NZ623293B2 - Neutralising antibodies to the major exotoxins tcda and tcdb of clostridium difficile - Google Patents

Abstract

Disclosed is a pharmaceutical composition comprising a monoclonal antibody suitable for reducing the duration and/or severity of diarrhoea or morbidity and/or mortality in a patient with Clostridium difficile infection or at risk of said infection wherein the antibody is specific to: antigen Clostridium difficile toxin A (TcdA) and binds 3 or more times in the range S1827-D2249 or G2205-R2608 of SEQ ID NO: 171 with an EC50 in the range 0.1 to 10 ng/ml when toxin is used at an LD80 or higher. dium difficile toxin A (TcdA) and binds 3 or more times in the range S1827-D2249 or G2205-R2608 of SEQ ID NO: 171 with an EC50 in the range 0.1 to 10 ng/ml when toxin is used at an LD80 or higher.

Description

NEUTRALISING ANTIBODIES TO THE MAJOR EXOTOXINS TCDA AND TCDB OF IDIUM DIFFICILE The present invention relates to antibodies to exotoxins of Closz‘rz‘dz’um dz‘fiicz'le, for example Tch and Tch, pharmaceutical compositions comprising the same, processes of producing said dies and compositions and use of the antibodies and compositions in treatment and/or prophylaxis, in particular treatment or prophylaxis of Clostrz‘dz'um difi‘icz'le infection, pseudomembranous colitis, fiilminant colitis and/or toxic mega colon.

The two major exotoxins Tch and Tch have been established as the major pathogenicity inants of Closz‘rz'dz'um difficz'le 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 ofthe role ofbinary toxin has yet to be ished (3).

Both toxins are - and cyto-toxic, but the balance of evidence suggests that Tch is a more powerfiil enterotoxin than Tch, whilst Tch is typically observed to be ~1000X more cytotoxic than Tch (4). Whilst both toxins are capable of inducing an inflammatory response, Tch appears to aid the migration ofthe more inflammatory Tch deeper into the gut mucosa (5).

In 10:0, a large collection of data ted for over 30 years support a model where both toxins are likely to be important in the human e process. It is probable that Tch initiates early (i.e. before Tch) and rapid (i.e. 1-3 hours) gut damage h 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 Tch to exert its superior molar potency (Tch is typically cited as being IOOOX more cytotoxic than Tch) more rapidly and effectively (i.e. deeper into tissue, alternative cellular targets and damaging ically accessed organs). Either toxin can be effective alone in vitro on human or s cells and s. Either toxin can be effective alone in vivo in animals depending upon other eliciting factors such as ical damage, barrier overload and host specific sensitivities. It is now clear that in hamsters at least either Tch or Tch alone delivered by a Clostridz'um diflicz'le 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 (~95%) of clinical strains are A+B+ hence drugs aimed at treating Closrrz'dz'um dz'flicz‘le infections (CD1) must be capable of neutralising the activities of and clearing both toxins effectively.

CDI is most typically a nosocomial infection of older patients or those with complicating co-morbidities. However, an increase in community acquired ions has been noted. Infection is almost always ated with or induced by use of broad spectrum antibiotics. Healthcare associated costs are estimated to be in excess of $1bn per annum in the US alone. These costs are primarily due to ts having longer hospitals stays. Current ies involve the use of antibiotics such as clindamycin, vancomycin or fidaxomicin which kill the Clostrz'a’ium dz'fiicz'le cells within the gut. Current therapies address the bacterial infection but do not deal with or prevent directly the significant pathogenesis caused by Tch and Tch which are major contributors to CD1 symptoms and mortality.

CDI ms 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 ts receiving current best care. Thus at the present time there is no c therapy available to patients to t the damage and injury caused by C. dz'flz‘cz'le toxins after infection.

Raising an antibody response through vaccination and parenteral stration of polyclonal and onal 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 Tch alone were all that was necessary for tion. However, use of strains fimctionally deleted for Tch or Tch demonstrate that either toxin is capable of causing disease in hamsters, but that both toxins together are more effective (1).

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 ts.

There have been a number of attempts to generate protective Mabs against Tch and Tch. The most ed ofthese in the clinic is a mixture of 2 lgGl Mabs, one against each Tch and Tch originally called CDAl and MDXl388 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 A 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 dies may only be useful for preventing recurrence of infection. Recurrence of infection results in approximately 25% ofpatients. Thus there likely to be a significant patient population in which these antibodies are not ive.

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

Summary of the Present Invention The present invention provides a pharmaceutical ition comprising 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 oea and death rate in humans suffering from Clostridium diflicile infection (CD1).

In one aspect there is provided a pharmaceutical composition comprising a onal antibody suitable for reducing the duration and/or severity of diarrhoea or morbidity and/or ity in a t with Clostridium difiicile infection or at risk of said infection wherein the antibody is specific to: antigen Clostrz‘dium diflicz’le toxin A (Tch) and binds 3 or more times in the range S1827-D2249 or R2608 of SEQ ID NO: 171 with an EC50 in the range 0.1 to 10 ng/ml when toxin is used at an LDgo or higher.

In one ment there is provided a monoclonal antibody specific to antigen Tch or Tch, wherein the antibody has high affinity for the target antigen and is suitable for ng the duration and/or severity of diarrhoea and morbidity in a patient with Closrria’ium difificile infection or at risk of said infection.

In one embodiment there is provided a Mab specific to Tch or Tch, or a population of at least two Mabs at least one of which is specific to Tch and at least one of which is specific to Tch, wherein the EC50 of the or each antibody or the combination of antibodies is ZOOng/ml or less, for example 150ng/ml or less such as lOOng/ml.

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

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

Detailed Description of the Present Invention 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.

Binding affinity may be measured by standard assays such as surface n nce, such as BIAcore. (followed by page 3a) In one embodiment the ECso is less than 75, 70, 60, 65, 55, 50, 45, 40, 35, 30, 25, 20, , 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 dies of the present disclosure have a significant and positive impact on survival of subjects receiving treatment.

As ed herein potency is the ability of the antibody to elicit an appropriate biological response, for example neutralisation of the deleterious toxin s, at a given dose [FOLLOWED BY PAGE 4] WO 38156 or concentration. Examples ofpotency include the percent maximal lisation of toxin activity (extent ofprotection), the lowest relative concentration ofMab to antigen (e.g. ECso), the speed and durability of neutralisation activity.

In cell culture assays lisation might be observed as one or more of the following: prevention ofbinding oftoxin to cells, immunoprecipitation oftoxin from solution, prevention of loss of cell form and shape, prevention of loss of cytoskeletal structures, prevention of loss of cell mono layer tight junctions and trans-epithelial electrical resistance, prevention of cell death, apoptosis and production ofpro-inflammatory cytokines such as TNFOL, IL- 1 [3, IL—6 and MIP let.

In tissue section and explant assays neutralisation may, for example be observed as prevention ofnecrosis and/or tous fluid accumulation.

In in viva assays neutralisation may be ed as one or more ofthe following: prevention of fluid accumulation in ligated ileal loops and prevention of gut tissue necrosis, diarrhoea, pseudo—membrane formation of death of animals, Thus in one embodiment there is provided an antibody (for example an oxin A antibody) comprising a CDR, such as l, 2, 3, 4, 5 or 6 CDRs, selected from: QASQS I SNALA SEQ ID NO: 1 SASSLAS SEQ ID NO: 2 QYTHYSHTSKNP SEQ ID N023 GFTISSYYMS SEQ ID NO: 4 I I SSGGHFTWYANWAKG SEQ ID NO: 5 AYVSGSSFNGYAL SEQ ID NO: 6 In one embodiment sequences 1 to 3 are in a light chain of the dy.

In one embodiment sequences 4 to 6 are in a heavy chain ofthe antibody.

In one embodiment SEQ ID NO: 1 is CDR Ll, SEQ ID NO: 2 is CDR L2 and SEQ ID NO; 3 is CDR L3.

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.

In one embodiment SEQ ID NO: 1 is CDR Ll, SEQ ID NO: 2 is CDR L2, SEQ ID NO; 3 is CDR L3, SEQ ID NO: 4 is CDR Hl, SEQ ID NO: 5 is CDR H2 and SEQ ID NO; 6 is CDR H3.

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: DPVMTQS PSTLSASVGDRVT I TCQASQS I SNALAWYQQKPGKAPKLLIYSAS PSRFK GSGSGTEFTLTISSLQPDDFATYYCQYTHYSHTSKNPFGGGTKVEIK wherein the CDRs are underlined and construct is referred to herein as 922.gl VK (ng).

The polynucleotide sequence encoding SEQ ID NO: 7 is shown in Figure l and SEQ ID NO: 8 therein.

In one ment there is ed 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: EVQLVESGGGLVQPGGSLRLSCAASGFT I S SYYMSWVRQAPGKGLEWIGI I S SGGHFTWYANW _§_RFT I S S DS TTVYLQMNSLRDEDTATYFCARAYVSG S S WGQGTLVTVS wherein the CDRs are underlined and construct is referred to herein as 922.gl VH (gH l) The polynucleotide sequence encoding SEQ ID NO: 9 is shown in Figure 1 and SEQ ID NO: 10 therein.

In one ment the antibody comprises the variable regions shown in SEQ ID NO: 7 and 9.

