Thermal Stability (Tm) Measured Via Thermofluor Assay

[0514] Since all of the molecules are IgG1, the Tm of the Fc domain (Tm(CH2)) are the same. The difference in thermal stability between the molecules can be determined by the Tm of the Fab' domain (Tm(Fab)).

[0515] For the anti-TcdA molecules, the rank order (most stable first) was CA922.gtoreq.997>923>995>1000 and for the anti-TcdB molecules (most stable first) was CA1151.g4>1125.g1,g4>1134.g4>1134.g5.gtoreq.1134.g6>1102.g1=1- 102.g4.

Stressing of Samples by Agitation It was possible to determine different aggregation stability between the different antibodies, FIG. 67 shows the effect of agitation via vortexing on different anti-TcdA IgG1 molecules in PBS, pH 7.4.

[0516] It was possible to determine a ranking order (most aggregation stable first): CA922.gtoreq.997>923.gtoreq.995>1000

[0517] FIG. 68 shows the effect of agitation via vortexing on different anti-TcdB molecules.

[0518] It was possible to rank the order of aggregation stability, such that the CA1125 grafts appeared more stable than the CA1134 molecules which were more stable than the CA1102 molecules.

[0519] A further study was performed to compare directly the aggregation stability of the anti-TcdB molecule (CA1151.g4) with the more stable molecule CA1125.g2 (see FIG. 2) and more aggregation stable anti-TcdA molecules (CA922.g1 and CA997.g1). The results can be seen in FIG. 69.

[0520] Further results for these 4 Mabs are also shown in FIGS. 67 and 68.

[0521] For the anti-TcdA molecules, CA922.g1 and CA977.g1, CA922 were preferable based on the analyses above, although apart from CA1000) all molecules could be considered suitable candidates for use as therapeutic IgG1.

[0522] For the anti-TcdB molecules, the biophysical characteristics could be grouped within the family of grafts based on the aggregation stability and Tm, such that the CA1125 grafts potentially proved more stable. The CA1102 grafts showed poorest Tm data and also showed the greatest tendency to aggregate via stress by agitation.

[0523] A study using CA1151.g4 showed that this molecule exhibited slightly increased aggregation stability relative to CA11125.g2 and seemed equivalent to the TcdA molecules (CA922.g1 and CA997.g1. All four molecules showed equivalent Tm values. CA997, CA1125 and CA1151 show very high levels of thermostability and very low levels of aggregate formation after agitation.

Example 9 Anti-C. difficile Toxin Mab Hamster Infection Study

[0524] The hamster infection study was performed by Ricerca Biosciences LLC, Cleveland, Ohio, USA. The study protocol was approved by the Ricerca IACUC committee. Active and control components (composition and dose) were blinded to Ricerca staff until after completion of the planned 28 day study period.

[0525] Golden Syrian male hamsters (weight 82-103 g, 54 days old) were individually housed in HEPA filtered disposable cages and fed Teklad Global Diet 2016 and water ad libitum. After acclimatisation, hamsters were pre-dosed (i.p.) with Mab mixtures or PBS (vehicle control) once a day for each of 4 days: days -3, -2, -1 and 0. Two doses of Mab were investigated: high dose=50 mg/kg each of anti-TcdA and anti-TcdB components and low dose 5 mg/kg each of anti-TcdA and anti-TcdB components.

[0526] The drug combination tested was composed of one anti-TcdA antibody (CA997.g1) which constituted 50% of the injected protein and two anti-TcdB antibodies (CA1125.g2 and CA1151.g4) which together constituted 50% of the injected protein but which alone constituted 25% of the injected protein. Hamsters were sensitised (day -1) with 50 mg/kg of Clindamycin phosphate in PBS (s.c.) before being challenged 1 day later (day 0) with 3.4.times.106 c.f.u. of vegetative cells from strain ATCC43596. Vancomycin was dosed at 5 mg/kg twice a day for 5 days (p.o.) on days 1, 2, 3, 4, 5.

[0527] Viability checks were performed on animals twice a day, animals found to be in extremis were euthanised and counted as dead. Body weights were determined on each day of dosing, then twice weekly and before euthanising survivors. Gross necropsy was performed on all animals. Survival curves were created by the method of Kaplan and Meier. Survival curves were analysed using the P value from the log rank test compared to the Bonferroni corrected threshold of P=0.005. The Vancomycin treated group were not included in the analysis. All statistical tests were done with Prism v5.04. All groups contained 11 animals, except the Vancomycin control group which contained 5 animals.

[0528] Survival curves can be seen in FIG. 63. Hamsters receiving PBS (control) all died on days +2 and +3, whilst those receiving vancomycin treatment for 5 days all died on days +10 and +11. Hamsters receiving the high dose of UCB Mab mixture all survived until day +11, thereafter only two animals died until the end of the 28 day study. Hamsters receiving the low dose of UCB Mab mixture all survived until day +3, thereafter animals were lost fairly steadily until day +16 when all had died. The data show exceptional levels and duration of protection when compared to published data for use of anti-toxin Mabs in hamsters (18). These in vivo data support the in vitro observations of very high level performance for neutralization and stability.

[0529] There is no apparent link between death and body weight during the acute phase (days 1-5) of the infection, FIGS. 64-65. Hence it may be supposed that hamsters die of overwhelming direct and indirect effects of TcdA and TcdB. Hamsters which survive the acute period due to partial protection (UCB low dose) of neutralizing Mabs lose weight, presumably due to gut damage and altered nutritional status. It was notable that many of the hamsters which went on to survive the 28 period of the study due to the protective effects of the UCB high dose Mabs recovered from weight loss and indeed even gained weight. This may be taken as evidence of the superior protective effects of the UCB Mabs enabling the gut to function as normal.

TABLE-US-00067 TABLE 15 Gross pathology scores Anogenital Black Dark red Red Pink Normal staining Red small Group caecum caecum caecum caecum caecum `wet-tail` intestine PBS 1 9 1 0 0 1 1 control UCB low 0 4 5 2 0 4 1 UCB high 0 0 1 1 9 3 0

[0530] It is clear that UCB Mabs were able to protect the large and small intestines from the bloody effusions caused by TcdA and TcdB.

[0531] The results are shown in FIGS. 63 to 66

[0532] The photographs in FIG. 66 show typical gross pathologies for the swelling and bloody effusions of caeca caused by TcdA and TcdB (left image, PBS control, animal death on day 2) and a normal stool filled caeca after protection by UCB high dose Mabs (right image, UCB high dose, animal surviving to day 28). These data show that after protection with a high dose of UCB Mabs the large intestine can return to normal morphology and function.

Example 10 Neutralisation of TcdA from Different Ribotyped Strains by Purified Mab

[0533] Clinical infections are caused by a variety of different strains. Strain differences are characterized using a number of different methods of which ribotyping is a key one. Different ribotype strains are observed to have different pathogenicity, infection and sporulation properties. All of the TcdA neutralization shown above used TcdA purified from strain known as VPI10463. However, the predominant aggressively pathogenic strain associated with out-breaks is called ribotype 027. Other key ribotypes include 078, 001, 106. Amino acid sequence difference have been observed between toxins produced by different ribotypes and hence it is important that Mabs are capable of neutralizing toxin from a diverse set of clinical isolates. CA922, 997 and 1000 were tested for their ability to neutralize TcdA from strains 027 and 078 and compared to their abilities against TcdA from VPI10463. Mabs were tested at 4 [TcdA] and found to be capable of neutralizing all toxins without significant difference at LD.sub.80, LD.sub.90 and LD.sub.95

TABLE-US-00068 TABLE 16 EC50 values (ng/ml) - TcdA strain VPI 10463 Antibody LD80 LD90 LD95 LDmax CA164_922 0.27 0.9 1.2 >500 CA164_997 1 2.5 3.5 25.4 CA164_1000 3.6 13.5 19.3 >500

TABLE-US-00069 TABLE 17 EC50 values (ng/ml) - TcdA ribotype 027 Antibody LD80 LD90 LD95 LDmax CA164_922 0.19 0.25 0.41 1.46 CA164_997 0.92 1.27 1.75 7.19 CA164_1000 2.25 2.49 3.52 16.32

TABLE-US-00070 TABLE 18 EC50 values (ng/ml) - TcdA ribotype 078 Antibody LD80 LD90 LD95 LDmax CA164_922 0.11 0.12 0.25 0.68 CA164_997 0.33 0.64 1.11 2.57 CA164_1000 2.04 2.41 5.03 14.16

Example 11 PK Data

[0534] A PK study of a human IgG1 (20 mg/kg) in healthy hamsters. The hamster PK was found a half-life similar to Mabs in mice or rats. (t1/2 6-8 days). i.p. and s.c. dosing were essentially the same. The pharmacokinetics and distribution to the gut of a hIgG1 Mab was studied in `normal` (non-infected) golden Syrian hamsters. Purified Mab was administered to male hamsters (120-135 g) by CARE Research LLC, Fort Collins, Colo., USA and samples were assayed by UCB Pharma. The study was approved by the CARE IACUC committee. Eight animals each received a single dose of 20 mg/kg of IgG1, four were dosed i.p., four were dosed s.c. Blood was collected at 1, 3, 8, 24, 48, 72, 103 and 168 hours post-dose, serum was separated before storage at -80.degree. C. Blood was also taken from two untreated hamsters in order to provide assay controls. Following euthanasia, a 2 cm length of colon was cut from the caeca junction onwards from each hamster. The colon section was flushed with wash buffer (50% (v/v) PBS containing 50% (v/v) Sigma protease inhibitor cocktail (P2714) before being opened and separation and removal of the mucosa from the underlying muscle. Mucosal samples were placed in 0.5 ml of wash buffer homogenized until visually uniform and stored at 4.degree. C. before immediate shipping on wet ice. For the anti-human IgG1 ELISA Nunc maxisorp 96 well plates were coated overnight in 0.1M NaHCO.sub.3 pH 8.3 with Goat F(ab')2 anti-human IgG-Fc.gamma. fragment (Jackson 109-006-098), plates were washed with PBS-Tween (PBS/0.1% (v/v) Tween 20) and then blocked with 1.0% (w/v) BSA & 0.1% (v/v) Tween in PBS. Serum samples were diluted in sample-conjugate buffer (1% (w/v) BSA, 0.2% Tween in PBS) and after washing were revealed with goat anti-human kappa-HRP (Cambridge Bioscience 2060-05) in sample-conjugate buffer and TMB with a 2.5M H2504 stop solution.

Gut, Mucosa and Serum Levels:

[0535] Serum samples collected from blood taken at 168 hour time point and colon samples were removed after this.

TABLE-US-00071 20mg/kg IP at 168 hour Sample ng/mL per cm mucosa serum .mu.g/mL 1001 23.2 75.0 1002 13.7 90.8 1003 21.8 70.5 1004 53.8 119.4

TABLE-US-00072 20mg/kg SC at 168 hour Sample ng/mL per cm mucosa serum .mu.g/mL 2001 41.4 108.7 2002 62.1 76.6 2003 35.6 163.7 2004 37.3 153.3

TABLE-US-00073 Serum Data Hamster i.p. Hamster s.c. SE of SE of Mean mean Mean mean C.sub.max: .mu.g/mL 202 12 186 21 T.sub.max: hr 36 7 76 16 AUC .sub.(last): hr .mu.g/mL 22626 1378 22371 2258 AUC .sub.(inf): hr .mu.g/mL 43287 7169 61290 17637 % Extrapolation: 43.7 9.2 54 11.7 CL/F mL/hr/kg 0.50 0.07 0.43 0.13 MRT.sub.inf h 223 53 310 88 t.sub.1/2,z: h 149.2 36.9 188.5 61.9

[0536] The data is also shown in FIGS. 70 and 71

TABLE-US-00074 Hamster ID Mean SE IP serum kinetics C.sub.max: .mu.g/mL 202 12 T.sub.max: hr 36 7 AUC.sub.(last): hr .mu.g/mL 22626 1378 AUC.sub.(inf): hr .mu.g/mL 43287 7169 % Extrapolation: 43.7 9.2 CL/F mL/hr/kg 0.50 0.07 MRT.sub.inf h 223 53 t.sub.1/2,z: h 149.2 36.9 SC serum kinetics Hamster ID Mean SE C.sub.max: .mu.g/mL 186 21 T.sub.max: hr 76 16 AUC.sub.(last): hr .mu.g/mL 22371 2258 AUC.sub.(inf): hr .mu.g/mL 61290 17637 % Extrapolation: 54 11.7 CL/F mL/hr/kg 0.43 0.13 MRT.sub.inf h 310 88 t.sub.1/2: h 188.5 61.9

[0537] It was also shown that hIgG1 could be found in `scrapings` of the gut i.e that hIgG1 gets into the vasculature of healthy gut--and so could be protective in `prophylactic dosing`. This effect would be even more profound in humans since they have a cognate hFcRn.

Example 12 Serum Levels in Hamsters with C. difficile Infection

[0538] This study was to determine the serum concentration of CA725.0, CA726.0, CA997.g1 CA1125.g2, and CA01151.g4 following i.p. administration (various doses detailed below) in the Golden Syrian Hamster.

[0539] Humanised Mabs were quantified using liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis following tryptic digestion. Quantitation was achieved by comparison to authentic standard material spiked at known concentrations into blank matrix, with spiked horse myoglobin used as the internal standard.

[0540] A unique ("proteotypic") peptide common to all of the humanised Mabs investigated was selected (DTLMISR, a CH2 region peptide) and both samples and calibration samples were tryptically digested as outlined. Tryptic digest of 5 .mu.l serum samples was performed overnight using sequencing grade modified Trypsin (Promega, Southampton, UK) following denaturation/reduction with acetonitrile/Tris (2-carboxyethyl) phosphine and carbamido-methylation with iodoacetamide (Sigma-Aldrich, Poole, UK).

[0541] The LC-MS/MS system consisted of a CTC HTS-x Autosampler (CTC Analytics, Zwingen, Switzerland), a Agilent 1290 LC system (Agilent Technologies, Stockport, UK) and a Sciex 5500 QTrap MS system (AB Sciex, Warrington, UK), equipped with a Turbo V ion source operated in electrospray mode. Analytes were separated using an Onyx Monolithic C18 column (100.times.4.6 mm, Phenomenex, Macclesfield, UK) with a gradient of 2 to 95% (v/v) water/acetonitrile (0.1% formic acid) delivered at 1.5 mL/min over 6 minutes. The injection volume was 10 .mu.L; all of the eluent was introduced into the mass spectrometer source. The source temperature of the mass spectrometer was maintained at 600.degree. C. and other source parameters (e.g. collision energy, declustering potential, curtain gas pressure etc.) were optimized to achieve maximum sensitivity for the peptide of interest. Selective transitions for each proteotypic peptide of interest were monitored. Unique ("proteotypic) peptides were selected for all of the analytes of interest; samples were analysed following tryptic digestion.

[0542] Plasma concentrations calculated based on the peptides monitored are outlined below.

[0543] For CA164_00997 and CA164_01151, interfering peaks were observed in the MRM traces. For this reason, these two analytes could not be quantified in the samples.

[0544] Total h-IgG was quantified in all samples using a peptide common to all analytes of interest. This was done using a combined standard curve of all five analytes. The validity of this approach is demonstrated by the fact that the sum of the concentrations observed for CA164_00725 and CA164_00726 are in good agreement (within experimental error) of the concentration observed for total h-IgG.

[0545] Using this approach, the total concentration of h-IgG in the samples of animals dosed with CA164_00997, CA164_01125 and CA164_01151 was determined.

[0546] Overall the data obtained indicate that the exposure of all five analytes of interest was similar for a given dose.