Thus in one embodiment there is provided an antibody (for example an anti-toxin A antibody) comprising a CDR, such as l, 2, 3, 4, 5 or 6 CDRs, selected from: QASQSISNYLA SEQ ID NO: 11 SASTLAS SEQ ID NO: 12 QYSI—IYGTGVFGA SEQ ID NO: 13 AFSLSNYYMS SEQ ID NO: 14 IISSGSNALKWYASWPKG SEQ ID NO: 15 NYVGSGSYYGMDL SEQ ID NO: 16 In one embodiment sequences 11 to 13 are in a light chain ofthe antibody.

In one embodiment sequences 14 to 16 are in a heavy chain ofthe antibody.

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.

In one embodiment SEQ ID NO: 14 is CDR Hl, SEQ ID NO: 15 is CDR H2 and SEQ ID NO; 16 is CDR H3.

In one embodiment SEQ ID NO: 1 1 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.

In one embodiment there is provided a variable region, such as a light chain variable region with the ing sequence (Antibody 923 oxin A antibody; Light chain Variable region sequence) SEQ ID NO: 17: 2012/052222 DVVMTQSPSSLSASVGDRVTITCQASQS I SNYLAWYQQKPGKVPKLLIYSASTLASGVPSRFK GSGSGTQFTLTI SSLQPEDVATYYCQYSHYGTGVFGAFGGGTKVEIK wherein the CDRs are ined and construct is referred to herein as g1 ng The cleotide sequence encoding SEQ ID NO: 17 is shown in Figure 1 and SEQ ID NO: 18 therein.

In one ment 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: SGGGLVQPGGSLRLSCAASAFSLSNYYMSWVRQAPGKGLEWIGI I S SGSNALKWYAS WPKGRFTI SKDSTTVYLQMNSLRAEDTATYFCARNYVGSGSYYGMDLWGQGTLVTVS wherein the CDRs are underlined and construct is referred to herein as CA923.g1 gH1 The polynucleotide sequence encoding SEQ ID NO: 19 is shown in Figure 2 and SEQ ID NO: 20 therein.

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

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: QASQSISSYFS SEQ ID NO: 21 GASTLAS SEQ ID NO: 22 QCTDYSGIYFGG SEQ ID NO: 23 GFSLSSYYMS SEQ ID NO: 24 STTFTWYASWAKG SEQ ID NO: 25 AYVGSSSYYGFDP SEQ ID NO: 26 In one embodiment sequences 21 to 23 are in a light chain ofthe antibody.

In one embodiment sequences 24 to 26 are in a heavy chain ofthe antibody.

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.

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.

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.

In one ment 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: DVVMTQS PSTLSASVGDRVT ITCQASQS I S SYFSWYQQKPGKAPQLLIYGASTLASGVPSRFK GSGSGTELTLTISSLQPDDFATYYCQCTDYSGIYFGGFGGGTKVEIK wherein the CDRs are underlined and construct is referred to herein as CA993.g1 ng The polynucleotide sequence encoding SEQ ID NO: 27 is shown in Figure 2 and SEQ ID NO: 28 therein.

In one embodiment there is provided a variable region, such as a heavy chain le region with the following sequence ody 993 anti-toxin A antibody heavy chain variable region sequence) SEQ ID NO: 29: EVQLVE SGGGLVQPGGSLKLSCTASGFSLS SYYMSWVRQAPGKGLEWIGI I S SGS S TTF‘I‘WYA IO FT I SKT S TTVYLQMNSLKTEDTATYFCARAYVGS S SYYGFDPWGQGTLVTVS wherein the CDRs are underlined and construct is referred to herein as gl ng The polynucleotide sequence encoding SEQ ID NO: 29 is shown in Figure 2 and SEQ ID NO: 30 therein.

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

In one embodiment there is ed an antibody (for example an oxin A antibody) comprising a CDR, such as 1, 2, 3, 4, 5 or 6 CDRs, selected from: QASQSINNYFS SEQ ID NO: 31 GAANLAS SEQ ID NO: 32 QNNYGVHIYGAA SEQ ID NO: 33 GFSLSNYDMI SEQ ID NO: 34 FINTGGITYYASWAKG SEQ ID NO: 35 VDDYIGAWGAGL SEQ ID NO: 36 In one embodiment sequences 31 to 33 are in a light chain ofthe antibody.

In one embodiment ces 34 to 36 are in a heavy chain ofthe antibody.

In one embodiment SEQ ID NO: 3l is CDR Ll, SEQ ID NO: 32 is CDR L2 and SEQ ID NO; 33 is CDR L3.

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.

In one embodiment SEQ ID NO: 31 is CDR Ll, 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.

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: 2012/052222 S PSTLSASVGDRVT I TCQASQS INNYFSWYQQKPGKAPKLLI YGAANLASGVPSRFK GSGSGTEYTL‘I‘ I S SLQPDDFATYSCQNNYGVHIYGAAFGGGTKVE IK wherein the CDRs are underlined The polynucleotide sequence encoding SEQ ID NO: 37 is shown in Figure 3 and SEQ ID NO: 38 therein.

In one embodiment there is provided a variable region, such as a heavy chain variable region with the following sequence (Antibody 995 oxin A dy heavy chain variable region sequence) SEQ ID NO: 39 EVQLVESGGGLVQPGGSLRLSCTASGFSLSNYDMIWVRQAPGKGLEYIGFINTGGITYYASWA ERFT I SRDS STVYLQMNSLRAEDTATYFCARVDDY IGAWGAGLWGQGTLVTVS wherein the CDRs are underlined The polynucleotide sequence encoding SEQ ID NO: 39 is shown in Figure 3 and SEQ ID NO: 40 therein.

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

In one embodiment there is ed an antibody (for example an anti-toxin A dy) comprising a CDR, such as 1, 2, 3, 4, 5 or 6 CDRs, selected from: QASQSISSYLS SEQ ID NO: 41 RASTLAS SEQ ID NO: 42 LGVYGYSNDDGIA SEQ ID NO: 43 GIDLSSHHMC SEQ ID NO: 44 VIYHFGSTYYANWATG SEQ ID NO: 45 ASIAGYSAFDP SEQ ID NO: 46 In one embodiment sequences 41 to 43 are in a light chain ofthe antibody.

In one embodiment sequences 44 to 46 are in a heavy chain ofthe antibody.

In one embodiment SEQ ID NO: 41 is CDR Ll, SEQ ID NO: 42 is CDR L2 and SEQ ID NO; 43 is CDR L3.

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.

In one embodiment SEQ ID NO: 41 is CDR Ll, 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.

W0 20131‘038156 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: ALVMTQSPSSFSASTGDRVTI TCQASQS I SSYLSWYQQKPGKAPKLLIYRASTLASGVPSRFS GSGSGTEYTLTI SCLQSEDFA‘I‘YYCLGVYGYSNDDGIAFGGGTKVE IK wherein the CDRs are underlined The polynucleotide sequence encoding SEQ ID NO: 47 is shown in Figure 3 and SEQ ID NO: 48 therein.

In one embodiment there is provided a variable region, such as a heavy chain variable region with the ing ce (Antibody 997 anti—toxin A antibody heavy chain variable region sequence) SEQ ID NO: 49: EVQLVE SGGGLVQPGGSLRLSCTVSG I DL S SHHMCWVRQAPGKGLEY I GVIYHFGSTYYANWA ERF'I' I SKDS MNSLRAEDTATYFCARAS IAGYSAFDPWGQGTLVTVS wherein the CDRs are underlined The polynucleotide sequence ng SEQ ID NO: 49 is shown in Figure 4 and SEQ ID NO: 50 therein.

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

In one embodiment there is provided an antibody (for example an oxin A antibody) comprising a CDR, such as l, 2, 3, 4, 5 or 6 CDRs, ed from: QASQSIYSYLA SEQ ID NO: 51 DASTLAS SEQ ID NO: 52 QGNAYTSNSHDNA SEQ ID NO: 53 GIDLSSDAVG SEQ ID NO: 54 I IA‘I‘FDS‘I‘YYASWAKG SEQ ID NO: 55 TGSWYYISGWGSYYYGMDL SEQ ID NO: 56 In one embodiment sequences 51 to 53 are in a light chain ofthe antibody.

In one embodiment sequences 54 to 56 are in a heavy chain ofthe antibody.

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.

In one embodiment SEQ ID NO: 54 is CDR Hl, SEQ ID NO: 55 is CDR H2 and SEQ ID NO; 56 is CDR H3.

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.

In one embodiment there is provided a variable region, such as a light chain variable region with the following sequence (Antibody 1000 oxin A antibody; Light chain Variable region sequence) SEQ ID NO: 57: EIVMTQSPSTLSASVGDRVTITCQASQS IYSYLAWYQQKPGKAPKLLIYDASTLASGVPSRFK GSGSGTEFTLTISSLQPDDFATYYCQGNAYTSNSHDNAFGGGTKVEIK wherein the CDRs are underlined.

The polynucleotide sequence encoding SEQ ID NO: 57 is shown in Figure 4 and SEQ ID NO: 58 therein.

In one embodiment there is provided a variable region, such as a heavy chain variable region with the following ce (Antibody 1000 oxin A antibody heavy chain le region sequence) SEQ ID NO: 59: SGGGLIQPGGSLRLSCTVSGIDLS SDAVGWVRQAPGKGLEYIGI IATFDSTYYASWA ERFT I SKAS STTVYLQMNSLRAEDTATYFCARTGSWYY I SGWGSYYYGMDLWGQGTLVTVS wherein the CDRs are ined.

The polynucleotide sequence encoding SEQ ID NO: 59 is shown in Figure 4 and SEQ ID NO: 60 therein.

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

In one embodiment there is provided an antibody (for example an anti-toxin B antibody) comprising a CDR, such as l, 2, 3, 4, 5 or 6 CDRs, selected from: RASKSVSTLMH SEQ ID NO: 61 LASNLES SEQ ID NO: 62 QQTWNDPWT SEQ ID NO: 63 GFTFSNYGMA SEQ ID NO: 64 SISSSGGSTYYRDSVKG SEQ ID NO: 65 VIRGYVMDA SEQ ID NO: 66 In one embodiment sequences 61 to 63 are in a light chain ofthe antibody.

In one embodiment sequences 64 to 66 are in a heavy chain ofthe antibody.

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.

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.

In one embodiment SEQ ID NO: 61 is CDR Ll, 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.

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: DTVLTQSPATLSLSPGERATLSCRASKSVSTLMHWFQQKPGQAPKLLIYLASNLESGVPARFS GSGSGTDFTLTI SSLE PEDFAVYYCQQTWNDPWTFGGGTKVEIK wherein the CDRs are underlined.

The polynucleotide sequence encoding SEQ ID NO: 67 is shown in Figure 5 and SEQ ID NO: 68 therein.

In one ment there is provided a variable region, such as a heavy chain variable l0 region with the following sequence (Antibody 926 anti-toxin B antibody heavy chain variable region sequence) SEQ ID NO: 69: EVELLESGGGLVQPGGSLRLSCEASGFTFSNYGMAWVRQAPTKGLEWVTSI SSSGGSTYYRDS YERFTI SRDNAKS SLYLQMNSLRAEDTATYYCTTVIRGYVMDAWGQGTLVTVS wherein the CDRs are underlined.

The polynucleotide sequence ng SEQ ID NO: 69 is shown in Figure 5 and SEQ ID NO: 70 therein.

In one embodiment there is provided an antibody (for example an anti—toxin B dy) sing a CDR, such as 1, 2, 3, 4, 5 or 6 CDRs, selected from: RASGSVSTLMH SEQ ID NO: 71 KASNLAS SEQ ID NO: 72 HQSWNSDT SEQ ID NO: 73 GFTFSNYGMA SEQ ID NO: 74 RTTHYRDSVKG SEQ ID NO: 75 I SRSHYFDC SEQ ID NO: 76 In one embodiment sequences 71 to 73 are in a light chain ofthe dy.

In one embodiment sequences 74 to 76 are in a heavy chain ofthe antibody.

In one embodiment SEQ ID NO: 71 is CDR Ll, SEQ ID NO: 72 is CDR L2 and SEQ ID NO: 73 is CDR L3.

In one embodiment SEQ ID NO: 74 is CDR Hl, SEQ ID NO: 75 is CDR H2 and SEQ ID NO: 76 is CDR H3.

In one embodiment SEQ ID NO: 71 is CDR Ll, 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.

W0 20132‘038156 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: DTQMTQSPSTLSASVGDRVTITCRASGSVSTLMHWYQQKPGKAPKLLIYKASNLASGVPSRFS GSGSGTEFTLTISSLQPDDFATYYCHQSWNSDTFGQGTRLEIK wherein the CDRs are ined The polynucleotide sequence encoding SEQ ID NO: 77 is shown in Figure 5 and SEQ ID NO: 78 therein.

In one embodiment there is provided a variable , such as a heavy chain variable region with the following sequence (Antibody 927 oxin B antibody heavy chain variable region sequence) SEQ ID NO: 79: EVQLVE SGGGVVQPGRSLRLSCAASGFTFSNYGMAWVRQAPGKGLEWVAT INYDGRTTHYRDS V_K§RFTI SRDNSKSTLYLQMNSLRAEDTAVYYCTS I SRSHYFDCWGQGTLVTVS wherein the CDRs are underlined.

The cleotide sequence ng SEQ ID NO: 79 is shown in Figure 5 and SEQ ID NO: 80 therein.

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

In one embodiment there is provided an antibody (for example an anti-toxin B dy) comprising a CDR, such as l, 2, 3, 4, 5 or 6 CDRs, selected from: KASKSISNHLA SEQ ID NO: 81 SGSTLQS SEQ ID NO: 82 QQYDEYPYT SEQ ID NO: 83 GFSLQSYTIS SEQ ID NO: 84 AI SGGGSTYYNLPLKS SEQ ID NO: 85 PRWYPRSYFDY SEQ ID NO: 86 In one embodiment sequences 81 to 83 are in a light chain ofthe antibody.

In one embodiment sequences 84 to 86 are in a heavy chain ofthe antibody.

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.

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.

In one embodiment SEQ ID NO: 81 is CDR Ll, 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.

In one embodiment there is provided a variable region, such as a light chain variable region with the following sequence ody 1099 anti-toxin B antibody; Light chain Variable region sequence) SEQ ID NO: 87: DVQLTQSPSFLSASVGDRVTITCKASKS I SNHLAWYQEKPGKANKLLIHSGSTLQSGTPSRFS GSGSGTEFTLTISSLQPEDFATYYCQQYDEYPYTFGQGTRLEIKRT wherein the CDRs are underlined.

In one embodiment the last two amino acids (RT) of SEQ ID NO: 87 are d.

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

In one ment 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: EVQLQESGPGLVKPSETLSLTCTVSGFSLQSYTISWVRQPPGKGLEWIAAISGGGSTYYNLPL ERVT I SRDTSKSQVSLKLS SVTAADTAVYYCTRPRWYPRSYFDYWGRGTLVTVS wherein the CDRs are underlined The polynucleotide sequence encoding SEQ ID NO: 89 is shown in Figure 6 and SEQ ID NO: 90 therein.

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

In one embodiment there is provided an antibody (for example an anti-toxin B antibody) comprising a CDR, such as l, 2, 3, 4, 5 or 6 CDRS, ed from: RASQRISTSIH SEQ ID NO: 91 YASQSIS SEQ ID NO: 92 oosyssmr SEQ ID NO: 93 GFTFSDSYMA SEQ ID NO: 94 SISYGGTIIQYGDSVKG SEQ ID NO: 95 RQGTYARYLDF SEQ ID NO: 96 In one embodiment sequences 91 to93 are in a light chain ofthe antibody.

In one embodiment sequences 94 to 96 are in a heavy chain ofthe antibody.

In one ment SEQ ID NO: 91 is CDR Ll, SEQ ID NO: 92 is CDR L2 and SEQ ID NO; 93 is CDR L3.

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. 2012/052222 In one ment 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.

In one embodiment there is provided a le 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: NIVLTQSPATLSLSPGERATLSCRASQRI STS IHWYQQKPGQAPRLLIKYASQS I SGI PARFS GSGSGTDFTLTISSLE PEDFAVYYCQQSYSSLYTFGQGTKLEIK wherein the CDRs are underlined The polynucleotide sequence encoding SEQ ID NO: 97 is shown in Figure 6 and SEQ ID NO: 98 therein.

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: EVQLVESGGGLVQPGGSLRLSCAVSGFTFSDSYMAWVRQAPGKGLEWIAS I SYGGTI IQYGDS YERFT I SRDNAKS SLYLQMNSLRAEDTAVYYCARRQGTYARYLDFWGQGTLVTVS wherein the CDRs are underlined.

The polynucleotide sequence encoding SEQ ID NO: 99 is shown in Figure 7 and SEQ ID NO: 100 therein.

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

In one embodiment there is ed an antibody (for example an anti-toxin B antibody) comprising a CDR, such as 1, 2, 3, 4, 5 or 6 CDRs, selected from: RASESVSTLLH SEQ ID NO: 101 KASNLAS SEQ ID NO: 102 HQSWNSPPT SEQ ID NO: 103 GFTFSNYGMA SEQ ID NO: 104 I TTHYRDSVKG SEQ ID NO: 105 YGRSHYFDY SEQ ID NO: 106 In one embodiment sequences 101 to 103 are in a light chain ofthe antibody.

In one embodiment ces 104 to 106 are in a heavy chain ofthe antibody.

In one ment SEQ ID NO: 101 is CDR Ll, SEQ ID NO: 102 is CDR L2 and SEQ ID NO: 103 is CDR L3.

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.

W0 2013I038156 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.

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 dy; Light chain Variable region sequence) SEQ ID NO: 107: ATQMTQSPSSLSASVGDRVTITCRASESVSTLLHWYQQKPGKAPKLLIYKASNLASGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCHQSWNSPPTFGQGTKLEIK wherein the CDRs are underlined.

The polynucleotide sequence encoding SEQ ID NO: 107 is shown in Figure 7 and SEQ ID NO: 108 therein.

In one embodiment there is provided a le region, such as a heavy chain le region with the following ce (Antibody 1114 anti-toxin B antibody heavy chain variable region sequence) SEQ ID NO: 109: EVQLVE SGGGLVQPGGSLRLSCAASGFTFSNYGMAWVRQAPGKGLEWVAI INYDAB '1‘THYRDS YERFT I SRDNAKS SLYLQMNSLRAEDTAVYYCTRYGRSHYFDYWGQGTLVTVS wherein the CDRs are underlined.

The polynucleotide sequence encoding SEQ ID NO: 109 is shown in Figure 7 and SEQ ID NO: 110 n. 2O In one ment an dy according to the invention comprises variable regions shown in SEQ ID NO: 107 and SEQ ID NO: 109.

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: RASESVSTLLH SEQ ID NO: 111 KASNLAS SEQ ID NO: 112 HQSWNSPPT SEQ ID NO: 113 GFTFSNYGMA SEQ ID NO: 114 IINYDASTTHYRDSVK SEQ ID NO: 115 YGRSHYFDY SEQ ID NO: 116 In one embodiment sequences 111 to 113 are in a light chain ofthe antibody.