TABLE-US-00075 Study groups Blinded labels Treatment components Grp Treatment Actual Treatments Dose days Anti-toxin A Anti-toxin B 4 Treatment3 Vehicle PBS 5 mL/kg i.p. 3, -2, -1, 0 2 Vancomycin Vancomycin 5 1, 2, 3, 4, 5 mg/kg b.i.d. p.o. 1 Treatment1 UCB LD* 3, -2, -1, 0 CA997.g1_P3 CA1125.g2_P3 CA1151.g4_P3 5 mg/kg A 5 mg/kg i.p. 5 mg/kg 2.5 mg/kg 2.5 mg/kg 5 Treatment4 UCB HD* 3, -2, -1, 0 CA997.g1_P3 CA1125.g2_P3 CA1151.g4_P3 50 mg/kg A 50 mg/kg i.p. 50 mg/kg 25 mg/kg 25 mg/kg 6 Treatment5 CompetitorLD* 3, -2, -1, 0 CA726_P3 CA725_P3 5 mg/kg A 5 mg/kg i.p. 5 mg/kg 5 mg/kg 3 Treatment2 CompetitorHD* 3, -2, -1, 0 CA726_P3 CA725_P3 50 mg/kg A 50 mg/kg i.p. 50 mg/kg 50 mg/kg

TABLE-US-00076 TABLE 19 Group/ Animal Serum conc time Day No Dose .mu.g/mLtotal h-IgG 1 1 44 5 mg/kg 997, 280 1 45 2.5 mg/kg 302 1 46 1125,2.5 182 6 45 mg/kg 1151 61 6 47 71 6 49 45 3 1 60 50 mg/kg 3040 1 61 725, 3330 1 62 50 mg/kg 2990 6 62 726 583 6 63 913 6 64 1240 28 64 199 28 65 36 4 1 71 Vehicle nd 1 72 nd 1 73 nd 5 1 82 50 mg/kg 3050 1 83 997, 2790 1 84 25 mg/kg 2370 6 82 1125,25 838 6 83 mg/kg 1151 645 6 84 855 28 82 116 28 83 65 28 84 66 28 85 44 28 86 101 28 87 89 28 88 27 28 89 31 28 90 66 6 1 93 5 mg/kg 725, 335 1 94 5 mg/kg 726 322 1 95 260 6 200 103 6 202 62 6 203 79 28 203 nd nd - not detected (LOQ = 2.5 .mu.g/mL for all analytes na - not analysed: interference in the sample was observed for 997 and 1151

TABLE-US-00077 TABLE 20 Antibody CA725 is prior art antibody MDX1388. Antibody CA726 is prior art antibody CDA1 as described (15) A summary of this data is presented in FIG. 72. Small intestine Caecal pathology pathology Dark Dark Group Black Red Red Pink Normal Red Red PBS control 1 9 1 0 0 0 1 MDX high 0 1 4 4 2 1 0 50 mg/Kg x4 UCB high 0 0 1 1 9 0 0 50 mg/Kg x4

REFERENCES

[0547] 1. Kuehne, S et al., "The role of toxin A and toxin B in Clostridium difficile Infection" Nature (2010) 467: 711-713.

[0548] 2. Davies A H et al., "Super toxins from a super bug: structure and function of Clostridium difficile toxins" Biochem. J (2011) 436: 517-526.

[0549] 3. Rothman, S et al., "Differential Cytotoxic Effects of Toxins A and B Isolated from Clostridium difficile" Infect. Imm. (1984) 46: 324-331.

[0550] 4. Du, T and Alfa, M J "Translocation of Clostridium difficile toxin B across polarized Caco-2 cell monolayers is enhanced by toxin A" Can J Infect Dis. (2004) 15: 83-88.

[0551] 5. Kim, Iaconis and Rolfe. "Immunization of Adult Hamsters against Clostridium difficile-Associated Ileocecitis and Transfer of Protection to Infant Hamsters" Infect. Imm. (1987) 55:2984-2992

[0552] 6. Rupnik J C M (2003) 41:1118-1125

[0553] 7. Chaves-Olarte J B C (1999) 274:11046-11052.

[0554] 8. Lylerly, D M et al., "Passive Immunization of Hamsters against Disease Caused by Clostridium difficile by Use of Bovine Immunoglobulin G Concentrate" Infection and Immunity (1991) 59:2215-2218.

[0555] 9. Lylerly, D M et al., "Vaccination against Lethal Clostridium difficile Enterocolitis with a Nontoxic Recombinant Peptide of Toxin A" Current Microbiology (1990) 21:29-32.

[0556] 10. Lylerly, D M et al., "Characterization of Toxins A and B of Clostridium difficile with Monoclonal Antibodies" Infect. Imm. (1986) 54:70-76.

[0557] 11. Corthier et al., "Protection against Experimental Pseudomembranous Colitis in Gnotobiotic Mice by Use of Monoclonal Antibodies against Clostridium difficile Toxin A" Infect. Imm. (1991) 59: 1192-1195.

[0558] 12. Kink J A and Williams J A, "Antibodies to Recombinant Clostridium difficile Toxins A and B Are an Effective Treatment and Prevent Relapse of C. difficile-Associated Disease in a Hamster Model of Infection" Infect. Imm. (1998) 66:2018-2025.

[0559] 13. Ma D, et al., Progenics inc. ASM Poster 27 May 2010

[0560] 14. Hansen, G and Demarest, S J. WO 2006/0718877 A2

[0561] 15. Babcock G J, et al., "Human monoclonal antibodies directed against toxins A and B prevent Clostridium difficile-induced mortality in hamster" Infect. Imm. (2006) 74:6339-6347.

[0562] 16. Lowy I et al., "Treatment with Monoclonal Antibodies against Clostridium difficile Toxins" NEJM (2010) 362: 197-205.

[0563] 17. Zubler, R. H., Erard, F., Lees, R. K., Van, L. M., Mingari, C., Moretta, L. & MacDonald, H. R. (1985). Mutant EL-4 thymoma cells polyclonally activate murine and human B cells via direct cell interaction. J. Immunol. 134, 3662-3668

[0564] 18. Babcook, J. S., Leslie, K. B., Olsen, O. A., Salmon, R. A. & Schrader, J. W. (1996). A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities. Proc. Natl. Acad. Sci. U.S.A 93, 7843-7848

[0565] 19. Tickle, S., Adams, R., Brown, D., Griffiths, M., Lightwood, D. & Lawson, A. (2010). High-Throughput Screening for High Affinity Antibodies., pp. 303-307.

[0566] 20. Demarest et al., mAbs (2010) 2:190-198

[0567] 21. Yoshida et al., J. Clin. Invest. (2006) 116: 2142-2151

[0568] 22. Xu et al., Vaccine (2005) 23:2658-2664.

[0569] 23. Yousaf et al., Clin. Exp. Immunol. (1986) 66:654-660

[0570] 24. Mannik et al., J. Exp. Med. (1971) 133: 713-739

[0571] 25. Nusrat et al., Infection and Immunity (2001) 69:1329-1336

[0572] 26. Lima et al., Infect Immun (1988) 56:582-588

[0573] 27. Ravichandran et al J of Pharmacology and Experimental Therapeutics. (2006) 318: 1343-1351

[0574] 28. Takahashi et al., (2009) Vaccine 27:2616-2619

[0575] 29. Cohen et al., Infect. Cont. and Hosp. Epidem. (2010) 31: 431-455

[0576] 30. Barbut et al., J. Clin. Microbiol. (2000) 38: 2386-2388

[0577] 31. Wilcox et al., J. Hospital Infection (1998) 38: 93-100.

Sequence CWU 1

1

194111PRTArtificialAntibody CDR 1Gln Ala Ser Gln Ser Ile Ser Asn Ala Leu Ala1 5 1027PRTArtificialAntibody CDR 2Ser Ala Ser Ser Leu Ala Ser1 5312PRTArtificialAntibody CDR 3Gln Tyr Thr His Tyr Ser His Thr Ser Lys Asn Pro1 5 10410PRTArtificialAntibody CDR 4Gly Phe Thr Ile Ser Ser Tyr Tyr Met Ser1 5 10517PRTArtificialAntibody CDR 5Ile Ile Ser Ser Gly Gly His Phe Thr Trp Tyr Ala Asn Trp Ala Lys1 5 10 15Gly613PRTArtificialAntibody CDR 6Ala Tyr Val Ser Gly Ser Ser Phe Asn Gly Tyr Ala Leu1 5 107110PRTArtificialAntibody variable region 7Asp Pro Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Ser Asn Ala 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Tyr Thr His Tyr Ser His Thr 85 90 95Ser Lys Asn Pro Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 1108330DNAArtificialPolynucleotide encoding variable region of anti-TcdA antibody 922.g1 8gaccctgtga tgacccagag tccgagcact ctttctgcct ccgtgggaga ccgcgtgacc 60attacatgtc aggcttcaca aagtatctcc aatgctctgg cctggtatca gcagaaaccc 120ggcaaagccc ctaagctgct catctactct gcatcaagcc tggctagcgg cgtgccaagc 180cgattcaagg ggagcggttc tggcactgag tttacgctga ccatcagtag cttgcagcct 240gacgattttg caacctatta ctgccagtac acacactact cccatacatc taaaaaccca 300ttcggagggg gtactaaggt cgaaataaag 3309119PRTArtificialAntibody variable region for anti-TcdA antibody 922 9Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Ser Tyr 20 25 30Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Ile Ile Ser Ser Gly Gly His Phe Thr Trp Tyr Ala Asn Trp Ala 50 55 60Lys Gly Arg Phe Thr Ile Ser Ser Asp Ser Thr Thr Val Tyr Leu Gln65 70 75 80Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg 85 90 95Ala Tyr Val Ser Gly Ser Ser Phe Asn Gly Tyr Ala Leu Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser 11510357DNAArtificialPolynucleotide encoding variable region of anti-TcdA antibody 922 (heavy chain variable region) 10gaagtgcaat tggtggaaag tggcggagga ctggtgcaac ccgggggtag tctgcgactg 60agctgtgctg cctccggctt taccattagc tcctactata tgagctgggt tcgacaggcc 120cctggaaaag gactcgaatg gatcggcatc atatcttccg gtgggcattt cacctggtac 180gcaaactggg ctaaggggag attcacgatt agcagcgact ccacaaccgt gtacctgcaa 240atgaacagcc tgagggatga ggacactgcc acatatttct gcgcacgcgc ttacgtgagc 300ggaagctcat ttaatggcta tgcactgtgg gggcaaggaa cactcgtgac tgtctcg 3571111PRTArtificialAntibody CDR 11Gln Ala Ser Gln Ser Ile Ser Asn Tyr Leu Ala1 5 10127PRTArtificialAntibody CDR 12Ser Ala Ser Thr Leu Ala Ser1 51312PRTArtificialAntibody CDR 13Gln Tyr Ser His Tyr Gly Thr Gly Val Phe Gly Ala1 5 101410PRTArtificialAntibody CDR 14Ala Phe Ser Leu Ser Asn Tyr Tyr Met Ser1 5 101518PRTArtificialAntibody CDR 15Ile Ile Ser Ser Gly Ser Asn Ala Leu Lys Trp Tyr Ala Ser Trp Pro1 5 10 15Lys Gly1613PRTArtificialAntibody CDR 16Asn Tyr Val Gly Ser Gly Ser Tyr Tyr Gly Met Asp Leu1 5 1017110PRTArtificialAnitbody ariable region of anti-TcdA antibody 923 17Asp Val Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Ser Asn Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly 50 55 60Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Val Ala Thr Tyr Tyr Cys Gln Tyr Ser His Tyr Gly Thr Gly 85 90 95Val Phe Gly Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 11018330DNAArtificialPolynucleotide encoding antibody variable region for anti-TcdA antibody 923.g1 18gacgtcgtga tgactcagag cccatctagt ctgagcgcta gcgtcggaga ccgagtcaca 60attacctgtc aagcctccca gagcatctcc aactacctgg cctggtacca acagaaacct 120ggcaaggtgc ccaagctgct gatctatagt gcttccacac tcgcaagcgg cgttccgtca 180cgctttaagg gatctggctc tggcactcag ttcaccttga cgatctcaag cctgcagcca 240gaagatgtgg ccacctatta ctgccagtat tcccactacg ggactggggt gttcggtgcc 300tttggaggtg ggaccaaagt ggagataaag 33019120PRTArtificialAntibody variable region for anti-TcdA antibody 923 19Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Ala Phe Ser Leu Ser Asn Tyr 20 25 30Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Ile Ile Ser Ser Gly Ser Asn Ala Leu Lys Trp Tyr Ala Ser Trp 50 55 60Pro Lys Gly Arg Phe Thr Ile Ser Lys Asp Ser Thr Thr Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Phe Cys Ala 85 90 95Arg Asn Tyr Val Gly Ser Gly Ser Tyr Tyr Gly Met Asp Leu Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser 115 12020360DNAArtificialPolynucleotide encoding antibody variable region for anti-TcdA antibody 923.g1 20gaagttcaac ttgtggaatc tggaggcggg ctcgtgcagc ctggtggaag ccttagactg 60agctgcgctg catccgcatt ttccctgtcc aactactaca tgagctgggt gcgacaagca 120ccaggcaagg gactggaatg gattggcatc ataagctccg gttccaatgc cctgaaatgg 180tacgcatcat ggccgaaagg ccgctttacc ataagcaagg actccaccac cgtctatctg 240cagatgaact cattgcgtgc cgaggacact gcaacgtact tctgtgctcg caactacgtg 300ggaagcggat cttattatgg catggatctg tggggacaag gtacactcgt gaccgtctcg 3602111PRTArtificialAntibody CDR 21Gln Ala Ser Gln Ser Ile Ser Ser Tyr Phe Ser1 5 10227PRTArtificialAntibody CDR 22Gly Ala Ser Thr Leu Ala Ser1 52312PRTArtificialAntibody CDR 23Gln Cys Thr Asp Tyr Ser Gly Ile Tyr Phe Gly Gly1 5 102410PRTArtificialAntibody CDR 24Gly Phe Ser Leu Ser Ser Tyr Tyr Met Ser1 5 102519PRTArtificialAntibody CDR 25Ile Ile Ser Ser Gly Ser Ser Thr Thr Phe Thr Trp Tyr Ala Ser Trp1 5 10 15Ala Lys Gly2613PRTArtificialAntibody CDR 26Ala Tyr Val Gly Ser Ser Ser Tyr Tyr Gly Phe Asp Pro1 5 1027110PRTArtificialAntibody variable region for anti-TcdA antibody 993 27Asp Val Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Phe Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Gln Leu Leu Ile 35 40 45Tyr Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly 50 55 60Ser Gly Ser Gly Thr Glu Leu Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Cys Thr Asp Tyr Ser Gly Ile 85 90 95Tyr Phe Gly Gly Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 11028330DNAArtificialPolynucleotide encoding antibody variable region for anti-TcdA antibody 933.g1 28gatgtcgtga tgactcagtc cccctctaca ttgagtgcct ctgtcggtga tcgagttacc 60atcacctgtc aagcaagcca gagcatcagc tcctacttct cttggtacca gcaaaagccg 120ggaaaagccc ctcaactgct gatttatggg gcctcaacac tggcttctgg cgtgccatca 180agattcaagg gatctggctc cggcactgag cttacactga ccattagctc cctgcaacct 240gacgattttg ctacctacta ctgccagtgc accgactata gtgggatata tttcggcgga 300tttgggggag ggacgaaagt ggaaatcaag 33029121PRTArtificialAntibody variable region for anti-TcdA antibody 993 (heavy chain) 29Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Thr Ala Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Ile Ile Ser Ser Gly Ser Ser Thr Thr Phe Thr Trp Tyr Ala Ser 50 55 60Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95Ala Arg Ala Tyr Val Gly Ser Ser Ser Tyr Tyr Gly Phe Asp Pro Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser 115 12030363DNAArtificialPolynucleotide encoding antibody variable region for anti-TcdA antibody 993.g1 30gaagttcagc tggtcgagag cggaggcgga ctggtgcaac ctggtggtag cctgaaactc 60tcttgtactg cctccgggtt ttccctgagc tcttactata tgtcatgggt gagacaggct 120cccgggaaag gattggaatg gatcgggatt atctcctccg gctcttccac cactttcaca 180tggtacgcct catgggcaaa ggggaggttt accataagca agacaagcac gaccgtgtat 240cttcagatga actccctgaa gacggaggat actgccacct acttttgcgc tcgggcctat 300gtgggctcaa gctcttacta tggcttcgac ccatggggac agggcacact tgtgaccgtc 360tcg 3633111PRTArtificialAntibody CDR 31Gln Ala Ser Gln Ser Ile Asn Asn Tyr Phe Ser1 5 10327PRTArtificialAntibody CDR 32Gly Ala Ala Asn Leu Ala Ser1 53312PRTArtificialAntibody CDR 33Gln Asn Asn Tyr Gly Val His Ile Tyr Gly Ala Ala1 5 103410PRTArtificialAntibody CDR 34Gly Phe Ser Leu Ser Asn Tyr Asp Met Ile1 5 103516PRTArtificialAntibody CDR 35Phe Ile Asn Thr Gly Gly Ile Thr Tyr Tyr Ala Ser Trp Ala Lys Gly1 5 10 153612PRTArtificialAntibody CDR 36Val Asp Asp Tyr Ile Gly Ala Trp Gly Ala Gly Leu1 5 1037110PRTArtificialAntibody variable region for anti-TcdA antibody 995 37Asp Val Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Asn Asn Tyr 20 25 30Phe Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Gly Ala Ala Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly 50 55 60Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Asp Asp Phe Ala Thr Tyr Ser Cys Gln Asn Asn Tyr Gly Val His Ile 85 90 95Tyr Gly Ala Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 11038330DNAArtificialPoly nucleotide encoding antibody variable region for anti-TcdA antibody 995. g1 38gacgtcgtga tgacacagag cccttcaaca ctgtctgcaa gcgtgggcga tagggtcacc 60ataacgtgcc aggcctctca atccatcaac aactatttta gctggtacca gcagaagcca 120ggcaaggctc cgaaacttct gatctacgga gctgccaacc tggcaagtgg cgtgccatca 180cggttcaagg gatccgggag cggtactgag tataccctga ccatttcatc tctccaaccc 240gacgatttcg ccacctactc ctgccagaat aattacggcg tgcacatcta tggagctgcc 300tttggcggtg ggacaaaagt ggaaattaag 33039117PRTArtificialAntibody variable region for anti-TcdA antibody 995 (heavy chain) 39Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Ser Leu Ser Asn Tyr 20 25 30Asp Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile 35 40 45Gly Phe Ile Asn Thr Gly Gly Ile Thr Tyr Tyr Ala Ser Trp Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Ser Ser Thr Val Tyr Leu Gln Met65 70 75 80Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Val 85 90 95Asp Asp Tyr Ile Gly Ala Trp Gly Ala Gly Leu Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser 11540351DNAArtificialPolynucleotide encoding antibody variable region for anti-TcdA antibody 40gaagttcagc tggtcgagag tgggggaggg cttgtgcaac ctggtggctc cctccgtctg 60agctgtactg cttctggatt ctcactgagc aattacgaca tgatctgggt gcgacaggca 120cccggcaaag gactggagta cattggcttc atcaacaccg ggggtataac gtactatgcc 180tcatgggcta aggggcgctt tacaattagt agggattcct ctaccgtgta cctgcagatg 240aactcactga gagccgagga cactgccaca tatttctgcg ctcgggtgga tgactatatc 300ggggcctggg gcgccggatt gtggggccaa ggaacactgg tcaccgtctc g 3514111PRTArtificialAntibody CDR 41Gln Ala Ser Gln Ser Ile Ser Ser Tyr Leu Ser1 5 10427PRTArtificialAntibody CDR 42Arg Ala Ser Thr Leu Ala Ser1 54313PRTArtificialAntibody CDR 43Leu Gly Val Tyr Gly Tyr Ser Asn Asp Asp Gly Ile Ala1 5 104410PRTArtificialAntibody CDR 44Gly Ile Asp Leu Ser Ser His His Met Cys1 5 104516PRTArtificialAntibody CDR 45Val Ile Tyr His Phe Gly Ser Thr Tyr Tyr Ala Asn Trp Ala Thr Gly1 5 10 154611PRTArtificialAntibody CDR 46Ala Ser Ile Ala Gly Tyr Ser Ala Phe Asp Pro1 5 1047111PRTArtificialAntibody variable region for anti-TcdA antibody 997 47Ala Leu Val Met Thr Gln Ser Pro Ser Ser Phe Ser Ala Ser Thr Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Cys Leu Gln Ser65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Val Tyr Gly Tyr Ser Asn 85 90 95Asp Asp Gly Ile Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 11048333DNAArtificialPolynucleotide sequence encoding antibody variable region for anti-TcdA antibody 997.g1 48gcactcgtga tgacacagag cccgagtagc tttagtgctt caaccggtga tagggtcact 60attacttgcc aagcctctca gagtatatct agctatctga gctggtacca gcaaaagccc 120gggaaggctc ctaaactgct gatctaccgg gcttccacat tggcctccgg cgttccctca 180cgctttagcg gctccggatc cggaaccgag tacaccctga ctatctcttg cctgcaatct 240gaggacttcg caacctacta ttgtctgggc gtctacggat atagcaacga tgacgggatc 300gccttcggcg gcggtaccaa agtggaaatt aag 33349116PRTArtificialAntibody variable region for anti-TcdA antibody 997 (heavy chain) 49Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Ile Asp Leu Ser Ser His 20 25 30His Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile 35 40 45Gly Val Ile Tyr His Phe Gly Ser Thr Tyr Tyr Ala Asn Trp Ala Thr 50 55 60Gly Arg Phe Thr Ile Ser Lys Asp Ser Thr Thr Val Tyr Leu Gln Met65 70 75 80Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Ala 85 90 95Ser Ile Ala Gly Tyr Ser Ala Phe Asp Pro Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser 11550348DNAArtificialPolynucleotide sequence encoding antibody variable region for anti-TcdA anitbody 997.g1 (heavy chain) 50gaggtgcaac ttgtggaaag cgggggagga ctggtgcagc ctgggggctc attgagactg 60agctgcaccg tttctggtat tgacctgagc tcccatcata tgtgctgggt gcgccaggca 120cccggaaaag