In one embodiment sequences 114 to 116 are in a heavy chain ofthe antibody.

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.

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.

W0 20132‘038156 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.

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: SPSSLSASVGDRVTITCRASESVSTLLHWYQQKPGKAPKLLIYKASNLASGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCHQSWNSPPTFGQGTKLEIK wherein the CDRs are underlined.

The polynucleotide sequence encoding SEQ ID NO: 117 is shown in Figure 8 and SEQ ID NO: 118 n.

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: EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMAWVRQAPGKGLEWVAI INYDASTTHYRDS EGRFT I SRDNAKS SLYLQMNSLRAEDTAVYYCTRYGRSHYFDYWGQGTLVTVS wherein the CDRs are underlined.

The polynucleotide sequence encoding SEQ ID NO: 119 is shown in Figure 8 and SEQ ID NO: 120 therein.

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

In one embodiment there is provided an dy (for example an oxin B antibody) sing a CDR, such as 1, 2, 3, 4, 5 or 6 CDRs, selected from: KASQNIYMYLN SEQ ID NO: 121 NTNKLHT SEQ ID NO: 122 LQHKSFPYT SEQ ID NO: 123 SFMA SEQ ID NO: 124 SISYEGDKTYYGDSVKG SEQ ID NO: 125 LTITTSGDS SEQ ID NO: 126 3O In one embodiment sequences 121 to 123 are in a light chain ofthe antibody.

In one embodiment sequences 124 to 126 are in a heavy chain ofthe dy.

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.

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.

In one embodiment SEQ ID NO: 121 is CDR Ll, 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.

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: DIQMTQSPSSLSASVGDRVTI TCKASQNIYMYLNWYQQKPGKAPKRLIYNTNKLHTGVPSRFS GSGSGTEYTLTISSLQPEDFATYYCLQHKSFPYTFGQGTKLEIK wherein the CDRs are ined.

The polynucleotide sequence encoding SEQ ID NO: 127 is shown in Figure 8 and SEQ ID NO: 128 therein.

In one embodiment there is provided a variable region, such as a heavy chain variable region with the ing sequence (Antibody 1125 anti-toxin B antibody heavy chain variable region sequence) SEQ ID NO: 129: EVQLVESGGGLVQPGGSLRLSCAASGFTFRDSFMAWVRQAPGKGLEWVAS I SYEGDKTYYGDS ERFT I SRDNAKNSLYLQMNSLRAEDTAVYYCARLT I TT SGDSWGQGTMVTVS S wherein the CDRs are ined.

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

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

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

In one embodiment there is provided antibody (for e an anti—toxin B dy) comprising a CDR, such as 1, 2, 3, 4, 5 or 6 CDRs, selected fiom: KASQHVGTNVD SEQ ID NO: 131 GAS IRYT SEQ ID NO: 132 LQYNYNPYT SEQ ID NO: 133 GFIFSNFGMS SEQ ID NO: 134 SISPSGGNAYYRDSVKG SEQ ID NO: 135 RAYSSPFAF SEQ ID NO: 136 In one embodiment sequences 131 to 133 are in a light chain ofthe dy.

In one embodiment sequences 134 to 136 are in a heavy chain ofthe antibody.

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.

In one embodiment SEQ ID NO: 134 is CDR Hl, SEQ ID NO: 135 is CDR H2 and SEQ ID NO: 136 is CDR H3.

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.

In one embodiment there is provided a variable , such as alight chain variable region with the following sequence (Antibody 1129 anti-toxin B antibody; Light chain le region sequence) SEQ ID NO: 137: DTQMTQS PS GDRVT I 'I'CKASQHVGTNVDWYQQKPGKVPKLL IYGAS IRYTGVPDRFT GSGSGTDFTLTISSLQPEDVATYYCLQYNYNPYTFGQGTKLEIK n the CDRs are underlined.

The polynucleotide sequence encoding SEQ ID NO: 137 is shown in Figure 8 and SEQ ID NO: 138 therein.

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: EVQLVESGGGVVQPGRSLRLSCATSGFI FSNFGMSWVRQAPGKGLEWVAS I S PSGGNAYYRDS mRFTISRDNSKTTLYLQMNSLRAEDTAVYYCTRRAY S S PFAFWGQGTLVTVS S wherein the CDRs are underlined.

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

The polynucleotide sequence ng SEQ ID NO: 139 is shown in Figure 8 and SEQ ID NO: 140 therein. In one ment the codon AGC encoding the last amino acid S is omitted.

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

In one embodiment there is provided an antibody (for e an anti-toxin B antibody) comprising a CDR, such as l, 2, 3, 4, 5 or 6 CDRs, selected from: KASKSISNHLA SEQ ID NO: 141 SGSTLQP SEQ ID NO: 142 QQYDEYPYT SEQ ID NO: 143 GFSLNSYTIT SEQ ID NO: 144 AISGGGSTYFNSALKS SEQ ID NO: 145 PRWYPRSYFDY SEQ ID NO: 146 In one embodiment sequences 141 to 143 are in a light chain ofthe antibody.

In one embodiment sequences 144 to 146 are in a heavy chain ofthe antibody.

W0 2013(038156 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.

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.

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.

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): DVQLTQSPSFLSASVGDRVTITCKASKS ISNHLAWYQEKPGKANKLLIHSGSTLQPGT PSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYDEYPYTFGQGTRLEIK SEQ ID NO: 147 wherein the CDRs are underlined.

The polynucleotide sequence encoding SEQ ID NO: 147 is shown in Figure 9 and SEQ ID NO: 148 n.

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 le region sequence) SEQ ID NO: 149: EVQLQESGPGLVKPSETLSLTCTVSGFSLNSYTITWVRQPPGKGLEWIAAISGGGSTYFNSAL K_SRVTI SRDTSKSQVSLKLSSVTAADTAVYYCTRPRWYPRSYFDYWGRGTLVTVS n the CDRS are underlined The polynucleotide sequence encoding SEQ ID NO: 149 is shown in Figure 9 and SEQ ID NO: 150 therein.

In one ment an antibody ing to the invention comprises variable regions shown in SEQ ID NO 147: and SEQ ID NO: 149.

In one embodiment there is provided dy (for example an oxin B antibody) comprising a CDR, such as 1, 2, 3, 4, 5 or 6 CDRs, selected fiom: KASQNVGNNVA SEQ ID NO: 151 YASNRFT SEQ ID NO: 152 QRVYQSTWT SEQ ID NO: 153 GFSLTSYYVH SEQ ID NO: 154 CIRTGGNTEYQSEFKS SEQ ID NO: 155 GNYGFAY SEQ ID NO: 156 In one embodiment sequences 151 to 153 are in a light chain ofthe antibody.

W0 38156 In one embodiment sequences 154 to 156 are in a heavy chain ofthe antibody.

In one embodiment SEQ ID NO: 151 is CDR Ll, SEQ ID NO: 152 is CDR L2 and SEQ ID NO: 153 is CDR L3.

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.

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.

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: AI QM'I‘QS PS GDRVT I TCKASQNVGNNVAWYQHKPGKAPKLL I YYASNRFTGVPSRFT GGGYGTDFTLTI SSLQPEDFATYYCQRVYQSTWTFGQGTKVEIK wherein the CDRs are underlined.

The polynucleotide sequence encoding SEQ ID NO: 157 is shown in Figure 9 and SEQ ID NO: 158 therein.

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: EVQLQESGPGLVKPSETLSLTCTVSGFSLTSYYVHWVRQPPGKGLEWMGCIRTGGNTEYQSEF ERV‘I' I SRDT SKNQVSLKLS SVTAADTAVYYCARGNYGFAYWGQGTLVTVS n the CDRS are underlined.

The polynucleotide sequence encoding SEQ ID NO: 159 is shown in Figure 9 and SEQ ID NO: 160 therein.

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

In one embodiment there is provided an antibody (for example an oxin B antibody) comprising a CDR, such as l, 2, 3, 4, 5 or 6 CDRS, selected from: KASQNINKYLD SEQ ID NO: 161 3O NIQSLHT SEQ ID NO: 162 FQHNSGW SEQ ID NO: 163 AAMF SEQ ID NO: 164 RISTKSNNFATYYPDSVKG SEQ ID NO: 165 PAYYYDG’I‘VPFAY SEQ ID NO: 166 In one embodiment ces 161 to 163 are in a light chain ofthe antibody.

In one embodiment ces 164 to 166 are in a heavy chain ofthe antibody.

In one embodiment SEQ ID NO: 161 is CDR Ll, SEQ ID NO: 162 is CDR L2 and SEQ ID NO: 163 is CDR L3.

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.

In one embodiment SEQ ID NO: 161 is CDR Ll, 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.

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: DIQMTQSPSSLSASVGDRVTITCKASQNINKYLDWYQQKPGKVPKLLIYNIQSLHTGI PSRFS GSGSGTDFTLTISSLQPEDVATYYCFQHNSGWTFGQGTRLEIK wherein the CDRs are underlined.

The polynucleotide sequence encoding SEQ ID NO: 167 is shown in Figure 10 and SEQ ID NO: 168 therein.

In one embodiment there is provided a variable region, such as a heavy chain le region with the following sequence (Antibody 1153 anti-toxin B antibody heavy chain variable region sequence) SEQ ID NO: 169: EVQLVE SGGGLVQPGGSLKLSCAASGFTFTQAAMFWVRQASGKGLEGIARI STKSNNFATYYP DSVKGRFT I SRDDSKNTVYLQMNSLKTEDTAVYYCTAPAYYYDGTVPFAYWGQGTLVTVS wherein the CDRs are underlined.