gactggaata catcggcgtc atataccact ttggctctac atactatgcc 180aactgggcaa ctgggcgatt cacaattagc aaggactcaa ctaccgttta cctgcaaatg 240aatagcctga gggctgagga tactgccacc tatttctgtg cccgggcttc aatcgccggc 300tattctgcct ttgatccatg ggggcaagga acactcgtga ccgtctcg 3485111PRTArtificialAntibody CDR 51Gln Ala Ser Gln Ser Ile Tyr Ser Tyr Leu Ala1 5 10527PRTArtificialAntibody CDR 52Asp Ala Ser Thr Leu Ala Ser1 55313PRTArtificialAntibody CDR 53Gln Gly Asn Ala Tyr Thr Ser Asn Ser His Asp Asn Ala1 5 105410PRTArtificialAntibody CDR 54Gly Ile Asp Leu Ser Ser Asp Ala Val Gly1 5 105516PRTArtificialAntibody CDR 55Ile Ile Ala Thr Phe Asp Ser Thr Tyr Tyr Ala Ser Trp Ala Lys Gly1 5 10 155619PRTArtificialAntibody CDR 56Thr Gly Ser Trp Tyr Tyr Ile Ser Gly Trp Gly Ser Tyr Tyr Tyr Gly1 5 10 15Met Asp Leu57111PRTArtificialAntibody variable region for anti-TcdA antibody 1000 57Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Tyr Ser Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asp Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gly Asn Ala Tyr Thr Ser Asn 85 90 95Ser His Asp Asn Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 11058333DNAArtificialPolynucleotide encoding antibody variable region for anti-TcdA antibody 1000.g1 58gaaatcgtga tgacgcagtc accaagcaca ctgagcgctt ctgtgggaga tcgggtcaca 60ataacctgtc aggcctccca gagcatctac tcttatctgg catggtacca gcagaagcca 120gggaaagctc ccaagctgct gatttatgac gccagcactt tggcttccgg tgttcctagt 180aggttcaaag gctccggaag cggtaccgag tttaccctga ccatctcatc tctgcaaccc 240gatgactttg ccacatacta ttgccagggg aatgcctaca cttccaactc acacgacaac 300gcattcgggg gaggcaccaa agtcgaaatt aag 33359125PRTArtificialAntibody variable region for anti-TcdA antibody 1000 (heavy chain) 59Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Ile Asp Leu Ser Ser Asp 20 25 30Ala Val Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile 35 40 45Gly Ile Ile Ala Thr Phe Asp Ser Thr Tyr Tyr Ala Ser Trp Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Lys Ala Ser Ser Thr Thr Val Tyr Leu Gln65 70 75 80Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg 85 90 95Thr Gly Ser Trp Tyr Tyr Ile Ser Gly Trp Gly Ser Tyr Tyr Tyr Gly 100 105 110Met Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 12560375DNAArtificialPolynucleotide encoding antibody variable region for anti-TcdA antibody 1000.g1 (heavy chain) 60gaagttcagc tggtcgagag cggagggggt ttgattcagc ccggtggctc acttagattg 60agctgcaccg tgtccggaat cgatctgtca tctgatgccg tgggctgggt gcgacaggca 120cctgggaaag gactggagta tatagggatc atcgccacct tcgactccac atactacgct 180agctgggcaa aagggcgctt tacgattagc aaggcctcct ctactaccgt gtacctccaa 240atgaactcac tgagggccga ggacactgcc acttatttct gtgctcggac cggtagctgg 300tactacatct ctggctgggg ctcctactat tatggcatgg acctgtgggg acaggggaca 360ctcgtgaccg tctcg 3756111PRTArtificialAntibody CDR 61Arg Ala Ser Lys Ser Val Ser Thr Leu Met His1 5 10627PRTArtificialAntibody CDR 62Leu Ala Ser Asn Leu Glu Ser1 5639PRTArtificialAntibody CDR 63Gln Gln Thr Trp Asn Asp Pro Trp Thr1 56410PRTArtificialAntibody CDR 64Gly Phe Thr Phe Ser Asn Tyr Gly Met Ala1 5 106517PRTArtificialAntibody CDR 65Ser Ile Ser Ser Ser Gly Gly Ser Thr Tyr Tyr Arg Asp Ser Val Lys1 5 10 15Gly669PRTArtificialAntibody CDR 66Val Ile Arg Gly Tyr Val Met Asp Ala1 567107PRTArtificialAntibody variable region for anti-TcdB antibody 926 67Asp Thr Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Leu 20 25 30Met His Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu Ile 35 40 45Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Trp Asn Asp Pro Trp 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 10568321DNAArtificialPolynucleotide encoding antibody variable region for anti-TcdB antibody 926.g1 68gataccgtgc tgacccagag ccctgctaca ttgtcactga gccccgggga gagggccaca 60ttgagctgcc gggcttcaaa atccgtgtcc accctcatgc actggtttca gcaaaagccc 120gggcaggccc caaaactgct gatctacctc gcatctaacc ttgaatctgg cgtgccggcc 180cgctttagtg gctccggaag cggaaccgac ttcacactga cgattagctc cctggagcct 240gaggatttcg ccgtgtacta ttgccagcaa acttggaatg acccttggac tttcgggggc 300ggtactaagg tcgaaataaa g 32169117PRTArtificialAntibody variable region for anti-TcdB antibody 926 (heavy chain) 69Glu Val Glu Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30Gly Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val 35 40 45Thr Ser Ile Ser Ser Ser Gly Gly Ser Thr Tyr Tyr Arg Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95Thr Thr Val Ile Arg Gly Tyr Val Met Asp Ala Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser 11570351DNAArtificialPolynucleotide encoding antibody variable region for anti-TcdBantibody 926.g1 (heavy chain) 70gaggtggaac tgctcgaatc tggtggtggg ctggtgcagc ccggtggatc tctgagattg 60tcatgcgagg catccggctt taccttttcc aactacggaa tggcctgggt gagacaggcc 120ccaacgaagg ggctcgaatg ggttacaagc atcagctctt ctgggggatc tacttactat 180cgcgatagcg tcaaaggccg gtttaccatt agccgagata atgccaaatc aagcctgtat 240ctgcaaatga acagcctgag ggctgaggac accgccacat actattgtac aaccgtgata 300aggggctacg tgatggacgc atggggacag gggacattgg ttaccgtctc g 3517111PRTArtificialAntibody CDR 71Arg Ala Ser Gly Ser Val Ser Thr Leu Met His1 5 10727PRTArtificialAntibody CDR 72Lys Ala Ser Asn Leu Ala Ser1 5738PRTArtificialAntibody CDR 73His Gln Ser Trp Asn Ser Asp Thr1 57410PRTArtificialAntibody CDR 74Gly Phe Thr Phe Ser Asn Tyr Gly Met Ala1 5 107517PRTArtificialAntibody CDR 75Thr Ile Asn Tyr Asp Gly Arg Thr Thr His Tyr Arg Asp Ser Val Lys1 5 10 15Gly769PRTArtificialAntibody CDR 76Ile Ser Arg Ser His Tyr Phe Asp Cys1 577106PRTArtificialAntibody variable region for anti-TcdB antibody 927 77Asp Thr Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gly Ser Val Ser Thr Leu 20 25 30Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Lys Ala Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Asp Asp Phe Ala Thr Tyr Tyr Cys His Gln Ser Trp Asn Ser Asp Thr 85 90 95Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 10578318DNAArtificialPolynucleotide sequence encoding antbody variable region for anti-TcdB antibody 927.g2 78gacacacaga tgacccagag cccatccact ttgtctgcat ccgtgggcga ccgagtgaca 60atcacctgta gagcaagcgg ttccgtgagc acactgatgc attggtacca gcagaagcct 120gggaaggctc ccaagctgct gatctacaaa gccagcaacc ttgcctccgg cgttccaagc 180cggtttagcg gttccggatc tggaaccgag ttcaccctga ccatatcaag cctgcaaccc 240gacgacttcg ccacctacta ttgccaccag agctggaata gcgacacgtt cgggcaaggc 300acaaggctgg aaatcaaa 31879117PRTArtificialAntibody variable region for anti-TcdB antibody 927 (heavy chain) 79Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30Gly Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Thr Ile Asn Tyr Asp Gly Arg Thr Thr His Tyr Arg Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ser Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Ser Ile Ser Arg Ser His Tyr Phe Asp Cys Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser 11580351DNAArtificialPolynucleotide encoding antibody variable region for anti-TcdB antibody 927.g2 (heavy chain) 80gaggtgcaac ttgtggaaag cggagggggc gtggtccaac ccggaagaag tctccgtctt 60tcttgcgccg caagtggctt caccttttcc aactacggaa tggcctgggt tcgacaagct 120cctgggaaag gattggagtg ggtggccact atcaactatg acggacgcac gacacactac 180cgagactctg ttaaggggcg ctttacgatt tcccgcgaca atagcaagag caccctctac 240ctgcaaatga atagcctccg ggccgaggat actgctgtgt actattgtac ctccatctca 300cggagccact acttcgattg ctggggacaa ggcacactcg tgactgtctc g 3518111PRTArtificialAntibody CDR 81Lys Ala Ser Lys Ser Ile Ser Asn His Leu Ala1 5 10827PRTArtificialAntibody CDR 82Ser Gly Ser Thr Leu Gln Ser1 5839PRTArtificialAntibody CDR 83Gln Gln Tyr Asp Glu Tyr Pro Tyr Thr1 58410PRTArtificialAntibody CDR 84Gly Phe Ser Leu Gln Ser Tyr Thr Ile Ser1 5 108516PRTArtificialAntibody CDR 85Ala Ile Ser Gly Gly Gly Ser Thr Tyr Tyr Asn Leu Pro Leu Lys Ser1 5 10 158611PRTArtificialAntibody CDR 86Pro Arg Trp Tyr Pro Arg Ser Tyr Phe Asp Tyr1 5 1087109PRTArtificialAntibody variable region for anti-TcdB antibody 1099 87Asp Val Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Lys Ser Ile Ser Asn His 20 25 30Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Ala Asn Lys Leu Leu Ile 35 40 45His Ser Gly Ser Thr Leu Gln Ser Gly Thr Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Glu Tyr Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr 100 10588327DNAArtificialPolynucleotide encoding antibody variable region for anti-TcdB antibody 1099.g2 88gacgtccagc tcactcaatc tccctccttt ctgtctgctt ctgtgggcga tcgcgtgaca 60ataacctgca aggcctccaa atcaattagc aaccatctgg catggtatca ggagaagcct 120ggcaaagcca ataagctgct gatccactcc ggctcaactc tgcaatccgg taccccaagc 180cgatttagcg gatctgggag cggaaccgag ttcacactta ccattagctc cctgcaaccg 240gaggacttcg ccacctatta ctgccagcaa tacgacgaat acccctatac gttcggccaa 300gggacaagat tggaaatcaa gcgtacg 32789118PRTArtificialAntibody variable region for anti-TcdB antibody 1099 (heavy chain) 89Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Gln Ser Tyr 20 25 30Thr Ile Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Ala Ala Ile Ser Gly Gly Gly Ser Thr Tyr Tyr Asn Leu Pro Leu Lys 50 55 60Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Ser Gln Val Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Thr 85 90 95Arg Pro Arg Trp Tyr Pro Arg Ser Tyr Phe Asp Tyr Trp Gly Arg Gly 100 105 110Thr Leu Val Thr Val Ser 11590354DNAArtificialPolynucleotide encoding antibody variable region for anti-TcdB antibody 1099.g2 (heavh chain) 90gaagttcagc tgcaggaatc tggacctggc ttggtgaaac caagcgagac acttagtctc 60acttgcaccg tttccggctt ctcccttcaa tcctacacga tctcttgggt gcggcaacca 120cccgggaaag gactggaatg gatcgcagcc attagcgggg gagggagcac ctattacaac 180ttgcctctca agagccgcgt gaccatatcc cgtgacacaa gcaagagcca ggtttccctg 240aagctgagct ccgtgactgc tgccgatacg gctgtttact attgcacccg acctcgctgg 300tatccccgtt cctatttcga ctactgggga agaggcacac tggttaccgt ctcg 3549111PRTArtificialAntibody CDR 91Arg Ala Ser Gln Arg Ile Ser Thr Ser Ile His1 5 10927PRTArtificialAntibody CDR 92Tyr Ala Ser Gln Ser Ile Ser1 5939PRTArtificialAntibody CDR 93Gln Gln Ser Tyr Ser Ser Leu Tyr Thr1 59410PRTArtificialAntibody CDRs 94Gly Phe Thr Phe Ser Asp Ser Tyr Met Ala1 5 109517PRTArtificialAntibody CDR 95Ser Ile Ser Tyr Gly Gly Thr Ile Ile Gln Tyr Gly Asp Ser Val Lys1 5 10 15Gly9611PRTArtificialAntibody CDR 96Arg Gln Gly Thr Tyr Ala Arg Tyr Leu Asp Phe1 5 1097107PRTArtificialAntibody variable region for anti-TcdB antibody 1102 97Asn Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Arg Ile Ser Thr Ser 20 25 30Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Tyr Ser Ser Leu Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 10598321DNAArtificialPolynucleotide encoding antibody variable region for anti-TcdB antibody 1102.g4 98aacatcgtgc tgacacagtc tcctgcaacc ctttcactgt ctccaggtga acgagcaacc 60ctgagttgta gagccagtca gaggatctcc acgagcattc actggtatca gcaaaagcct 120gggcaagctc ccagactctt gatcaagtac gcctctcaga gcataagtgg cattccagct 180aggtttagcg gctcaggctc aggaacagac ttcactctga ccatcagctc cctggaaccg 240gaggactttg ccgtctatta ctgccagcaa tcctactcca gtctgtacac cttcgggcag 300ggtactaaac tggagataaa g 32199119PRTArtificialAntibody variabl region for anti-TcdB antibody 1102 (heavy chain) 99Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30Tyr Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Ala Ser Ile Ser Tyr Gly Gly Thr Ile Ile Gln Tyr Gly Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Gln Gly Thr Tyr Ala Arg Tyr Leu Asp Phe Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser 115100357DNAArtificialPolynucleotide encoding antibody variable region for anti-TcdB antibody 1002.g4 (heavy chain) 100gaagtgcagc

tggtcgaatc cgggggaggt ttggtgcaac caggtggctc actgagactg 60agctgtgccg tttccggctt tacgttctca gacagttata tggcctgggt gcgtcaagca 120cctggaaaag ggctggagtg gattgccagt atcagctatg gtgggaccat aatccagtac 180ggcgatagcg tcaagggcag gtttactatc tccagggaca acgccaagtc aagcctttac 240ctgcagatga attctctccg cgcagaggat accgctgtgt attactgcgc tagacggcag 300ggaacctacg ctcgatacct ggacttctgg ggtcagggaa cactcgttac agtctcg 35710111PRTArtificialAntibody CDR 101Arg Ala Ser Glu Ser Val Ser Thr Leu Leu His1 5 101027PRTArtificialAntibody CDR 102Lys Ala Ser Asn Leu Ala Ser1 51039PRTArtificialAntibody CDR 103His Gln Ser Trp Asn Ser Pro Pro Thr1 510410PRTArtificialAnitbody CDR 104Gly Phe Thr Phe Ser Asn Tyr Gly Met Ala1 5 1010517PRTArtificialAntibody CDR 105Ile Ile Asn Tyr Asp Ala Ser Thr Thr His Tyr Arg Asp Ser Val Lys1 5 10 15Gly1069PRTArtificialAntibody CDR 106Tyr Gly Arg Ser His Tyr Phe Asp Tyr1 5107107PRTArtificialAntibody variable region for anti-TcdB anitbody 1114 107Ala Thr Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Thr Leu 20 25 30Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Lys Ala Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Ser Trp Asn Ser Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105108321DNAArtificialPolynucleotide sequence encoding antibody variable region for anti-TcdB antibody 1114.g2 108gcgacgcaaa tgactcagtc gccctcatcg cttagcgcgt ccgtcggaga tagagtgacg 60atcacctgcc gcgcatcaga gtcggtgtcc acactcctcc actggtatca gcagaaaccg 120gggaaggcac caaaactctt gatctacaaa gccagcaacc ttgcgtccgg tgtcccgtca 180aggttctccg ggagcggttc ggggacagac tttactttga ccatttcgtc gcttcagccg 240gaggacttcg ccacctatta ctgtcatcag tcatggaact cacctcccac atttggccag 300ggaacgaaac tcgaaatcaa g 321109117PRTArtificialAntibody variable region for anti-TcdB antibody 1114 (heavy chain) 109Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30Gly Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ile Ile Asn Tyr Asp Ala Ser Thr Thr His Tyr Arg Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Arg Tyr Gly Arg Ser His Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser 115110351DNAArtificialPolynucleotide encoding anitbody variable region for anti-TcdB antibody 1114.g2 (heavy chain) 110gaagtacaac tcgtagagtc agggggtggg ctggtccaac ctggcggctc ccttcggctt 60tcgtgtgccg cctcgggatt cacgtttagc aattacggta tggcctgggt gaggcaggca 120ccagggaagg gtcttgagtg ggtagcgatc atcaactatg atgcaagcac cacccactac 180agggatagcg tcaagggacg ctttactatc agccgggata atgcgaaatc ctcgctctat 240ctgcagatga actccctcag agccgaggac accgcagtgt actattgcac acgatacgga 300cgctcgcact atttcgacta ttggggacag gggacgctcg taactgtctc g 35111111PRTArtificialAntibody CDR 111Arg Ala Ser Glu Ser Val Ser Thr Leu Leu His1 5 101127PRTArtificialAnitbody CDR 112Lys Ala Ser Asn Leu Ala Ser1 51139PRTArtificialAntibody CDR 113His Gln Ser Trp Asn Ser Pro Pro Thr1 511410PRTArtificialAntibody CDR 114Gly Phe Thr Phe Ser Asn Tyr Gly Met Ala1 5 1011516PRTArtificialAntibody CDR 115Ile Ile Asn Tyr Asp Ala Ser Thr Thr His Tyr Arg Asp Ser Val Lys1 5 10 151169PRTArtificialAntibody CDR 116Tyr Gly Arg Ser His Tyr Phe Asp Tyr1 5117107PRTArtificialAntibody variable region for anti-TcdB antibody 1114 graft 8 117Asp Thr Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Thr Leu 20 25 30Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Lys Ala Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Ser Trp Asn Ser Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105118321DNAArtificialPolynucleotide encoding antibody variable region for anti-TcdB antibody 1114.g8 118gacacggtcc tgactcagtc gccctcatcg cttagcgcgt ccgtcggaga tagagtgacg 60atcacctgcc gcgcatcaga gtcggtgtcc acactcctcc actggtatca gcagaaaccg 120gggaaggcac caaaactctt gatctacaaa gccagcaacc ttgcgtccgg tgtcccgtca 180aggttctccg ggagcggttc ggggacagac tttactttga ccatttcgtc gcttcagccg 240gaggacttcg ccacctatta ctgtcatcag tcatggaact cacctcccac atttggccag 300ggaacgaaac tcgaaatcaa g 321119117PRTArtificialAntibody variable region for anti-TcdB antibody 1114 graft 8 (heavy chain) 119Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30Gly Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ile Ile Asn Tyr Asp Ala Ser Thr Thr His Tyr Arg Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Arg Tyr Gly Arg Ser His Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser 115120351DNAArtificialPolynucleotide encoding antibody variable region for anti-TcdB antibody 1114.g8 120gaagtacaac tcgtagagtc agggggtggg ctggtccaac ctggcggctc ccttcggctt 60tcgtgtgccg cctcgggatt cacgtttagc aattacggta tggcctgggt gaggcaggca 120ccagggaagg gtcttgagtg ggtagcgatc atcaactatg atgcaagcac cacccactac 180agggatagcg tcaagggacg ctttactatc agccgggata atgcgaaatc ctcgctctat 240ctgcagatga actccctcag agccgaggac accgcagtgt actattgcac acgatacgga 300cgctcgcact atttcgacta ttggggacag gggacgctcg taactgtctc g 35112111PRTArtificialAntibody CDR 121Lys Ala Ser Gln Asn Ile Tyr Met Tyr Leu Asn1 5 101227PRTArtificialAntibody CDR 122Asn Thr Asn Lys Leu His Thr1 51239PRTArtificialAntibody CDR 123Leu Gln His Lys Ser Phe Pro Tyr Thr1 512410PRTArtificialAntibody CDR 124Gly Phe Thr Phe Arg Asp Ser Phe Met Ala1 5 1012517PRTArtificialAntibody CDR 125Ser Ile Ser Tyr Glu Gly Asp Lys Thr Tyr Tyr Gly Asp Ser Val Lys1 5 10 15Gly1269PRTArtificialAntibody CDR 126Leu Thr Ile Thr Thr Ser Gly Asp Ser1 5127107PRTArtificialAntibody variable region for anti-TcdB antibody 1125 127Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Ile Tyr Met Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45Tyr Asn Thr Asn Lys Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Lys Ser Phe Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105128321DNAArtificialPolynucleotide sequence encoding antibody variable region for anti-TcdB antibody 1125.g2 128gatatacaaa tgactcagag ccctagctca ctgagcgctt ctgtgggcga tcgtgtgaca 60atcacttgca aagcaagcca gaacatctat atgtacctga attggtacca gcaaaaaccg 120ggaaaagctc ccaagcgcct gatttacaac accaataagc tgcataccgg cgtgccaagc 180cgttttagcg gatctggctc tggaaccgaa tatacactga ccataagctc cctgcaaccg 240gaagactttg caacttacta ttgcctccag cacaaatcct tcccctatac gttcggacaa 300gggaccaaac tggaaatcaa a 321129118PRTArtificialAntibody variable region for anti-TcdB antibody 1125 (heavy chain) 129Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp Ser 20 25 30Phe Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Ile Ser Tyr Glu Gly Asp Lys Thr Tyr Tyr Gly Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Thr Ile Thr Thr Ser Gly Asp Ser Trp Gly Gln Gly Thr 100 105 110Met Val Thr Val Ser Ser 115130354DNAArtificialPolynucleotide encoding antibody variable region for anti-TcdB antibody 1125.g2 (heavy chain) 130gaagtgcagc tggtcgaaag cggcggagga ttggtgcaac ctggtggctc tcttcgcctg 60tcttgcgctg caagcggctt tacgttccgc gatagcttta tggcttgggt gcgacaagct 120cctgggaaag ggctggaatg ggtcgctagc ataagctacg aaggcgacaa gacttactat 180ggggactctg tgaaaggccg attcaccatt agccgagaca acgcaaagaa ctccctgtac 240ctgcagatga actccctgcg tgccgaagat accgccgtgt actattgcgc taggctgacg 300atcactacaa gcggagatag ctggggacaa gggacaatgg tgaccgtctc gagc 35413111PRTArtificialAntibody CDR 131Lys Ala Ser Gln His Val Gly Thr Asn Val Asp1 5 101327PRTArtificialAntibody CDR 132Gly Ala Ser Ile Arg Tyr Thr1 51339PRTArtificialAntibody CDR 133Leu Gln Tyr Asn Tyr Asn Pro Tyr Thr1 513410PRTArtificialAntibody CDR 134Gly Phe Ile Phe Ser Asn Phe Gly Met Ser1 5 1013517PRTArtificialAntibody CDR 135Ser Ile Ser Pro Ser Gly Gly Asn Ala Tyr Tyr Arg Asp Ser Val Lys1 5 10 15Gly1369PRTArtificialAntibody CDR 136Arg Ala Tyr Ser Ser Pro Phe Ala Phe1 5137107PRTArtificialAntibody variable region for anti-TcdB antibody 1129 137Asp Thr Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln His Val Gly Thr Asn 20 25 30Val Asp Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45Tyr Gly Ala Ser Ile Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Val Ala Thr Tyr Tyr Cys Leu Gln Tyr Asn Tyr Asn Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105138321DNAArtificialPolynucleotide sequence encoding antibody variable region for anti-TcdB antibody 1129.g1 138gacacccaga tgactcagtc tccgtcaagc ctttctgcct ctgttggaga tcgagtcaca 60attacgtgca aggcaagcca acacgtgggt accaacgtgg actggtatca acagaagcca 120gggaaggtcc ccaaactgct gatctacggt gccagtattc gctataccgg cgtgcctgat 180cgcttcaccg gaagcgggtc agggaccgat ttcacactga caatcagctc cctgcaacct 240gaagacgtgg ctacttacta ctgcctgcag tacaactata atccctacac ctttggccag 300ggcaccaaac tggagataaa g 321139118PRTArtificialAntibody variable region for anti-TcdB antibody 1129 (heavy chain) 139Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Ile Phe Ser Asn Phe 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Ile Ser Pro Ser Gly Gly Asn Ala Tyr Tyr Arg Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Thr Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Arg Arg Ala Tyr Ser Ser Pro Phe Ala Phe Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser 115140354DNAArtificialPolynucleotide sequence encoding antibody variable region for anti-TcdB antibody 1129.g1(heavy chain) 140gaggtgcaac ttgtggaatc aggaggtggc gtggttcagc ccggtagatc acttcgtctg 60agttgtgcaa caagcggctt tatcttctcc aacttcggga tgtcttgggt tagacaggct 120cctggtaagg gcctcgaatg ggtggctagt attagcccaa gcgggggaaa cgcctactat 180agggacagcg tgaaaggacg cttcactatc agccgagata actccaagac cacgctgtat 240ctgcagatga atagtctgag ggccgaggat accgcagtgt actactgcac tcgacgggcc 300tattcttccc cttttgcctt ttggggacag gggactctgg tgacagtctc gagc 35414111PRTArtificialAntibody CDR 141Lys Ala Ser Lys Ser Ile Ser Asn His Leu Ala1 5 101427PRTArtificialAntibody CDR 142Ser Gly Ser Thr Leu Gln Pro1 51439PRTArtificialAntibody CDR 143Gln Gln Tyr Asp Glu Tyr Pro Tyr Thr1 514410PRTArtificialAntibody CDR 144Gly Phe Ser Leu Asn Ser Tyr Thr Ile Thr1 5 1014516PRTArtificialAntibody CDR 145Ala Ile Ser Gly Gly Gly Ser Thr Tyr Phe Asn Ser Ala Leu Lys Ser1 5 10 1514611PRTArtificialAntibody CDR 146Pro Arg Trp Tyr Pro Arg Ser Tyr Phe Asp Tyr1 5 10147107PRTArtificialAntibody variable region for anti-TcdB antibody 1134 147Asp Val Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Lys Ser Ile Ser Asn His 20 25 30Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Ala Asn Lys Leu Leu Ile 35 40 45His Ser Gly Ser Thr Leu Gln Pro Gly Thr Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Glu Tyr Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105148321DNAArtificialPolynucleotide encoding antibody variable region for anti-TcdB antibody 1134.g5 148gacgtccagc tcactcaatc tccctccttt ctgtctgctt ctgtgggcga tcgcgtgaca 60ataacctgca aggcctccaa atcaattagc aaccatctgg catggtatca ggagaagcct 120ggcaaagcca ataagctgct gatccactcc ggctcaactc tgcaacccgg taccccaagc 180cgatttagcg gatctgggag cggaaccgag ttcacactta ccattagctc cctgcaaccg 240gaggacttcg ccacctatta ctgccagcaa tacgacgaat acccctatac gttcggccaa 300gggacaagat tggaaatcaa g 321149118PRTArtificialAntibody variable region for anti-TcdB antibody 1134 (heavy chain) 149Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Asn Ser Tyr 20 25 30Thr Ile Thr Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Ala Ala Ile Ser Gly Gly Gly Ser Thr Tyr Phe Asn Ser Ala Leu Lys 50 55 60Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Ser Gln Val Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Thr 85 90 95Arg Pro Arg Trp Tyr Pro Arg Ser Tyr Phe Asp Tyr Trp Gly Arg Gly 100 105 110Thr Leu Val Thr Val Ser 115150354DNAArtificialPolynucleotide encoding antibody variable region for anti-TcdB antibody 1134.g5 (heavy chain) 150gaagttcagc tgcaggaatc tggacctggc ttggtgaaac caagcgagac