The polynucleotide sequence encoding SEQ ID NO: 169 is shown in Figure 10 and SEQ ID NO: 170 n.

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

In one embodiment there is provided antibody comprising 6 CDRs independently selected from SEQ ID NOS 1,2, 3, 4,5,6,ll,12,13,l4,15,16, 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, ,102,103,104,105,106,111,112, 113, 114,115, 116, 2,123,124,125,126, 131, 132, 133,134,135,136, 141,142,143, 144,145,146,151,152,153,154,155,156,161,162,l63,164,165 and 166.

In one embodiment there is provided an anti-Tch dy 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.

In one embodiment there is provided an anti—Tch 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,l63,164,165 and 166.

In one ment there is provided an antibody which comprises two variable regions ndently 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.

In one embodiment there is provided an antibody which comprises two variable regions independently ed fiom SEQ ID NOS: 7, 9, 17, 19, 27, 29, 37, 39, 47, 49, 57 and 59.

In one embodiment there is provided an dy which comprises two variable regions independently selected from SEQ ID N08: 67, 69, 77, 79, 87, 89, 97, 99, 107, 109, 117, 119, 127, 129, 137, 139, 147, 149, 157 and 159.

In one ment the antibodies according to the invention are humanized.

In one embodiment the antibody or antibodies are directed to the C terminal “cell binding” portion ofthe Tch and/or Tch toxin.

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

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

The inventors have established by using internal comparisons n antibodies discovered in this application and by comparison against antibodies well described in the art (Babcock et a1. 2006; Lowy et a1., 2010) that some antibodies have the desirable characteristic ofmaintaining effective neutralization (for e low ECso 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.

Effective toxin concentrations can be defined as a ‘lethal dose’ (LD) in titration studies in the absence of lizing antibodies. Neutralisation assays are typically ted at an 3O ID of50% ofcomplete cell killing (226. an LD50) but may be more rigorously conducted at an LDgo.

Assays may also be performed under considerably more challenging conditions such as LD90, LD95 and/or LDmax (LDmax 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. dz'fiicile growth in the bowel is rampant and diarrhea and other symptoms lead one to esise that toxin concentrations are at their highest. Antibodies which effectively neutralize ng 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 dy or antibodies of the present disclosure have useful, for example low ECso values and/or high % protection from cell death for one or more the LDgo, LD90, LD95 and/or LDmaX. In one embodiment the EC50 in the one or more ofthe latter situations is lSng/ml or less, for example l or less, such as Sng/ml or less, in particular lng/ml or less. In one embodiment the % protection from cell death is >90%, or >75% or >50%.

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.

The harmful effect n may, for example be measured in a suitable in vitro assay.

In one embodiment the lization 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 ofthe antibody is ined in the ce of high levels of toxin.

Toxin A is used interchangeably with Tch.

Toxin B is used interchangeably with Tch.

In one embodiment an antibody according to the invention is a monoclonal antibody or binding fiagment thereof.

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

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

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

In one embodiment a monoclonal antibody according to the invention is capable of neutralising Tch with very high potency and y and high y and high valency of binding. y ofbinding as employed herein refers to the ability for a monoclonal antibody to bind to an antigen multiple times. High valency ofbinding hence results in high levels of tion of n with antibodies and / or high levels of cross-linking oftoxin molecules, which is thought to be advantageous.

Anti—Tch Mabs according to the present disclosure may be suitable for neutralising the early effects ofTch, for example on cells such as loss of tight junctions.

Tight junction as employed herein is intended to refer to impermeable zone of connection between cells within a mono layer or anatomical tissue structure. Fluid loss does not occur when tight ons retain their structural and functional ity. 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 ofTch and Tch (25) and results in loss of fluid containing serum, immunoglobulin and ions (26, 3). Loss oftight junctions is thought to be a first step on the onset of oea in humans.

The TEER assay system, can be used to measure the loss oftight junction in vz'z‘ro.

TEER is an m 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 ing 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.

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 t context. The resistance of the membrane changes when the membrane has been compromised.

Generally the antibody will be pre-incubated with the toxin before addition to the TEER system to establish ifthe dy 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 ished that the 4 hour time point is particularly discriminating for therapeutically useful antibodies.

The concentration n employed in the TEER assay is generally in the range 100- 200ng/ml, most preferably l25ng/m1 The concentration of antibody (for example IgG 1) employed in the TEER assay is generally in the range of 4 to 2000ng/ml, for example 50 to lOOOng/ml, such as 100 to SOOng/ml.

In one embodiment the EC50 ofthe antibody in the TEER assay employed in said condition is at least 200ng/ml, for e less than lOOng/ml, such as about 60-80ng/ml.

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

In one aspect there is ed a method of screening an antibody in a TEER assay for the ability to slow or t loss of tight junctions. In one embodiment the antibody or antibodies screened are anti-Tch antibodies. In one embodiment the antibody or antibodies screened are anti—Tch antibodies. In one embodiment the antibody or antibodies screened are a combination of anti-Tch and anti-Tch antibodies. In one embodiment the method comprises the step of identifying an appropriate dy or antibodies and expressing suitable quantities of same. In one embodiment the method comprises the r step of formulating said antibody or antibodies in a pharmaceutical ation. In one embodiment the method comprises the further step of administering said antibody or antibodies or said formulation to a patient in need thereof.

In one embodiment multiple antibodies ofthe disclosure may be capable ofbinding to the target toxin (Tch or Tch), which may aid immune clearance ofthe toxin.

Multiple antibodies as employed herein is ed 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.

In one embodiment the antibodies according to the invention are specific to the target antigen, for e specific to an epitope in the target antigen.

In one embodiment the antibodies ofthe ion 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 e 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 e multiple times in ent locations in the n.

In one embodiment the antibody binds to the given antigen le times, for example 2 to 20 times such as 3, 4, 5, 6, 7, 8, 9, 10, ll, 12, l3, I4, 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 lisation 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 tion with 3 or more Fc fragments is important in ring rapid clearance ofthe toxin (24) primarily via the liver and spleen (27, 28).

In one embodiment the anti-Tch antibody binds 3 or more times, for example 3 to 16 times.

In one embodiment the anti-Tch antibody binds 12 times.

In one embodiment the anti-Tch antibody binds 2 times.

In one embodiment an anti-Tch antibody binds in the catalytic-terminal cell binding domain ofTch.

In one embodiment the anti-Ted B antibody binds 2 or more times, for example 2 times.

W0 2013i038156 In one embodiment an anti—Tch antibody binds in the catalytic—terminal cell binding domain ofTch.

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 ofthe antibody binds a epitope in a different toxin molecule, thereby forming a sort ofimmune complex. The formation of the latter may also tate activation of the immune system to clear the relate toxin and thereby minimise the deleterious in vivo effects ofthe same.

In one embodiment an innate immune response, such as complement is ted.

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

In one embodiment dies against Tch may have an EC50 in the range of 0.1 — lOOng/ml, such as l to l and a maximal inhibition in the range of % at toxin concentrations of 5, for example against toxins fiom strains ofribotypes 003, 027 and 078.

In one embodiment antibodies against Tch may have an EC50 in the range of 0.1 — 100ng/ml, such as l to 10ng/ml and a maximal tion in the range of 60- 100%, 70— 100%, 80- 100% or 90-100% at toxin concentrations of LDgo_95, for example against toxins from strains of pes 003, 027 and 078.

In one embodiment dies against Tch may have EC50 in the range of 0.1 — lOOng/ml, such as 1 to lOng/ml and a maximal inhibition in the range of 50— 100% at toxin concentrations of LD30_95, for example against toxins from strains ofribotype 003.

In one embodiment antibodies against Tch may have EC50 in the range of 0.1 — lOOng/ml, such as 1 to 10ng/ml and a maximal inhibition in the range of60- 100%, 70-100%, 80~100% or % at toxin concentrations of LD80_95, for example against toxins from strains of ribotype 003.

In one embodiment there are provided ations of antibodies according to the invention, for example combinations of antibodies specific to Tch, combinations of antibodies specific to Tch or combinations of antibodies to specific to Tch and antibodies specific to Tch.

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

W0 20131038156 2012/052222 ations of antibodies specific to Tch will lly refer to combinations of antibodies directed to different epitopes on the target antigen Tch, or at least with different g properties.

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.

In one embodiment there is provided a combination of one anti-Tch antibody and two anti-Tch, for example wherein the anti—Tch antibody is 997 and where the anti-Tch antibodies are 1125 and 1151 In particular there is ed a combination ofone anti-Tch 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-Tch 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: l59 and light variable region shown in SEQ ID NO: 157.

Distinct antibodies as employed herein is intended to refer to antibodies with ent amino acid sequences, which may bind the same e or different es on the target antigen.

Also provided by the present invention is a specific region or e ofTch 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.

Also provided by the present invention is a specific region or epitope ofTch which is bound by an antibody provided by the t 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.

This specific region or epitope ofthe Tch or Tch toxins can be identified by any suitable epitope mapping method known in the art in combination with any one of the antibodies ed by the present invention. Examples of such methods include screening peptides of 3O varying lengths d 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 ofthe epitope recognised by the antibody. The peptides may be produced synthetically or by proteolytic digestion ofthe 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 dy of the present invention.

WO 38156 2012/052222 Once fied, the epitopic fragment which binds an antibody of the present invention can be used, ifrequired, as an imrnunogen to obtain additional antagonistic dies which bind the same epitope.