acttagtctc 60acttgcaccg tttccggctt ctcccttaat tcctacacga tcacttgggt gcggcaacca 120cccgggaaag gactggaatg gatcgcagcc attagcgggg gagggagcac ctatttcaac 180tcggctctca agagccgcgt gaccatatcc cgtgacacaa gcaagagcca ggtttccctg 240aagctgagct ccgtgactgc tgccgatacg gctgtttact attgcacccg acctcgctgg 300tatccccgtt cctatttcga ctactgggga agaggcacac tggttaccgt ctcg 35415111PRTArtificialAntibody CDR 151Lys Ala Ser Gln Asn Val Gly Asn Asn Val Ala1 5 101527PRTArtificialAntibody CDR 152Tyr Ala Ser Asn Arg Phe Thr1 51539PRTArtificialAntibody CDR 153Gln Arg Val Tyr Gln Ser Thr Trp Thr1 515410PRTArtificialAntibody CDR 154Gly Phe Ser Leu Thr Ser Tyr Tyr Val His1 5 1015516PRTArtificialAntibody CDR 155Cys Ile Arg Thr Gly Gly Asn Thr Glu Tyr Gln Ser Glu Phe Lys Ser1 5 10 151567PRTArtificialAntibody CDR 156Gly Asn Tyr Gly Phe Ala Tyr1 5157107PRTArtificialAntibody variable region for anti-TcdB antibody 1151 157Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Asn Asn 20 25 30Val Ala Trp Tyr Gln His Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Tyr Ala Ser Asn Arg Phe Thr Gly Val Pro Ser Arg Phe Thr Gly 50 55 60Gly Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Arg Val Tyr Gln Ser Thr Trp 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105158321DNAArtificialPolynucleotide encoding antibody variable region for anti-TcdB antibody 1151.g1 158gcgattcaaa tgactcagtc gccctcatcg cttagcgcgt ccgtcggaga tagagtgacg 60atcacgtgca aagcatcaca aaatgtcggg aacaatgtgg catggtatca gcataaaccg 120gggaaggcac caaaactctt gatctactac gccagcaaca ggtttactgg tgtcccgtca 180aggttcacgg gagggggtta cgggacagac tttactttga ccatttcgtc gcttcagccg 240gaggacttcg ccacctatta ctgtcagagg gtctaccagt caacgtggac atttggccag 300ggaacgaaag tggaaatcaa g 321159114PRTArtificialAntibody variable region for anti-TcdB antibody 1151 (heavy chain) 159Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30Tyr Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Cys Ile Arg Thr Gly Gly Asn Thr Glu Tyr Gln Ser Glu Phe Lys 50 55 60Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Gly Asn Tyr Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110Val Ser160342DNAArtificialPolynucleotide sequence encoding antibody variable region for anti-TcdB antibody 1151.g4 (heavy chain) 160gaagtacaac tccaagagtc ggggcctggt ctggtcaagc cgtccgaaac actttcgctg 60acgtgtacgg tatcaggatt ctcacttaca tcatactacg tccactgggt gaggcagcca 120cccgggaagg gtcttgagtg gatgggctgc attagaaccg gagggaatac cgagtaccag 180agcgaattta agagccgcgt cactatcagc cgggatacgt ccaaaaacca ggtgtcgctc 240aaattgtcct ccgtgacggc cgctgacacc gcagtgtact attgcgcgcg aggaaactat 300ggctttgcgt attggggaca ggggacgctc gtaactgtct cg 34216111PRTArtificialAntibody CDR 161Lys Ala Ser Gln Asn Ile Asn Lys Tyr Leu Asp1 5 101627PRTArtificialAntibody CDR 162Asn Ile Gln Ser Leu His Thr1 51637PRTArtificialAntibody CDR 163Phe Gln His Asn Ser Gly Trp1 516410PRTArtificialAntibody CDR 164Gly Phe Thr Phe Thr Gln Ala Ala Met Phe1 5 1016519PRTArtificialAntibody CDR 165Arg Ile Ser Thr Lys Ser Asn Asn Phe Ala Thr Tyr Tyr Pro Asp Ser1 5 10 15Val Lys Gly16613PRTArtificialAntibody CDR 166Pro Ala Tyr Tyr Tyr Asp Gly Thr Val Pro Phe Ala Tyr1 5 10167106PRTArtificialAntibody variable region for anti-TcdB antibody 1153.g8 167Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Ile Asn Lys Tyr 20 25 30Leu Asp Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45Tyr Asn Ile Gln Ser Leu His Thr Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Val Ala Thr Tyr Tyr Cys Phe Gln His Asn Ser Gly Trp Thr 85 90 95Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105168318DNAArtificialPolynucleotide sequence encoding antibody variable region for anti-TcdB antibody 1153.g8 168gatatacaga tgactcagtc cccttctagc ctttcagctt ccgtgggcga tagagtgact 60atcacgtgta aggctagtca gaacattaac aagtatctgg actggtacca gcagaaaccc 120gggaaggttc ccaagctgct gatctacaac atccagtccc tgcatacagg cattcctagc 180cggtttagcg gatctggttc agggaccgac ttcaccctga caatcagctc tctgcaacca 240gaagacgtgg ccacctatta ctgcttccag cacaatagtg gctggacttt tggacaaggt 300accaggctgg agatcaaa 318169123PRTArtificialAntibody variable region for anti-TcdB antibody 1153 (graft 8 heavy chain) 169Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Gln Ala 20 25 30Ala Met Phe Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Gly Ile 35 40 45Ala Arg Ile Ser Thr Lys Ser Asn Asn Phe Ala Thr Tyr Tyr Pro Asp 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr65 70 75 80Val Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Thr Ala Pro Ala Tyr Tyr Tyr Asp Gly Thr Val Pro Phe Ala 100 105 110Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120170369DNAArtificialPolynucleotide sequence encoding antibody variable region for anti-TcdB antibody 1153.g8 (heavy chain) 170gaggttcagc tggtggaatc aggagggggt ctggtgcaac caggaggctc cctgaaactg 60tcttgcgccg caagcggctt tacgtttacc caggccgcta tgttctgggt taggcaggcc 120agtgggaagg gtcttgaagg catcgcaaga atcagcacca agagcaacaa tttcgctacg 180tactatccgg actccgtgaa aggccggttt accatttctc gcgatgacag caagaacacc 240gtgtacctgc agatgaacag tctcaagacc gaggacacag ccgtgtacta ttgtactgct 300cccgcctatt attacgatgg cacagtgcct ttcgcatact ggggacaggg tactttggtg 360actgtctcg 3691712710PRTClostridia 171Met Ser Leu Ile Ser Lys Glu Glu Leu Ile Lys Leu Ala Tyr Ser Ile1 5 10 15Arg Pro Arg Glu Asn Glu Tyr Lys Thr Ile Leu Thr Asn Leu Asp Glu 20 25 30Tyr Asn Lys Leu Thr Thr Asn Asn Asn Glu Asn Lys Tyr Leu Gln Leu 35 40 45Lys Lys Leu Asn Glu Ser Ile Asp Val Phe Met Asn Lys Tyr Lys Thr 50 55 60Ser Ser Arg Asn Arg Ala Leu Ser Asn Leu Lys Lys Asp Ile Leu Lys65 70 75 80Glu Val Ile Leu Ile Lys Asn Ser Asn Thr Ser Pro Val Glu Lys Asn 85 90 95Leu His Phe Val Trp Ile Gly Gly Glu Val Ser Asp Ile Ala Leu Glu 100 105 110Tyr Ile Lys Gln Trp Ala Asp Ile Asn Ala Glu Tyr Asn Ile Lys Leu 115 120 125Trp Tyr Asp Ser Glu Ala Phe Leu Val Asn Thr Leu Lys Lys Ala Ile 130 135 140Val Glu Ser Ser Thr Thr Glu Ala Leu Gln Leu Leu Glu Glu Glu Ile145 150 155 160Gln Asn Pro Gln Phe Asp Asn Met Lys Phe Tyr Lys Lys Arg Met Glu 165 170 175Phe Ile Tyr Asp Arg Gln Lys Arg Phe Ile Asn Tyr Tyr Lys Ser Gln 180 185 190Ile Asn Lys Pro Thr Val Pro Thr Ile Asp Asp Ile Ile Lys Ser His 195 200 205Leu Val Ser Glu Tyr Asn Arg Asp Glu Thr Val Leu Glu Ser Tyr Arg 210 215 220Thr Asn Ser Leu Arg Lys Ile Asn Ser Asn His Gly Ile Asp Ile Arg225 230 235 240Ala Asn Ser Leu Phe Thr Glu Gln Glu Leu Leu Asn Ile Tyr Ser Gln 245 250 255Glu Leu Leu Asn Arg Gly Asn Leu Ala Ala Ala Ser Asp Ile Val Arg 260 265 270Leu Leu Ala Leu Lys Asn Phe Gly Gly Val Tyr Leu Asp Val Asp Met 275 280 285Leu Pro Gly Ile His Ser Asp Leu Phe Lys Thr Ile Ser Arg Pro Ser 290 295 300Ser Ile Gly Leu Asp Arg Trp Glu Met Ile Lys Leu Glu Ala Ile Met305 310 315 320Lys Tyr Lys Lys Tyr Ile Asn Asn Tyr Thr Ser Glu Asn Phe Asp Lys 325 330 335Leu Asp Gln Gln Leu Lys Asp Asn Phe Lys Leu Ile Ile Glu Ser Lys 340 345 350Ser Glu Lys Ser Glu Ile Phe Ser Lys Leu Glu Asn Leu Asn Val Ser 355 360 365Asp Leu Glu Ile Lys Ile Ala Phe Ala Leu Gly Ser Val Ile Asn Gln 370 375 380Ala Leu Ile Ser Lys Gln Gly Ser Tyr Leu Thr Asn Leu Val Ile Glu385 390 395 400Gln Val Lys Asn Arg Tyr Gln Phe Leu Asn Gln His Leu Asn Pro Ala 405 410 415Ile Glu Ser Asp Asn Asn Phe Thr Asp Thr Thr Lys Ile Phe His Asp 420 425 430Ser Leu Phe Asn Ser Ala Thr Ala Glu Asn Ser Met Phe Leu Thr Lys 435 440 445Ile Ala Pro Tyr Leu Gln Val Gly Phe Met Pro Glu Ala Arg Ser Thr 450 455 460Ile Ser Leu Ser Gly Pro Gly Ala Tyr Ala Ser Ala Tyr Tyr Asp Phe465 470 475 480Ile Asn Leu Gln Glu Asn Thr Ile Glu Lys Thr Leu Lys Ala Ser Asp 485 490 495Leu Ile Glu Phe Lys Phe Pro Glu Asn Asn Leu Ser Gln Leu Thr Glu 500 505 510Gln Glu Ile Asn Ser Leu Trp Ser Phe Asp Gln Ala Ser Ala Lys Tyr 515 520 525Gln Phe Glu Lys Tyr Val Arg Asp Tyr Thr Gly Gly Ser Leu Ser Glu 530 535 540Asp Asn Gly Val Asp Phe Asn Lys Asn Thr Ala Leu Asp Lys Asn Tyr545 550 555 560Leu Leu Asn Asn Lys Ile Pro Ser Asn Asn Val Glu Glu Ala Gly Ser 565 570 575Lys Asn Tyr Val His Tyr Ile Ile Gln Leu Gln Gly Asp Asp Ile Ser 580 585 590Tyr Glu Ala Thr Cys Asn Leu Phe Ser Lys Asn Pro Lys Asn Ser Ile 595 600 605Ile Ile Gln Arg Asn Met Asn Glu Ser Ala Lys Ser Tyr Phe Leu Ser 610 615 620Asp Asp Gly Glu Ser Ile Leu Glu Leu Asn Lys Tyr Arg Ile Pro Glu625 630 635 640Arg Leu Lys Asn Lys Glu Lys Val Lys Val Thr Phe Ile Gly His Gly 645 650 655Lys Asp Glu Phe Asn Thr Ser Glu Phe Ala Arg Leu Ser Val Asp Ser 660 665 670Leu Ser Asn Glu Ile Ser Ser Phe Leu Asp Thr Ile Lys Leu Asp Ile 675 680 685Ser Pro Lys Asn Val Glu Val Asn Leu Leu Gly Cys Asn Met Phe Ser 690 695 700Tyr Asp Phe Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu Leu Leu Ser705 710 715 720Ile Met Asp Lys Ile Thr Ser Thr Leu Pro Asp Val Asn Lys Asn Ser 725 730 735Ile Thr Ile Gly Ala Asn Gln Tyr Glu Val Arg Ile Asn Ser Glu Gly 740 745 750Arg Lys Glu Leu Leu Ala His Ser Gly Lys Trp Ile Asn Lys Glu Glu 755 760 765Ala Ile Met Ser Asp Leu Ser Ser Lys Glu Tyr Ile Phe Phe Asp Ser 770 775 780Ile Asp Asn Lys Leu Lys Ala Lys Ser Lys Asn Ile Pro Gly Leu Ala785 790 795 800Ser Ile Ser Glu Asp Ile Lys Thr Leu Leu Leu Asp Ala Ser Val Ser 805 810 815Pro Asp Thr Lys Phe Ile Leu Asn Asn Leu Lys Leu Asn Ile Glu Ser 820 825 830Ser Ile Gly Asp Tyr Ile Tyr Tyr Glu Lys Leu Glu Pro Val Lys Asn 835 840 845Ile Ile His Asn Ser Ile Asp Asp Leu Ile Asp Glu Phe Asn Leu Leu 850 855 860Glu Asn Val Ser Asp Glu Leu Tyr Glu Leu Lys Lys Leu Asn Asn Leu865 870 875 880Asp Glu Lys Tyr Leu Ile Ser Phe Glu Asp Ile Ser Lys Asn Asn Ser 885 890 895Thr Tyr Ser Val Arg Phe Ile Asn Lys Ser Asn Gly Glu Ser Val Tyr 900 905 910Val Glu Thr Glu Lys Glu Ile Phe Ser Lys Tyr Ser Glu His Ile Thr 915 920 925Lys Glu Ile Ser Thr Ile Lys Asn Ser Ile Ile Thr Asp Val Asn Gly 930 935 940Asn Leu Leu Asp Asn Ile Gln Leu Asp His Thr Ser Gln Val Asn Thr945 950 955 960Leu Asn Ala Ala Phe Phe Ile Gln Ser Leu Ile Asp Tyr Ser Ser Asn 965 970 975Lys Asp Val Leu Asn Asp Leu Ser Thr Ser Val Lys Val Gln Leu Tyr 980 985 990Ala Gln Leu Phe Ser Thr Gly Leu Asn Thr Ile Tyr Asp Ser Ile Gln 995 1000 1005Leu Val Asn Leu Ile Ser Asn Ala Val Asn Asp Thr Ile Asn Val 1010 1015 1020Leu Pro Thr Ile Thr Glu Gly Ile Pro Ile Val Ser Thr Ile Leu 1025 1030 1035Asp Gly Ile Asn Leu Gly Ala Ala Ile Lys Glu Leu Leu Asp Glu 1040 1045 1050His Asp Pro Leu Leu Lys Lys Glu Leu Glu Ala Lys Val Gly Val 1055 1060 1065Leu Ala Ile Asn Met Ser Leu Ser Ile Ala Ala Thr Val Ala Ser 1070 1075 1080Ile Val Gly Ile Gly Ala Glu Val Thr Ile Phe Leu Leu Pro Ile 1085 1090 1095Ala Gly Ile Ser Ala Gly Ile Pro Ser Leu Val Asn Asn Glu Leu 1100 1105 1110Ile Leu His Asp Lys Ala Thr Ser Val Val Asn Tyr Phe Asn His 1115 1120 1125Leu Ser Glu Ser Lys Lys Tyr Gly Pro Leu Lys Thr Glu Asp Asp 1130 1135 1140Lys Ile Leu Val Pro Ile Asp Asp Leu Val Ile Ser Glu Ile Asp 1145 1150 1155Phe Asn Asn Asn Ser Ile Lys Leu Gly Thr Cys Asn Ile Leu Ala 1160 1165 1170Met Glu Gly Gly Ser Gly His Thr Val Thr Gly Asn Ile Asp His 1175 1180 1185Phe Phe Ser Ser Pro Ser Ile Ser Ser His Ile Pro Ser Leu Ser 1190 1195 1200Ile Tyr Ser Ala Ile Gly Ile Glu Thr Glu Asn Leu Asp Phe Ser 1205 1210 1215Lys Lys Ile Met Met Leu Pro Asn Ala Pro Ser Arg Val Phe Trp 1220 1225 1230Trp Glu Thr Gly Ala Val Pro Gly Leu Arg Ser Leu Glu Asn Asp 1235 1240 1245Gly Thr Arg Leu Leu Asp Ser Ile Arg Asp Leu Tyr Pro Gly Lys 1250 1255 1260Phe Tyr Trp Arg Phe Tyr Ala Phe Phe Asp Tyr Ala Ile Thr Thr 1265 1270 1275Leu Lys Pro Val Tyr Glu Asp Thr Asn Ile Lys Ile Lys Leu Asp 1280 1285 1290Lys Asp Thr Arg Asn Phe Ile Met Pro Thr Ile Thr Thr Asn Glu 1295 1300 1305Ile Arg Asn Lys Leu Ser Tyr Ser Phe Asp Gly Ala Gly Gly Thr 1310 1315 1320Tyr Ser Leu Leu Leu Ser Ser Tyr Pro Ile Ser Thr Asn Ile Asn 1325 1330 1335Leu Ser Lys Asp Asp Leu Trp Ile Phe Asn Ile Asp Asn Glu Val 1340 1345 1350Arg Glu Ile Ser Ile Glu Asn Gly Thr Ile Lys Lys Gly Lys Leu 1355 1360 1365Ile Lys Asp Val Leu Ser Lys Ile Asp Ile Asn Lys Asn Lys Leu 1370 1375 1380Ile Ile Gly Asn Gln Thr Ile Asp Phe Ser Gly Asp Ile Asp Asn 1385 1390 1395Lys Asp Arg Tyr Ile Phe Leu Thr Cys Glu Leu Asp Asp Lys Ile 1400

1405 1410Ser Leu Ile Ile Glu Ile Asn Leu Val Ala Lys Ser Tyr Ser Leu 1415 1420 1425Leu Leu Ser Gly Asp Lys Asn Tyr Leu Ile Ser Asn Leu Ser Asn 1430 1435 1440Thr Ile Glu Lys Ile Asn Thr Leu Gly Leu Asp Ser Lys Asn Ile 1445 1450 1455Ala Tyr Asn Tyr Thr Asp Glu Ser Asn Asn Lys Tyr Phe Gly Ala 1460 1465 1470Ile Ser Lys Thr Ser Gln Lys Ser Ile Ile His Tyr Lys Lys Asp 1475 1480 1485Ser Lys Asn Ile Leu Glu Phe Tyr Asn Asp Ser Thr Leu Glu Phe 1490 1495 1500Asn Ser Lys Asp Phe Ile Ala Glu Asp Ile Asn Val Phe Met Lys 1505 1510 1515Asp Asp Ile Asn Thr Ile Thr Gly Lys Tyr Tyr Val Asp Asn Asn 1520 1525 1530Thr Asp Lys Ser Ile Asp Phe Ser Ile Ser Leu Val Ser Lys Asn 1535 1540 1545Gln Val Lys Val Asn Gly Leu Tyr Leu Asn Glu Ser Val Tyr Ser 1550 1555 1560Ser Tyr Leu Asp Phe Val Lys Asn Ser Asp Gly His His Asn Thr 1565 1570 1575Ser Asn Phe Met Asn Leu Phe Leu Asp Asn Ile Ser Phe Trp Lys 1580 1585 1590Leu Phe Gly Phe Glu Asn Ile Asn Phe Val Ile Asp Lys Tyr Phe 1595 1600 1605Thr Leu Val Gly Lys Thr Asn Leu Gly Tyr Val Glu Phe Ile Cys 1610 1615 1620Asp Asn Asn Lys Asn Ile Asp Ile Tyr Phe Gly Glu Trp Lys Thr 1625 1630 1635Ser Ser Ser Lys Ser Thr Ile Phe Ser Gly Asn Gly Arg Asn Val 1640 1645 1650Val Val Glu Pro Ile Tyr Asn Pro Asp Thr Gly Glu Asp Ile Ser 1655 1660 1665Thr Ser Leu Asp Phe Ser Tyr Glu Pro Leu Tyr Gly Ile Asp Arg 1670 1675 1680Tyr Ile Asn Lys Val Leu Ile Ala Pro Asp Leu Tyr Thr Ser Leu 1685 1690 1695Ile Asn Ile Asn Thr Asn Tyr Tyr Ser Asn Glu Tyr Tyr Pro Glu 1700 1705 1710Ile Ile Val Leu Asn Pro Asn Thr Phe His Lys Lys Val Asn Ile 1715 1720 1725Asn Leu Asp Ser Ser Ser Phe Glu Tyr Lys Trp Ser Thr Glu Gly 1730 1735 1740Ser Asp Phe Ile Leu Val Arg Tyr Leu Glu Glu Ser Asn Lys Lys 1745 1750 1755Ile Leu Gln Lys Ile Arg Ile Lys Gly Ile Leu Ser Asn Thr Gln 1760 1765 1770Ser Phe Asn Lys Met Ser Ile Asp Phe Lys Asp Ile Lys Lys Leu 1775 1780 1785Ser Leu Gly Tyr Ile Met Ser Asn Phe Lys Ser Phe Asn Ser Glu 1790 1795 1800Asn Glu Leu Asp Arg Asp His Leu Gly Phe Lys Ile Ile Asp Asn 1805 1810 1815Lys Thr Tyr Tyr Tyr Asp Glu Asp Ser Lys Leu Val Lys Gly Leu 1820 1825 1830Ile Asn Ile Asn Asn Ser Leu Phe Tyr Phe Asp Pro Ile Glu Phe 1835 1840 1845Asn Leu Val Thr Gly Trp Gln Thr Ile Asn Gly Lys Lys Tyr Tyr 1850 1855 1860Phe Asp Ile Asn Thr Gly Ala Ala Leu Thr Ser Tyr Lys Ile Ile 1865 1870 1875Asn Gly Lys His Phe Tyr Phe Asn Asn Asp Gly Val Met Gln Leu 1880 1885 1890Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr Phe Ala Pro Ala 1895 1900 1905Asn Thr Gln Asn Asn Asn Ile Glu Gly Gln Ala Ile Val Tyr Gln 1910 1915 1920Ser Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Asp Asn 1925 1930 1935Asn Ser Lys Ala Val Thr Gly Trp Arg Ile Ile Asn Asn Glu Lys 1940 1945 1950Tyr Tyr Phe Asn Pro Asn Asn Ala Ile Ala Ala Val Gly Leu Gln 1955 1960 1965Val Ile Asp Asn Asn Lys Tyr Tyr Phe Asn Pro Asp Thr Ala Ile 1970 1975 1980Ile Ser Lys Gly Trp Gln Thr Val Asn Gly Ser Arg Tyr Tyr Phe 1985 1990 1995Asp Thr Asp Thr Ala Ile Ala Phe Asn Gly Tyr Lys Thr Ile Asp 2000 2005 2010Gly Lys His Phe Tyr Phe Asp Ser Asp Cys Val Val Lys Ile Gly 2015 2020 2025Val Phe Ser Thr Ser Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn 2030 2035 2040Thr Tyr Asn Asn Asn Ile Glu Gly Gln Ala Ile Val Tyr Gln Ser 2045 2050 2055Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asn 2060 2065 2070Ser Lys Ala Val Thr Gly Leu Gln Thr Ile Asp Ser Lys Lys Tyr 2075 2080 2085Tyr Phe Asn Thr Asn Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr 2090 2095 2100Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ala Glu Ala 2105 2110 2115Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn 2120 2125 2130Thr Asn Thr Ala Ile Ala Ser Thr Gly Tyr Thr Ile Ile Asn Gly 2135 2140 2145Lys His Phe Tyr Phe Asn Thr Asp Gly Ile Met Gln Ile Gly Val 2150 2155 2160Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr 2165 2170 2175Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Leu Tyr Gln Asn Glu 2180 2185 2190Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp Ser 2195 2200 2205Lys Ala Val Thr Gly Trp Arg Ile Ile Asn Asn Lys Lys Tyr Tyr 2210 2215 2220Phe Asn Pro Asn Asn Ala Ile Ala Ala Ile His Leu Cys Thr Ile 2225 2230 2235Asn Asn Asp Lys Tyr Tyr Phe Ser Tyr Asp Gly Ile Leu Gln Asn 2240 2245 2250Gly Tyr Ile Thr Ile Glu Arg Asn Asn Phe Tyr Phe Asp Ala Asn 2255 2260 2265Asn Glu Ser Lys Met Val Thr Gly Val Phe Lys Gly Pro Asn Gly 2270 2275 2280Phe Glu Tyr Phe Ala Pro Ala Asn Thr His Asn Asn Asn Ile Glu 2285 2290 2295Gly Gln Ala Ile Val Tyr Gln Asn Lys Phe Leu Thr Leu Asn Gly 2300 2305 2310Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Ala Val Thr Gly Trp 2315 2320 2325Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn Thr Ala 2330 2335 2340Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr 2345 2350 2355Phe Asn Leu Asn Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile 2360 2365 2370Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe Ile Ala Ser 2375 2380 2385Thr Gly Tyr Thr Ser Ile Asn Gly Lys His Phe Tyr Phe Asn Thr 2390 2395 2400Asp Gly Ile Met Gln Ile Gly Val Phe Lys Gly Pro Asn Gly Phe 2405 2410 2415Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly 2420 2425 2430Gln Ala Ile Leu Tyr Gln Asn Lys Phe Leu Thr Leu Asn Gly Lys 2435 2440 2445Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Ala Val Thr Gly Leu Arg 2450 2455 2460Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ala Val 2465 2470 2475Ala Val Thr Gly Trp Gln Thr Ile Asn Gly Lys Lys Tyr Tyr Phe 2480 2485 2490Asn Thr Asn Thr Ser Ile Ala Ser Thr Gly Tyr Thr Ile Ile Ser 2495 2500 2505Gly Lys His Phe Tyr Phe Asn Thr Asp Gly Ile Met Gln Ile Gly 2510 2515 2520Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr Phe Ala Pro Ala Asn 2525 2530 2535Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Arg Tyr Gln Asn 2540 2545 2550Arg Phe Leu Tyr Leu His Asp Asn Ile Tyr Tyr Phe Gly Asn Asn 2555 2560 2565Ser Lys Ala Ala Thr Gly Trp Val Thr Ile Asp Gly Asn Arg Tyr 2570 2575 2580Tyr Phe Glu Pro Asn Thr Ala Met Gly Ala Asn Gly Tyr Lys Thr 2585 2590 2595Ile Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gln Ile 2600 2605 2610Gly Val Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Ala Pro Ala 2615 2620 2625Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Arg Tyr Gln 2630 2635 2640Asn Arg Phe Leu His Leu Leu Gly Lys Ile Tyr Tyr Phe Gly Asn 2645 2650 2655Asn Ser Lys Ala Val Thr Gly Trp Gln Thr Ile Asn Gly Lys Val 2660 2665 2670Tyr Tyr Phe Met Pro Asp Thr Ala Met Ala Ala Ala Gly Gly Leu 2675 2680 2685Phe Glu Ile Asp Gly Val Ile Tyr Phe Phe Gly Val Asp Gly Val 2690 2695 2700Lys Ala Pro Gly Ile Tyr Gly 2705 27101722366PRTClostridia 172Met Ser Leu Val Asn Arg Lys Gln Leu Glu Lys Met Ala Asn Val Arg1 5 10 15Phe Arg Thr Gln Glu Asp Glu Tyr Val Ala Ile Leu Asp Ala Leu Glu 20 25 30Glu Tyr His Asn Met Ser Glu Asn Thr Val Val Glu Lys Tyr Leu Lys 35 40 45Leu Lys Asp Ile Asn Ser Leu Thr Asp Ile Tyr Ile Asp Thr Tyr Lys 50 55 60Lys Ser Gly Arg Asn Lys Ala Leu Lys Lys Phe Lys Glu Tyr Leu Val65 70 75 80Thr Glu Val Leu Glu Leu Lys Asn Asn Asn Leu Thr Pro Val Glu Lys 85 90 95Asn Leu His Phe Val Trp Ile Gly Gly Gln Ile Asn Asp Thr Ala Ile 100 105 110Asn Tyr Ile Asn Gln Trp Lys Asp Val Asn Ser Asp Tyr Asn Val Asn 115 120 125Val Phe Tyr Asp Ser Asn Ala Phe Leu Ile Asn Thr Leu Lys Lys Thr 130 135 140Val Val Glu Ser Ala Ile Asn Asp Thr Leu Glu Ser Phe Arg Glu Asn145 150 155 160Leu Asn Asp Pro Arg Phe Asp Tyr Asn Lys Phe Phe Arg Lys Arg Met 165 170 175Glu Ile Ile Tyr Asp Lys Gln Lys Asn Phe Ile Asn Tyr Tyr Lys Ala 180 185 190Gln Arg Glu Glu Asn Pro Glu Leu Ile Ile Asp Asp Ile Val Lys Thr 195 200 205Tyr Leu Ser Asn Glu Tyr Ser Lys Glu Ile Asp Glu Leu Asn Thr Tyr 210 215 220Ile Glu Glu Ser Leu Asn Lys Ile Thr Gln Asn Ser Gly Asn Asp Val225 230 235 240Arg Asn Phe Glu Glu Phe Lys Asn Gly Glu Ser Phe Asn Leu Tyr Glu 245 250 255Gln Glu Leu Val Glu Arg Trp Asn Leu Ala Ala Ala Ser Asp Ile Leu 260 265 270Arg Ile Ser Ala Leu Lys Glu Ile Gly Gly Met Tyr Leu Asp Val Asp 275 280 285Met Leu Pro Gly Ile Gln Pro Asp Leu Phe Glu Ser Ile Glu Lys Pro 290 295 300Ser Ser Val Thr Val Asp Phe Trp Glu Met Thr Lys Leu Glu Ala Ile305 310 315 320Met Lys Tyr Lys Glu Tyr Ile Pro Glu Tyr Thr Ser Glu His Phe Asp 325 330 335Met Leu Asp Glu Glu Val Gln Ser Ser Phe Glu Ser Val Leu Ala Ser 340 345 350Lys Ser Asp Lys Ser Glu Ile Phe Ser Ser Leu Gly Asp Met Glu Ala 355 360 365Ser Pro Leu Glu Val Lys Ile Ala Phe Asn Ser Lys Gly Ile Ile Asn 370 375 380Gln Gly Leu Ile Ser Val Lys Asp Ser Tyr Cys Ser Asn Leu Ile Val385 390 395 400Lys Gln Ile Glu Asn Arg Tyr Lys Ile Leu Asn Asn Ser Leu Asn Pro 405 410 415Ala Ile Ser Glu Asp Asn Asp Phe Asn Thr Thr Thr Asn Thr Phe Ile 420 425 430Asp Ser Ile Met Ala Glu Ala Asn Ala Asp Asn Gly Arg Phe Met Met 435 440 445Glu Leu Gly Lys Tyr Leu Arg Val Gly Phe Phe Pro Asp Val Lys Thr 450 455 460Thr Ile Asn Leu Ser Gly Pro Glu Ala Tyr Ala Ala Ala Tyr Gln Asp465 470 475 480Leu Leu Met Phe Lys Glu Gly Ser Met Asn Ile His Leu Ile Glu Ala 485 490 495Asp Leu Arg Asn Phe Glu Ile Ser Lys Thr Asn Ile Ser Gln Ser Thr 500 505 510Glu Gln Glu Met Ala Ser Leu Trp Ser Phe Asp Asp Ala Arg Ala Lys 515 520 525Ala Gln Phe Glu Glu Tyr Lys Arg Asn Tyr Phe Glu Gly Ser Leu Gly 530 535 540Glu Asp Asp Asn Leu Asp Phe Ser Gln Asn Ile Val Val Asp Lys Glu545 550 555 560Tyr Leu Leu Glu Lys Ile Ser Ser Leu Ala Arg Ser Ser Glu Arg Gly 565 570 575Tyr Ile His Tyr Ile Val Gln Leu Gln Gly Asp Lys Ile Ser Tyr Glu 580 585 590Ala Ala Cys Asn Leu Phe Ala Lys Thr Pro Tyr Asp Ser Val Leu Phe 595 600 605Gln Lys Asn Ile Glu Asp Ser Glu Ile Ala Tyr Tyr Tyr Asn Pro Gly 610 615 620Asp Gly Glu Ile Gln Glu Ile Asp Lys Tyr Lys Ile Pro Ser Ile Ile625 630 635 640Ser Asp Arg Pro Lys Ile Lys Leu Thr Phe Ile Gly His Gly Lys Asp 645 650 655Glu Phe Asn Thr Asp Ile Phe Ala Gly Phe Asp Val Asp Ser Leu Ser 660 665 670Thr Glu Ile Glu Ala Ala Ile Asp Leu Ala Lys Glu Asp Ile Ser Pro 675 680 685Lys Ser Ile Glu Ile Asn Leu Leu Gly Cys Asn Met Phe Ser Tyr Ser 690 695 700Ile Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu Leu Leu Lys Val Lys705 710 715 720Asp Lys Ile Ser Glu Leu Met Pro Ser Ile Ser Gln Asp Ser Ile Ile 725 730 735Val Ser Ala Asn Gln Tyr Glu Val Arg Ile Asn Ser Glu Gly Arg Arg 740 745 750Glu Leu Leu Asp His Ser Gly Glu Trp Ile Asn Lys Glu Glu Ser Ile 755 760 765Ile Lys Asp Ile Ser Ser Lys Glu Tyr Ile Ser Phe Asn Pro Lys Glu 770 775 780Asn Lys Ile Thr Val Lys Ser Lys Asn Leu Pro Glu Leu Ser Thr Leu785 790 795 800Leu Gln Glu Ile Arg Asn Asn Ser Asn Ser Ser Asp Ile Glu Leu Glu 805 810 815Glu Lys Val Met Leu Thr Glu Cys Glu Ile Asn Val Ile Ser Asn Ile 820 825 830Asp Thr Gln Ile Val Glu Glu Arg Ile Glu Glu Ala Lys Asn Leu Thr 835 840 845Ser Asp Ser Ile Asn Tyr Ile Lys Asp Glu Phe Lys Leu Ile Glu Ser 850 855 860Ile Ser Asp Ala Leu Cys Asp Leu Lys Gln Gln Asn Glu Leu Glu Asp865 870 875 880Ser His Phe Ile Ser Phe Glu Asp Ile Ser Glu Thr Asp Glu Gly Phe 885 890 895Ser Ile Arg Phe Ile Asn Lys Glu Thr Gly Glu Ser Ile Phe Val Glu 900 905 910Thr Glu Lys Thr Ile Phe Ser Glu Tyr Ala Asn His Ile Thr Glu Glu 915 920 925Ile Ser Lys Ile Lys Gly Thr Ile Phe Asp Thr Val Asn Gly Lys Leu 930 935 940Val Lys Lys Val Asn Leu Asp Thr Thr His Glu Val Asn Thr Leu Asn945 950 955 960Ala Ala Phe Phe Ile Gln Ser Leu Ile Glu Tyr Asn Ser Ser Lys Glu 965 970 975Ser Leu Ser Asn Leu Ser Val Ala Met Lys Val Gln Val Tyr Ala Gln 980 985 990Leu Phe Ser Thr Gly Leu Asn Thr Ile Thr Asp Ala Ala Lys Val Val 995 1000 1005Glu Leu Val Ser Thr Ala Leu Asp Glu Thr Ile Asp Leu Leu Pro 1010 1015 1020Thr Leu Ser Glu Gly Leu Pro Ile Ile Ala Thr Ile Ile Asp Gly 1025 1030 1035Val Ser Leu Gly Ala Ala Ile Lys Glu Leu Ser Glu Thr Ser Asp 1040 1045 1050Pro Leu Leu Arg Gln Glu Ile Glu Ala Lys Ile Gly Ile Met Ala 1055 1060 1065Val Asn Leu Thr Thr Ala Thr Thr Ala Ile Ile Thr Ser Ser Leu 1070 1075 1080Gly Ile Ala Ser Gly Phe Ser Ile Leu Leu Val Pro Leu Ala Gly 1085 1090 1095Ile Ser Ala Gly Ile Pro Ser Leu Val Asn Asn Glu Leu Val Leu 1100 1105 1110Arg Asp Lys Ala Thr Lys Val Val Asp Tyr Phe Lys His Val Ser 1115 1120 1125Leu Val Glu Thr Glu Gly Val Phe Thr Leu Leu Asp Asp Lys Ile 1130 1135 1140Met Met Pro Gln Asp Asp Leu Val Ile