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 Tch or Tch, which cross-blocks the binding of any one ofthe antibodies described above to Tch or Tch 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 e 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 blocking neutralising antibody of this aspect ofthe ion does not bind to the same epitope as an dy ofthe present invention or an epitope that borders and/or overlaps with said epitope. blocking antibodies can be identified using any suitable method in the art, for example by using competition ELISA or BIAcore assays where binding ofthe cross blocking antibody to Tch or Tch prevents the binding of an antibody ofthe present invention or vice versa.

In one embodiment there is provided a method of generating an anti—Tch or anti—Tch dy, in particular a neutralizing antibody and/or an antibody which cross—blocks the binding of an antibody described herein, said method sing the steps of immunizing a host with a suitable n, 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 e 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 able composition.

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.

In one embodiment the antibodies according to the invention have an affinity to the target antigen of 10nM or less, for example lnM or less such as 900pM, in particular SOOpM, 700pM, 600pM orSOOpM, such as 60pM.

In one embodiment the affinity is for Tch or Tch or a fragment thereof. In one example the fragment is Tch123 corresponding to residues S 1827-D2249 of Tch. In one example the fragment is Tch456 corresponding to residues G2205-R2608. In one embodiment the t is Tch 1234 corresponding to residues S 1833—E2366 ofTch.

In one embodiment antibodies according to the invention or a combination thereof have an ECso of200ng/ml or less, for example lSOng/ml or less such as 100ng/ml or less, such as in the range 0.1 to lOng/ml.

The individual component antibodies of mixtures are not required to have an EC50 in said range provided that when they are used in combination with one or more antibodies the combination has an EC50 in said range. ageously, the antibodies of the invention are stable, for e are thermally stable at temperatures above 50°C such as 60 or 70°C.

The antibodies and combinations according to the t invention also have one or more ofthe following advantageous properties: slow off rate, high affinity, high potency, the ability to bind multiple times to the target n, to neutralise the toxin by a mechanism which reduces the loss of able 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).

The residues in dy 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 ofImmunological 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.

The Kabat residue designations do not always correspond directly with the linear numbering ofthe 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 ning of, or insertion into, a structural component, whether framework or complementarity determining region (CDR), of the basic variable domain structure. The correct Kabat ing of residues may be determined for a given antibody by alignment of residues ofhomology in the sequence ofthe antibody with a ard” Kabat numbered sequence.

The CDRs ofthe heavy chain variable domain are located at residues 31-35 (CDR—Hl), residues 50-65 (CDR-HZ) and residues 95- 102 (CDR—H3) according to the Kabat numbering system. r, according to Chothia (Chothia, C. and Lesk, AM. J. Mol. Biol, 196, 901- 917 (1987)), the loop equivalent to CDR—Hl extends from residue 26 to residue 32. Thus unless indicated ise ‘CDR—Hl’ as employed herein is intended to refer to residues 26 to W0 2013l038156 , as described by a ation of the Kabat numbering system and a’s topological loop definition.

The CDRs ofthe light chain le domain are located at residues 24-34 (CDR—Ll), residues 50-56 (CDR-LZ) and residues 89-97 (CDR-L3) according to the Kabat numbering system.

Antibodies for use in the present invention may be obtained using any le method known in the art. The toxin A and/or toxin B polypeptide/protein including fusion proteins, for example toxin-Fe 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 tive thereof.

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” es es, polypeptides and proteins. These are used interchangeably unless otherwise specified. The ce for Tch from ribotype 027 is given in SEQ ID NO: 171 (Uniprot accession number C9YJ37) and the sequence for Tch from ribotype 027 is given is SEQ ID NO: 172 (Uniprot accession number C9YJ35).

The antigen polypeptide may in some ces be part of a larger protein such as a fusion n for example fused to an affinity tag.

Antibodies generated against the antigen polypeptide may be obtained, where sation of an animal is necessary, by administering the polypeptides to an animal, preferably a non—human animal, using well-known and routine ols, 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.

Monoclonal antibodies may be prepared by any method known in the art such as the hybridoma technique (Kohler & Milstein, l975, 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, pp77-96, Alan R Liss, Inc., 1985).

Antibodies for use in the invention may also be generated using single lymphocyte antibody methods by g and expressing immunoglobulin variable region cDNAs generated from single lymphocytes ed 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.

Humanised antibodies (which include CDR-grafted antibodies) are antibody molecules having one or more complementarity determining regions (CDRs) from a non—human s and a framework region fi'om a human immunoglobulin molecule (see, e.g. US 5,585,089; W09 1/09967). It will be appreciated that it may only be necessary to transfer the city determining residues ofthe CDRs rather than the entire CDR (see for example, Kashmiri et al., 2005, Methods, 36, 25-34). Humanised antibodies may ally further comprise one or more framework residues derived from the non-human species from which the CDRs were derived.

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) grafied into a heavy and/or light chain variable region framework of an acceptor dy (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 ofthe specificity determining residues from any one ofthe CDRs described herein above are transferred to the human antibody ork (see for example, Kashmiri et al., 2005, Methods, 36, 25-34). In one embodiment only the specificity ining residues from one or more of the CDRs described herein above are transferred to the human dy framework. In another embodiment only the specificity ining residues from each of the CDRs described herein above are transferred to the human antibody ork.

When the CDRs or specificity determining residues are grafted, any riate acceptor variable region framework sequence may be used having regard to the class/type of the donor antibody from which the CDRS are d, ing mouse, e and human framework regions. ly, 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.

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.

Examples ofhuman frameworks which can be used in the t 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.rnrc-cpe.cam.ac.uk/ In a humanised antibody of the present invention, the acceptor heavy and light chains do not necessarily need to be d from the same antibody and may, if desired, comprise composite chains having framework regions derived from different .

Also, in a humanised antibody of the present invention, the framework s need not have exactly the same sequence as those ofthe acceptor dy. For instance, unusual residues may be changed to more frequently-occurring residues for that acceptor chain class or type. Alternatively, selected es in the acceptor framework regions may be changed so that they correspond to the residue found at the same position in the donor dy (see Reichmann et a1., 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. lly the antibody sequences disclosed in the present specification are sed.

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.

"Identity", as used herein, indicates that at any ular position in the aligned sequences, the amino acid residue is cal 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 d to: - alanine, tyrosine and tryptophan (amino acids having aromatic side chains); — lysine, arginine and histidine (amino acids having basic side chains); - aspartate and glutamate (amino acids having acidic side chains); — asparagine and glutamine (amino acids having amide side chains); and — ne and methionine (amino acids having r-containing side chains).

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 ts, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A.M., and , 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 BLASTTM software available from NCBI (Altschul, S.F. et a1., 1990, J. Mol. Biol. 3—410; Gish, W. & States, DJ. 1993, Nature Genet. 31266-272. Madden, T.L. et a1., PCT/GBZOl2/052222 1996, Meth. Enzymol. 1-141; Altschul, S.F. et al., 1997, Nucleic Acids Res. 25:3389— 3402; Zhang, J. & Madden, TL. 1997, Genome Res. 7:649-656,).

The antibody molecules ofthe 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 dies (e.g. VH or VL or VHH), scFv, bi, tri or 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):l 126-1 136; Adair and Lawson, 2005, Drug Design Reviews - Online 2(3), 209-217). The methods for creating and manufacturing these antibody nts 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 t invention include the Fab and Fab’ fragments described in International patent applications WO2005/003169, WO2005/003 170 and W02005/003l71. valent antibodies may comprise multiple specificities e.g bispecific or may be monospecific (see for example WO 92/22853 and W005/113605). Bispecific and multispecific antibody variants are especially considered in this example since the aim is to lise two independent target proteins: Tch and Tch. Variable regions from antibodies disclosed herein may be configured in such a way as to produce a single antibody variant which is e of g to and neutralising Tch and Tch.

In one embodiment the antibody according to the present disclosure is provided as Tch or Tch binding antibody fusion protein which comprises an immunoglobulin , 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.

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 e as described in WO2010/035012.

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

In one embodiment the alent fusion protein according to the invention binds Tch and Tch.

In one embodiment the multivalent fusion protein according to the invention binds Tch in multiple ons, for e has distinct g regions specific for two different epitopes.

The constant region domains of the antibody molecule of the t invention, if t, 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 ons are required.

Alternatively, IgG2 and IgG4 isotypes may be used when the antibody le 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 (I), 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 ofien 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, RJ. Journal of Chromatography 705: 129—134, 1995).

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

Biological molecules, such as dies or fragments, contain acidic and/or basic functional groups, thereby giving the molecule a net positive or negative charge. The amount of l “observed” charge will depend on the absolute amino acid sequence ofthe entity, the local nment of the charged groups in the 3D structure and the environmental conditions ofthe molecule. The isoelectric point (pI) is the pH at which a particular molecule or solvent accessible surface thereof carries no net electrical . In one e, the dy and fragments ofthe 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 cation teristics.

Thus in one aspect the invention provides a humanised antibody engineered to have an isoelectric point different to that of the originally fied antibody from which it is derived.

The antibody may, for example be ered by replacing an amino acid residue such as replacing an acidic amino acid residue with one or more basic amino acid residues. atively, 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 pl, as required. It is important that when manipulating the p1 care must be taken to retain the desirable ty of the dy or fragment. Thus in one embodiment the engineered antibody or fragment has the same or substantially the same ty as the “unmodified” antibody or fragment. ms 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 ofthe antibody or fragment.

It will be appreciated that the affinity of antibodies provided by the t invention may be altered using any suitable method known in the art. The affinity ofthe antibodies or variants thereofmay be ed using any suitable method known in the art, including BIAcore, using an appropriate isolated natural or recombinant protein or a le fusion protein/polypeptide.