Ser Glu Ile Asp Phe Asn 1145 1150 1155Asn Asn Ser Ile Val Leu Gly Lys Cys Glu Ile Trp Arg Met Glu 1160 1165 1170Gly Gly Ser Gly His Thr Val Thr Asp Asp Ile Asp His Phe Phe 1175 1180 1185Ser Ala Pro Ser Ile Thr Tyr Arg Glu Pro His Leu Ser Ile Tyr 1190 1195 1200Asp Val Leu Glu Val Gln Lys Glu Glu Leu Asp Leu Ser Lys Asp 1205 1210 1215Leu Met Val Leu Pro Asn Ala Pro Asn Arg Val Phe Ala Trp Glu 1220 1225 1230Thr Gly Trp Thr Pro Gly Leu Arg Ser Leu Glu Asn Asp Gly Thr 1235 1240 1245Lys Leu Leu Asp Arg Ile Arg Asp Asn Tyr Glu Gly Glu Phe Tyr 1250 1255 1260Trp Arg Tyr Phe Ala Phe Ile Ala Asp Ala Leu Ile Thr Thr Leu 1265 1270 1275Lys Pro Arg Tyr Glu Asp Thr Asn Ile Arg Ile Asn Leu Asp Ser 1280 1285 1290Asn Thr Arg Ser Phe Ile Val Pro Ile Ile Thr Thr Glu Tyr Ile 1295 1300 1305Arg Glu Lys Leu Ser Tyr Ser Phe Tyr Gly Ser Gly Gly Thr Tyr 1310 1315 1320Ala Leu Ser Leu Ser Gln Tyr Asn Met Gly Ile Asn Ile Glu Leu 1325 1330 1335Ser Glu Ser Asp Val Trp Ile Ile Asp Val Asp Asn Val Val Arg 1340 1345 1350Asp Val Thr Ile Glu Ser Asp Lys Ile Lys Lys Gly Asp Leu Ile 1355 1360 1365Glu Gly Ile Leu Ser Thr Leu Ser Ile Glu Glu Asn Lys Ile Ile 1370 1375 1380Leu Asn Ser His Glu Ile Asn Phe Ser Gly Glu Val Asn Gly Ser 1385 1390 1395Asn Gly Phe Val Ser Leu Thr Phe Ser Ile Leu Glu Gly Ile Asn 1400 1405 1410Ala Ile Ile Glu Val Asp Leu Leu Ser Lys Ser Tyr Lys Leu Leu 1415 1420 1425Ile Ser Gly Glu Leu Lys Ile Leu Met Leu Asn Ser Asn His Ile 1430 1435 1440Gln Gln Lys Ile Asp Tyr Ile Gly Phe Asn Ser Glu Leu Gln Lys 1445 1450 1455Asn Ile Pro Tyr Ser Phe Val Asp Ser Glu Gly Lys Glu Asn Gly 1460 1465 1470Phe Ile Asn Gly Ser Thr Lys Glu Gly Leu Phe Val Ser Glu Leu 1475 1480 1485Pro Asp Val Val Leu Ile Ser Lys Val Tyr Met Asp Asp Ser Lys 1490 1495 1500Pro Ser Phe Gly Tyr Tyr Ser Asn Asn Leu Lys Asp Val Lys Val 1505 1510 1515Ile Thr Lys Asp Asn Val Asn Ile Leu Thr Gly Tyr Tyr Leu Lys 1520 1525 1530Asp Asp Ile Lys Ile Ser Leu Ser Leu Thr Leu Gln Asp Glu Lys 1535 1540 1545Thr Ile Lys Leu Asn Ser Val His Leu Asp Glu Ser Gly Val Ala 1550 1555 1560Glu Ile Leu Lys Phe Met Asn Arg Lys Gly Asn Thr Asn Thr Ser 1565 1570 1575Asp Ser Leu Met Ser Phe Leu Glu Ser Met Asn Ile Lys Ser Ile 1580 1585 1590Phe Val Asn Phe Leu Gln Ser Asn Ile Lys Phe Ile Leu Asp Ala 1595 1600 1605Asn Phe Ile Ile Ser Gly Thr Thr Ser Ile Gly Gln Phe Glu Phe 1610 1615 1620Ile Cys Asp Glu Asn Asp Asn Ile Gln Pro Tyr Phe Ile Lys Phe 1625 1630 1635Asn Thr Leu Glu Thr Asn Tyr Thr Leu Tyr Val Gly Asn Arg Gln 1640 1645 1650Asn Met Ile Val Glu Pro Asn Tyr Asp Leu Asp Asp Ser Gly Asp 1655 1660 1665Ile Ser Ser Thr Val Ile Asn Phe Ser Gln Lys Tyr Leu Tyr Gly 1670 1675 1680Ile Asp Ser Cys Val Asn Lys Val Val Ile Ser Pro Asn Ile Tyr 1685 1690 1695Thr Asp Glu Ile Asn Ile Thr Pro Val Tyr Glu Thr Asn Asn Thr 1700 1705 1710Tyr Pro Glu Val Ile Val Leu Asp Ala Asn Tyr Ile Asn Glu Lys 1715 1720 1725Ile Asn Val Asn Ile Asn Asp Leu Ser Ile Arg Tyr Val Trp Ser 1730 1735 1740Asn Asp Gly Asn Asp Phe Ile Leu Met Ser Thr Ser Glu Glu Asn 1745 1750 1755Lys Val Ser Gln Val Lys Ile Arg Phe Val Asn Val Phe Lys Asp 1760 1765 1770Lys Thr Leu Ala Asn Lys Leu Ser Phe Asn Phe Ser Asp Lys Gln 1775 1780 1785Asp Val Pro Val Ser Glu Ile Ile Leu Ser Phe Thr Pro Ser Tyr 1790 1795 1800Tyr Glu Asp Gly Leu Ile Gly Tyr Asp Leu Gly Leu Val Ser Leu 1805 1810 1815Tyr Asn Glu Lys Phe Tyr Ile Asn Asn Phe Gly Met Met Val Ser 1820 1825 1830Gly Leu Ile Tyr Ile Asn Asp Ser Leu Tyr Tyr Phe Lys Pro Pro 1835 1840 1845Val Asn Asn Leu Ile Thr Gly Phe Val Thr Val Gly Asp Asp Lys 1850 1855 1860Tyr Tyr Phe Asn Pro Ile Asn Gly Gly Ala Ala Ser Ile Gly Glu 1865 1870 1875Thr Ile Ile Asp Asp Lys Asn Tyr Tyr Phe Asn Gln Ser Gly Val 1880 1885 1890Leu Gln Thr Gly Val Phe Ser Thr Glu Asp Gly Phe Lys Tyr Phe 1895 1900 1905Ala Pro Ala Asn Thr Leu Asp Glu Asn Leu Glu Gly Glu Ala Ile 1910 1915 1920Asp Phe Thr Gly Lys Leu Ile Ile Asp Glu Asn Ile Tyr Tyr Phe 1925 1930 1935Asp Asp Asn Tyr Arg Gly Ala Val Glu Trp Lys Glu Leu Asp Gly 1940 1945 1950Glu Met His Tyr Phe Ser Pro Glu Thr Gly Lys Ala Phe Lys Gly 1955 1960 1965Leu Asn Gln Ile Gly Asp Tyr Lys Tyr Tyr Phe Asn Ser Asp Gly 1970 1975 1980Val Met Gln Lys Gly Phe Val Ser Ile Asn Asp Asn Lys His Tyr 1985 1990 1995Phe Asp Asp Ser Gly Val Met Lys Val Gly Tyr Thr Glu Ile Asp 2000 2005 2010Gly Lys His Phe Tyr Phe Ala Glu Asn Gly Glu Met Gln Ile Gly 2015 2020 2025Val Phe Asn Thr Glu Asp Gly Phe Lys Tyr Phe Ala His His Asn 2030 2035 2040Glu Asp Leu Gly Asn Glu Glu Gly Glu Glu Ile Ser Tyr Ser Gly 2045 2050 2055Ile Leu Asn Phe Asn Asn Lys Ile Tyr Tyr Phe Asp Asp Ser Phe 2060 2065 2070Thr Ala Val Val Gly Trp Lys Asp Leu Glu Asp Gly Ser Lys Tyr 2075 2080 2085Tyr Phe Asp Glu Asp Thr Ala Glu Ala Tyr Ile Gly Leu Ser Leu 2090 2095 2100Ile Asn Asp Gly Gln Tyr Tyr Phe Asn Asp Asp Gly Ile Met Gln 2105 2110 2115Val Gly Phe Val Thr Ile Asn Asp Lys Val Phe Tyr Phe Ser Asp 2120 2125 2130Ser Gly Ile Ile Glu Ser Gly Val Gln Asn Ile Asp Asp Asn Tyr 2135 2140 2145Phe Tyr Ile Asp Asp Asn Gly Ile Val Gln Ile Gly Val Phe Asp 2150 2155 2160Thr Ser Asp Gly Tyr Lys Tyr Phe Ala Pro Ala Asn Thr Val Asn 2165 2170 2175Asp Asn Ile Tyr Gly Gln Ala Val Glu Tyr Ser Gly Leu Val Arg 2180 2185 2190Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Thr Ile Glu 2195 2200 2205Thr Gly Trp Ile Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr Tyr 2210 2215 2220Phe Asn Pro Glu Thr Lys Lys Ala Cys Lys Gly Ile Asn Leu Ile 2225 2230 2235Asp Asp Ile Lys Tyr Tyr Phe Asp Glu Lys Gly Ile Met Arg Thr 2240 2245 2250Gly Leu Ile Ser Phe Glu Asn Asn Asn Tyr Tyr Phe Asn Glu Asn 2255 2260 2265Gly Glu Met Gln Phe Gly Tyr Ile Asn Ile Glu Asp Lys Met Phe 2270 2275 2280Tyr Phe Gly Glu Asp Gly Val Met Gln Ile Gly Val Phe Asn Thr 2285 2290 2295Pro Asp Gly Phe Lys Tyr Phe Ala His Gln Asn Thr Leu Asp Glu 2300 2305 2310Asn Phe Glu Gly Glu Ser Ile Asn Tyr Thr Gly Trp Leu Asp Leu 2315 2320 2325Asp Glu Lys Arg Tyr Tyr Phe Thr Asp Glu Tyr Ile Ala Ala Thr 2330 2335 2340Gly Ser Val Ile Ile Asp Gly Glu Glu Tyr Tyr Phe Asp Pro Asp 2345 2350 2355Thr Ala Gln Leu Val Ile Ser Glu 2360 236517318PRTArtificialFragment from C. difficile toxin TcdB 173Ser Pro Val Glu Lys Asn Leu His Phe Val Trp Ile Gly Gly Glu Val1 5 10 15Ser Asp17411PRTArtificialFragment from C. difficile toxin TcdB 174Asn Leu Ala Ala Ala Ser Asp Ile Val Arg Leu1 5 1017515PRTArtificialFragment of C. difficile toxin TcdB 175Cys Gly Gly Val Tyr Leu Asp Val Asp Met Leu Pro Gly Ile His1 5 10 1517620PRTArtificialFragment from C. difficile toxin TcdB 176Cys Gly Gly Val Tyr Leu Asp Val Asp Met Leu Pro Gly Ile His Ser1 5 10 15Asp Leu Phe Lys 2017714PRTArtificialFragment of C. difficile toxin TcdB 177Cys Trp Glu Met Ile Lys Leu Glu Ala Ile Met Lys Tyr Lys1 5 1017812PRTArtificialFragment of C. difficile toxin TcdB 178Cys Thr Asn Leu Val Ile Glu Gln Val Lys Asn Arg1 5 1017912PRTArtificialFragment of C. difficile toxin TcdB 179Pro Glu Ala Arg Ser Thr Ile Ser Leu Ser Gly Pro1 5 1018012PRTArtificialFragment of C. difficile toxin TcdB 180Cys Ser Asn Leu Ile Val Lys Gln Ile Glu Asn Arg1 5 1018114PRTArtificialFragment of C. difficile toxin TcdB 181Thr Glu Gln Glu Ile Asn Ser Leu Trp Ser Phe Asp Gln Ala1 5 1018225PRTArtificialFragment of C. difficile toxin TcdB 182Thr Glu Gln Glu Ile Asn Ser Leu Trp Ser Phe Asp Pro Glu Ala Arg1 5 10 15Ser Thr Ile Ser Leu Ser Gly Pro Cys 20 2518313PRTArtificialFragment of C. difficile toxin TcdB 183Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu Leu Leu Cys1 5 1018414PRTArtificialFragment of C. difficile toxin TcdB 184Cys Ala Asn Gln Tyr Glu Val Arg Ile Asn Ser Glu Gly Arg1 5 1018515PRTArtificialFragment of C. difficile toxin TcdB 185Val Asn Thr Leu Asn Ala Ala Phe Phe Ile Gln Ser Leu Ile Cys1 5 10 1518613PRTArtificialFragment of C. difficile toxin TcdB 186Tyr Ala Gln Leu Phe Ser Thr Gly Leu Asn Thr Ile Cys1 5 1018716PRTArtificialFragment of C. difficile toxin TcdB 187Cys Ala Gly Ile Ser Ala Gly Ile Pro Ser Leu Val Asn Asn Glu Leu1 5 10 1518816PRTArtificialFragment of C. difficile toxin TcdB 188Asp Asp Leu Val Ile Ser Glu Ile Asp Phe Asn Asn Asn Ser Ile Cys1 5 10 1518910PRTArtificialFragment of C. difficile toxin TcdB 189Met Glu Gly Gly Ser Gly His Thr Val Thr1 5 1019035PRTArtificialFragment of C. difficile toxin TcdB 190Ala Val Asn Asp Thr Ile Asn Val Leu Pro Thr Ile Thr Glu Gly Ile1 5 10 15Pro Ile Val Ser Thr Ile Leu Asp Gly Ile Asn Leu Gly Ala Ala Ile 20 25 30Lys Glu Leu 3519120PRTArtificialFragment of C. difficile toxin TcdB 191Cys Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile1 5 10 15Glu Gly Gln Ala 2019220PRTArtificialFragment of C. difficile toxin TcdB 192Cys Gly Tyr Lys Tyr Phe Ala Pro Ala Asn Thr Val Asn Asp Asn Ile1 5 10 15Tyr Gly Gln Ala 2019312PRTArtificialFragment of C. difficile toxin TcdB 193Cys Lys Tyr Tyr Phe Asn Thr Asn Thr Ala Glu Ala1 5 1019412PRTArtificialFragment of C. difficile toxin TcdB 194Cys Lys Tyr Tyr Phe Asp Glu Asp Thr Ala Glu Ala1 5 10