The present invention therefore also relates to variants of the antibody molecules of the present invention, which have an improved affinity for Tch or Tch, as riate. 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, l992), 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 a1. (supra) discusses these methods of affinity maturation.

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

If desired an antibody for use in the present invention may be ated 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 or molecule, this may be ed 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 les 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, 62:1 19-58 and Dubowchik et al., 1999, Pharmacology and Therapeutics, 83, 67-123). ular chemical procedures include, for example, those described in WO 93/06231, WO 92/22583, WO 95, WO 89/01476 and WOO3031581. 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 33 and EP0392745.

The term effector molecule as used herein includes, for e, 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 nts thereof, radionuclides, ularly radioiodide, radioisotopes, chelated metals, nanoparticles and reporter groups such as fluorescent nds or compounds which may be detected by NMR or ESR spectroscopy.

Other effector les may include chelated radionuclides such as 1 1 Mn and 90Y, Lul77, Bismuth213, Californium252, Iridiuml92 and Tungsten188/Rhenium188; or drugs such as but not limited to, hosphocholines, topoisomerase I inhibitors, taxoids and suramin.

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 e, but are not limited to, immunoglobulins, toxins such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin, a n such as insulin, tumour necrosis factor, ct-interferon, B—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, eukin—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.

Other effector molecules may include detectable substances useful for example in diagnosis. Examples of detectable substances include various enzymes, etic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive nuclides, positron emitting metals (for use in positron emission aphy), and nonradioactive gnetic metal ions. See generally US. Patent 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 avidin, avidin and biotin; le fluorescent als include umbelliferone, fluorescein, fluorescein isothiocyanate, ine, dichlorotriazinylamine W0 2013I038156 fluorescein, dansyl chloride and phycoerythrin; suitable luminescent materials include luminol; le bioluminescent materials include luciferase, luciferin, and aequorin; and suitable radioactive nuclides include 1251, 1311, lllIn and 99Tc.

In another example the effector molecule may increase the half-life of the antibody in vivo, and/or reduce immunogenicity ofthe antibody and/or enhance the delivery of an antibody across an epithelial r to the immune system. Examples of le effector molecules of this type include polymers, albumin, albumin binding ns or albumin binding nds such as those described in W005/117984.

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

Specific optional substituents which may be present on the above-mentioned synthetic polymers include one or more y, methyl or methoxy groups. c 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.

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

“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 h a linker segment to the polymer. It will be appreciated that the e of such a group will in some instances form part of the product as the linking group between the dy fragment and the polymer.

The size of the polymer may be varied as d, but will generally be in an average molecular weight range from 500Da to 50000Da, for example from 5000 to a such as from 20000 to a. The polymer size may in particular be selected on the basis ofthe intended use of the product for example y 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 t 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 5000Da.

For applications where the product remains in the ation, it may be advantageous to use a higher molecular weight polymer, for example having a molecular weight in the range from 20000Da to 40000Da.

Suitable polymers e a kylene polymer, such as a poly(ethyleneglycol) or, especially, a ypoly(ethyleneglycol) or a derivative thereof, and ally with a molecular weight in the range fi'om about lSOOODa to about 40000Da.

In one example antibodies for use in the present invention are ed to poly(ethyleneglycol) (PEG) moieties. In one particular example the antibody is an dy fragment and the PEG molecules may be attached through any available amino acid side-chain or terminal amino acid functional group d 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 e US 5,219,996; US 5,667,425; WO98/2597l, W02008/038024). In one e the antibody molecule of the present ion 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.

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 r 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 riately 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 ofpolymer—modified antibody fragments as described above. The activated r may be any polymer containing a thiol reactive group such as an a-halocarboxylic acid or ester, eg. iodoacetamide, an imide, e.g. maleimide, a vinyl sulphone or a disulphide. Such starting als may be obtained commercially (for example from Nektar, formerly Shearwater Polymers Inc., Huntsville, AL, 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).

In one ment, the antibody is a modified Fab fragment or diFab which is ted, i.e. has PEG (poly(ethyleneglycol)) ntly attached thereto, e.g. according to the method disclosed in EP 4 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, gton DC 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 ne 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 ofthe amine groups on the lysine residue may be attached a methoxypoly(ethyleneglycol) polymer having a molecular weight of approximately 20,000Da.

The total lar weight of the PEG attached to the Fab fragment may therefore be approximately 40,000Da.

Particular PEG molecules e 2~[3—(N-maleimido)propionamido]ethyl amide of N,N’-bis(methoxypoly(ethylene glycol) MW 20,000) modified lysine, also known as L40K (obtainable from Nektar, formerly Shearwater).

Alternative sources ofPEG linkers include NOF who supply GL2—400MA2 (wherein rn in the structure below is 5) and GL2-400MA (where m is 2) and n is imately 450: Mamasmergeiw HSCG-{CHECHEO}I;’/L\§ if: Q Q\fx“\\w_, SEXY/I {882% ‘xNx‘f’i‘ {E ,x’Lmif m is 2 or 5 That is to say each PEG is about 20,000Da.

Further alternative PEG effector molecules ofthe ing type: CHSO—(CH20H20)n CHZCHZO)n / are available from Dr Reddy, NOF and Jenkem. O In one embodiment there is provided an antibody which is PEGylated (for example with a PEG described ), 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).

In one embodiment one certain antibodies according to the present disclosure have the in; pro 0 erties: The t invention also provides compositions such as a pharmaceutical composition of antibody or combination of antibodies defined .

The present invention also provides a composition that comprises at least two antibodies according to the invention, for e wherein at least one antibody therein is specific to Tch and at least one antibody therein is specific to Tch or alternatively at least two antibodies c to Tch or at least two antibodies specific to Tch.

In one embodiment there is provided a composition that comprises multiple antibodies specific to Tch and optionally one or more antibodies c to Tch.

In one embodiment there is ed a composition that comprises multiple antibodies specific to Tch and optionally one or more antibodies specific to Tch.

Thus in one embodiment there is provided a composition sing 2, 3, 4, 5, 6, 7, 8, 9, 10, ll, l2, l3, 14 or 15 antibodies according to the invention i.e. distinct antibodies.

The ion describes one particular mixture comprising 3 Mabs, one Mab of which is specific for Tch and two Mabs of which are c for Tch. This mixture demonstrated very high levels of protection from death and gut inflammation from a lethal infective oral dose of Clostrz‘dz'um diflicz'le in hamsters.

In particular there is provided a composition comprising a combination of one anti— Tch 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- WO 38156 Tch 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.

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

In one embodiment there is provided a composition comprising 2, 3, 4 or 5 antibodies c to Tch and optionally l, 2, 3, 4 or 5 antibodies specific to Tch.

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

In one embodiment the composition of antibodies has an ECso of 200ng/ml or less, for example lSOng/ml or less, such as lOOng/ml or less, such as 0.1 to lOng/ml.

Advantageously the antibodies bed herein have very high levels of biophysical stability and so are le for inclusion in mixtures of antibodies.

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

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

Suitable forms for administration include forms suitable for eral stration, e.g. by injection or on, for example by bolus ion 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 sing agents. Alternatively, the antibody molecule may be in dry form, for reconstitution before use with an appropriate sterile liquid.

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 ments the compositions are adapted for administration to human subjects.

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 e if the pH ofthe 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.

In one embodiment the composition or formulation of the present disclosure comprises l-200mg/mL of antibodies, that this to say the combined antibody content, for example lSOmg/mL or less, such as lOOmg/mL or less, in particular 90, 80, 70 , 60, 50, 40, 30, 20, lOmg/mL or less.

In one embodiment a composition or ation according to the present disclosure ses 20mg/mL of each antibody therein.

In one embodiment there is provided a formulation comprising: 33mg/mL or less of one ch 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 28mg/mL or less of a first ch with a heavy variable region shown in SEQ ID NO: 129 and a light variable region shown in SEQ ID NO: 127, and 25mg/mL of a second anti-Tch with a heavy variable region shown in SEQ ID NO: 159 and light variable region shown in SEQ ID NO: 157.

In one embodiment the pharmaceutical formulation at a pH in the range of 4.0 to 7.0 comprises: 1 to 200mg/mL of an antibody according to the present disclosure, 1 to lOOmM of a buffer, 0.001 to 1% of a surfactant, a) 10 to SOOmM of a stabiliser, b) 5 to 500 mM of a tonicity agent or c) 10 to SOOmM of a stabiliser and 5 to 500 mM of a tonicity agent.

In one embodiment the composition or formulation according to the present disclosure comprises the buffer ate ed saline.

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

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

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.

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

It will be appreciated that the active ingredient in the composition will be an antibody molecule. As such, it will be tible 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 e the antibody once it has been absorbed from the intestinal tract.

A thorough discussion ofpharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack hing Company, NJ. 1991).

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 membranous colitis, bloating, abdominal pain and toxic megacolon. laxis can also be achieved by the administration ofpre-formed complexes of inactivated toxin antigen (toxoid) and antibody in order to create a vaccine.

In one embodiment the antibodies, combinations thereof and itions comprising the same according to the invention are suitable for treating infection with so-called super strains of C. difiicz’le, i.e. hypervirulent strains such as ribotype 027.

The antibodies and compositions according to the present invention are suitable for use in the ent or prophylaxis of acute and/or chronic effects of the relevant C. difi‘icile toxins during primary infection.