Claims

1-41. (canceled)

42. A pharmaceutical composition comprising one or more monoclonal antibodies specific to antigen TcdB1234 wherein the antibody has high affinity of 600 pM or less for the target antigen TcdB1234 and said one or more monoclonal antibodies are independently selected from: i) a heavy chain wherein the variable domain of the heavy chain comprises a CDR having the sequence given in SEQ ID NO:124 for CDR-H1, a CDR having the sequence given in SEQ ID NO: 125 for CDR-H2 and a CDR having the sequence given in SEQ ID NO: 126 for CDR-H3, and a light chain wherein the variable domain of the light chain comprises a CDR having the sequence given in SEQ ID NO:121 for CDR-L1, a CDR having the sequence given in in SEQ ID NO:122 for CDR-L2 and a CDR having the sequence given in SEQ ID NO:123 for CDR-L3; ii) a heavy chain wherein the variable domain of the heavy chain comprises a CDR having the sequence given in SEQ ID NO:154 for CDR-H1, a CDR having the sequence given in SEQ ID NO:155 for CDR-H2 and a CDR having the sequence given in SEQ ID NO:156 for CDR-H3, and a light chain wherein the variable domain of the light chain comprises a CDR having the sequence given in SEQ ID NO:151 for CDR-L1, a CDR having the sequence given in in SEQ ID NO:152 for CDR-L2 and a CDR having the sequence given in SEQ ID NO:153 for CDR-L3; iii) a heavy chain wherein the variable domain of the heavy chain comprises a CDR having the sequence given in SEQ ID NO:144 for CDR-H1, a CDR having the sequence given in SEQ ID NO:145 for CDR-H2 and a CDR having the sequence given in SEQ ID NO:146 for CDR-H3, and a light chain wherein the variable domain of the light chain comprises a CDR having the sequence given in SEQ ID NO:141 for CDR-L1, a CDR having the sequence given in in SEQ ID NO:142 for CDR-L2 and a CDR having the sequence given in SEQ ID NO:143 for CDR-L3; iv) a heavy chain wherein the variable domain of the heavy chain comprises a CDR having the sequence given in SEQ ID NO:164 for CDR-H1, a CDR having the sequence given in SEQ ID NO:165 for CDR-H2 and a CDR having the sequence given in SEQ ID NO:166 for CDR-H3, and a light chain wherein the variable domain of the light chain comprises a CDR having the sequence given in SEQ ID NO:161 for CDR-L1, a CDR having the sequence given in in SEQ ID NO:162 for CDR-L2 and a CDR having the sequence given in SEQ ID NO:163 for CDR-L3; v) a heavy chain wherein the variable domain of the heavy chain comprises a CDR having the sequence given in SEQ ID NO:64 for CDR-H1, a CDR having the sequence given in SEQ ID NO: 65 for CDR-H2 and a CDR having the sequence given in SEQ ID NO: 66 for CDR-H3, and a light chain wherein the variable domain of the light chain comprises a CDR having the sequence given in SEQ ID NO:61 for CDR-L1, a CDR having the sequence given in in SEQ ID NO:62 for CDR-L2 and a CDR having the sequence given in SEQ ID NO:63 for CDR-L3; vi) a heavy chain wherein the variable domain of the heavy chain comprises a CDR having the sequence given in SEQ ID NO:74 for CDR-H1, a CDR having the sequence given in SEQ ID NO: 75 for CDR-H2 and a CDR having the sequence given in SEQ ID NO: 76 for CDR-H3, and a light chain wherein the variable domain of the light chain comprises a CDR having the sequence given in SEQ ID NO:71 for CDR-L1, a CDR having the sequence given in in SEQ ID NO:72 for CDR-L2 and a CDR having the sequence given in SEQ ID NO:73 for CDR-L3; vii) a heavy chain wherein the variable domain of the heavy chain comprises a CDR having the sequence given in SEQ ID NO:84 for CDR-H1, a CDR having the sequence given in SEQ ID NO: 85 for CDR-H2 and a CDR having the sequence given in SEQ ID NO: 86 for CDR-H3, and a light chain wherein the variable domain of the light chain comprises a CDR having the sequence given in SEQ ID NO:81 for CDR-L1, a CDR having the sequence given in in SEQ ID NO:82 for CDR-L2 and a CDR having the sequence given in SEQ ID NO:83 for CDR-L3; viii) a heavy chain wherein the variable domain of the heavy chain comprises a CDR having the sequence given in SEQ ID NO:134 for CDR-H1, a CDR having the sequence given in SEQ ID NO: 135 for CDR-H2 and a CDR having the sequence given in SEQ ID NO: 136 for CDR-H3, and a light chain wherein the variable domain of the light chain comprises a CDR having the sequence given in SEQ ID NO:131 for CDR-L1, a CDR having the sequence given in in SEQ ID NO:132 for CDR-L2 and a CDR having the sequence given in SEQ ID NO:133 for CDR-L3.

43. A pharmaceutical composition according to claim 42, wherein said one or more monoclonal antibodies are independently selected from: i) a heavy chain wherein the variable domain of the heavy chain comprises a CDR having the sequence given in SEQ ID NO:124 for CDR-H1, a CDR having the sequence given in SEQ ID NO: 125 for CDR-H2 and a CDR having the sequence given in SEQ ID NO: 126 for CDR-H3, and a light chain wherein the variable domain of the light chain comprises a CDR having the sequence given in SEQ ID NO:121 for CDR-L1, a CDR having the sequence given in in SEQ ID NO:122 for CDR-L2 and a CDR having the sequence given in SEQ ID NO:123 for CDR-L3; ii) a heavy chain wherein the variable domain of the heavy chain comprises a CDR having the sequence given in SEQ ID NO:154 for CDR-H1, a CDR having the sequence given in SEQ ID NO:155 for CDR-H2 and a CDR having the sequence given in SEQ ID NO:156 for CDR-H3, and a light chain wherein the variable domain of the light chain comprises a CDR having the sequence given in SEQ ID NO:151 for CDR-L1, a CDR having the sequence given in in SEQ ID NO:152 for CDR-L2 and a CDR having the sequence given in SEQ ID NO:153 for CDR-L3; iii) a heavy chain wherein the variable domain of the heavy chain comprises a CDR having the sequence given in SEQ ID NO:144 for CDR-H1, a CDR having the sequence given in SEQ ID NO:145 for CDR-H2 and a CDR having the sequence given in SEQ ID NO:146 for CDR-H3, and a light chain wherein the variable domain of the light chain comprises a CDR having the sequence given in SEQ ID NO:141 for CDR-L1, a CDR having the sequence given in in SEQ ID NO:142 for CDR-L2 and a CDR having the sequence given in SEQ ID NO:143 for CDR-L3; and iv) a heavy chain wherein the variable domain of the heavy chain comprises a CDR having the sequence given in SEQ ID NO:164 for CDR-H1, a CDR having the sequence given in SEQ ID NO:165 for CDR-H2 and a CDR having the sequence given in SEQ ID NO:166 for CDR-H3, and a light chain wherein the variable domain of the light chain comprises a CDR having the sequence given in SEQ ID NO:161 for CDR-L1, a CDR having the sequence given in in SEQ ID NO:162 for CDR-L2 and a CDR having the sequence given in SEQ ID NO:163 for CDR-L3.

44. A pharmaceutical composition according to claim 42, wherein a monoclonal antibody which specifically binds TcdB1234 comprises a heavy chain wherein the variable domain of the heavy chain comprises a CDR having the sequence given in SEQ ID NO:124 for CDR-H1, a CDR having the sequence given in SEQ ID NO: 125 for CDR-H2 and a CDR having the sequence given in SEQ ID NO: 126 for CDR-H3, and a light chain wherein the variable domain of the light chain comprises a CDR having the sequence given in SEQ ID NO:121 for CDR-L1, a CDR having the sequence given in in SEQ ID NO:122 for CDR-L2 and a CDR having the sequence given in SEQ ID NO:123 for CDR-L3.

45. A pharmaceutical composition according to claim 44, wherein the monoclonal antibody has a heavy chain comprising the sequence given in SEQ ID NO:129 and a light chain comprising the sequence given in SEQ ID NO:127.

46. A pharmaceutical composition according to claim 42, wherein a monoclonal antibody which specifically binds TcdB1234 comprising a heavy chain wherein the variable domain of the heavy chain comprises a CDR having the sequence given in SEQ ID NO:154 for CDR-H1, a CDR having the sequence given in SEQ ID NO:155 for CDR-H2 and a CDR having the sequence given in SEQ ID NO:156 for CDR-H3, and a light chain wherein the variable domain of the light chain comprises a CDR having the sequence given in SEQ ID NO:151 for CDR-L1, a CDR having the sequence given in in SEQ ID NO:152 for CDR-L2 and a CDR having the sequence given in SEQ ID NO:153 for CDR-L3.

47. A pharmaceutical composition according to claim 46, wherein the monoclonal antibody has a heavy chain comprising the sequence given in SEQ ID NO:159 and a light chain comprising the sequence given in SEQ ID NO:157.

48. A pharmaceutical composition according to claim 42, wherein the monoclonal antibody has a heavy chain comprising the sequence given in SEQ ID NO:149 and a light chain comprising the sequence given in SEQ ID NO:147.

49. A pharmaceutical composition according to claim 42, wherein the monoclonal antibody has a heavy chain comprising the sequence given in SEQ ID NO:169 and a light chain comprising the sequence given in SEQ ID NO:167.

50. A pharmaceutical composition according to claim 42, wherein at least one of said monoclonal antibodies is a neutralizing antibody which is effective against ribotypes 003.

51. A pharmaceutical composition according to claim 42, further comprising two or more antibodies specific to TcdB.

52. A pharmaceutical composition according to claim 42, further comprising one or more antibodies specific to TcdA.

53. A pharmaceutical composition according to claim 52, wherein the antibody which specifically binds TcdA comprises a heavy chain sequence given in SEQ ID NO: 19 and a light chain sequence given in SEQ ID NO: 17.

54. A pharmaceutical composition according to claim 52, wherein the antibody which specifically binds TcdA comprises a heavy chain sequence given in SEQ ID NO: 29 and a light chain sequence given in SEQ ID NO: 27.

55. A pharmaceutical composition according to claim 52, wherein the antibody which specifically binds TcdA comprises a heavy chain sequence given in SEQ ID NO: 49 and a light chain sequence given in SEQ ID NO: 47.

56. A pharmaceutical composition according to claim 52, wherein the antibody which specifically binds TcdA comprises a heavy chain sequence given in SEQ ID NO: 59 and a light chain sequence given in SEQ ID NO: 57.

57. A pharmaceutical composition according to claim 52, wherein the antibody which specifically binds TcdA comprises a heavy chain sequence given in SEQ ID NO: 39 and a light chain sequence given in SEQ ID NO: 37.

58. A pharmaceutical composition according to claim 52, wherein the antibody which specifically binds TcdA comprises a heavy chain sequence given in SEQ ID NO: 9 and a light chain sequence given in SEQ ID NO: 7.

59. A pharmaceutical composition according to claim 52, which further comprises a pharmaceutically acceptable excipient.

60. A method of treatment or prophylaxis of Clostridium difficile infection or complications therefrom comprising administering a pharmaceutical composition as defined in claim 42 to a patient in need thereof.

61. A method according to claim 60, wherein the composition is co-administrated with a compound selected the group comprising metronidazole, vancomycin, clindamycin, fidaxomicin and combinations thereof.