The antibodies and compositions according to the present invention are suitable for use in the treatment or prophylaxis of effects ofthe relevant C. dficile toxins during ary infection or re-infection. International guidelines ne time intervals after a primary infection which hence defines a secondary (or ent) ion 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 pe as the primary infection. In such cases recurrence rather than relapse relies on agreed temporal constraints.

However, research also clearly shows that ary ion 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 ion (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).

In one embodiment the antibodies, combinations f and compositions comprising the same according to the invention are le for use in the prevention of damage, for example long term structural damage to the epithelium ofthe colon.

In one embodiment the antibodies, combinations and composition are suitable for preventing C. dz'fi‘icile infection ing recurrence of infection, in particular nosocomial infection.

In one embodiment the antibodies, combinations f and compositions comprising the same according to the invention are suitable for reducing the risk ofrecurrence of C. diflicz'le infection.

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

Advantageously the antibodies ofthe 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 t damage or injury to a patient by toxins A and/or B in the acute C. dz'flz‘cz'le infection stage.

Thus in one embodiment the antibodies, ations thereof and compositions comprising the same ing to the invention are suitable for ting damage to vital organs.

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.

The antibodies ofthe present disclosure have fast on-rates, which facilitates the rapid efi’ect in vivo.

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

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

PCT/G32012/052222 The antibodies according the invention may be employed in combination with otic treatment for e metronidazo 1e, vancomycin or fidaxomicin.

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 ties of anti-Tch and 50% molar quantities of anti- Tch components) was able to protect hamsters from a lethal CDI.

In one ment there is provided a method of treating a t 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. dz‘fiicz'le infection or complications associated with the same such as diarrhoea, s in particular pseudomembranous colitis, bloating, abdominal pain and toxic megacolon.

In one embodiment the antibody, combination or formulation is administered by a parenteral route, for example subcutaneously, intraperitoneally, intravenously or uscularly. 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 .

In one embodiment the antibody, combination or formulation is stered orally, for example an enterically coated formulation.

In one embodiment there is provided use of an dy, combination or formulation as described herein for the manufacture of a medicament for the treatment or prophylaxis of C. dz'fifzcile infection.

In one embodiment the dose administered is in the range 1 to lOOOmg/Kg, for example to 75mg/Kg, such 20 to 50mg/Kg.

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.

In one ment the antibody or antibodies are given as one dose only.

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

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

In one embodiment there is provided complex sing Tch or an immunogenic fragment thereof, complexed with one or more anti-Tch dies 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.

In one embodiment there is ed complex comprising Tch or an immunogenic fragment thereof, complexed with one or more anti-Tch dies defined herein. The WO 38156 complex may be ed as the antigen in a vaccine formulation, for example suitable for generating protective antibodies to toxin B in Vivo after administration to a human. pe immunostimulants which may be formulated to produce adjuvants le for use in the t ion include and are not restricted to the following.

In one embodiment there is ed a complex comprising Tch or an immunogenic fragment thereof and Tch 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.

Antibodyzantigen complexes are known to be taken up by the immune system in an Fc receptor mediated s (27, 28) and pre-formed complexes of antibodyzantigen complexes have been successfully use as es in human clinical trials (22).

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

Monophosphoryl lipid A, in particular 3—de-O—acylated monophosphoryl lipid A (3D- MPL), is a preferred Thl-type immunostimulant for use in the invention. 3D-MPL is a well known adjuvant manufactured by Ribi Immunochem, Montana. Chemically it is often ed 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 (US 6,005,099 and EP 0 729 473 Bl; Hilgers er a1., 1986, IntArchAZlergylmmunol., 79(4):392—6; Hilgers et al., 1987, Immunology, 60(1): 141-6; and EP 0 549 074 B l). A preferred form of3D-MPL is in the form of a particulate formulation having a small particle size less than 0.2mm in diameter, and its method ofmanufacture is disclosed in EP 0 689 454.

Saponins are also preferred Th1 immunostirnulants 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 ofthe South American tree Quillaja Saponaria Molina), and fractions thereof, are described in US 540 and ins as e adjuvants”, Kensil, C. R., Crit Rev Ther Drug Carrier Syst, 1996, 12 (l- 2):1—55; and EP 0 362 279 B1. The haemolytic saponins QS21 and Q8 17 (HPLC purified fractions of Quil A) have been described as potent systemic nts, and the method of their production is disclosed in US Patent No. 5,057,540 and EP 0 362 279 B 1. Also bed 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 er al. (1991. J.

WO 38156 Immunology vol 146, 431—437). Combinations of QS21 and polysorbate or cyclodextrin are also known (W0 99/ 10008). Particulate adjuvant systems comprising ons of QuilA, such as QS21 and QS7 are described in W0 96/33739 and W0 96/ l 171 1. One such system is known as an Iscorn and may contain one or more saponins.

Another preferred immunostirnulant is an immunostimulatory oligonucleotide ning 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., Jlmmunol, 1998, l60(2):870~876; McCluskie and Davis, J.Immunol., 1998, 16 l(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 ng stimulatory effects (both in vitro and in vivo). The authors of these studies concluded that certain palindromic sequences, including a l CG motif, carried this activity. The central role of the CG motif in immunostimulation was later elucidated in a ation by Krieg, Nature 374, p546 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 stimulatory sequence is ofien: 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 t invention.

In certain combinations ofthe six tides a palindromic sequence is present.

Several of these motifs, either as s of one motif or a combination of different motifs, can be present in the same oligonucleotide. The presence of one or more ofthese immunostimulatory sequences containing oligonucleotides can te 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.

CpG when formulated into vaccines, is generally administered in free solution er with free antigen (W0 96/02555; McCluskie and Davis, supra) or covalently conjugated to an antigen (W0 98/ 16247), or formulated with a carrier such as aluminium ide titis surface antigen) Davis et al. supra ; Brazolot—Millan er al., Proc.Natl.Acad.Sci., USA, 1998, 95(26), 15553-8).

Such immunostimulants as described above may be formulated together with carriers, such as for e 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 (W0 95/ 172 10); QSZl may be advantageously formulated with cholesterol containing liposomes (WO 96/33739), oil in water emulsions (WO 95/17210) or alum (W0 98/ 15287); CpG may be formulated with alum (Davis et (2/. supra ; Brazolot-Millan supra) or with other cationic carriers.

Combinations of immunostimulants are also preferred, in particular a combination of a monophosphoryl lipid A and a saponin derivative (W0 53; WO 95/17210; WO 96/33739; WO 98/56414; WO 99/12565; WO 99/11241), more particularly the combination of 0821 and 3D—MPL as disclosed in WO 94/00153. Alternatively, a combination of CpG plus a saponin such as QSZl also forms a potent nt for use in the present invention.

Alternatively the n may be formulated in a liposome or in an Iscorn and combined with an immunostimulatory oligonucleotide.

Thus, suitable adjuvant systems include, for example, a combination of monophosphoryl lipid A, ably , er with an aluminium salt.

Thus is one ment the adjuvant is a combination ofQS21 and 3D—MPL in an oil in water or liposomal formulation.

An enhanced system involves the ation of a monophosphoryl lipid A and a saponin derivative particularly the combination of QSZl and 3D-MPL as sed in WO 94/00153, or a less reactogenic composition where the QS21 is quenched in cholesterol containing liposomes (DQ) as disclosed in W0 96/33739. This combination may additionally comprise an immunostimulatory oligonucleotide.

A particularly potent adjuvant formulation involving Q82 1, 3D-MPL & tocopherol in an oil in water emulsion is described in WO 95/17210 and is r preferred formulation for use in the invention. r preferred formulation ses a CpG oligonucleotide alone or together with an aluminium salt.

In a further aspect of the present invention there is ed 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.

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 iii) Alum + QS21 in a liposome + 3D-MPL iv) Alum + CpG PCT/G32012/052222 V) 3D-MPL + Q82] + oil in water emulsion Vi) CpG As used herein, the term “comprising” in context of the present specification should be reted as “including”.

Embodiments and preferences may be combined as technically appropriate.

The disclosure herein describes embodiments comprising certain integers. The disclosure also extends to the same embodiments consisting or consisting ially of said integers.

FIGURES Fig 1-10 shows various antibody and fragment sequences Fig 11 shows sera titres for Tch and Tch Fig 12 shows anti Tch (Ribotype 003) in-Vitro neutralization data for single Mabs Fig 13 shows anti Tch ype 003) in-vitro neutralization data for single and paired Mabs Fig 14-15 shows anti Tch (Ribotype 003) ro neutralization data for paired Mabs Fig 16-18 shows anti Tch (Ribotype 003) in-vitro neutralization data for three Mab mixtures Fig 19-20 shows anti Tch (Ribotype 003) in-vitro neutralization data for four and five Mab mixtures Fig 21-22 shows anti Tch (Ribotype 003) in-Vitro neutralization data for single and paired Mabs at different Tch concentrations Fig 23-24 shows anti Tch (Ribotype 003) in-vitro neutralization data for single and to five Mab ess at different Tch concentrations Fig 25-26 shows anti Tch (Ribotype 003) in-Vitro lization data for single Mabs Fig 27-30 shows anti Tch (Ribotype 003) in—Vitro neutralization data for paired Mabs Fig 31-33 shows anti Tch (Ribotype 003) in—Vitro lization data for three Mab mixtures Fig 34-40 shows anti Tch (Ribotype 003) in-Vitro neutralization data for two Mab mixtures at different toxin concentrations Fig 41-45 shows anti Tch (Ribotype 003) in—vitro neutralization data for two Mab es at different ve Mab ratios and different toxin concentrations Fig 46-59 shows Tch neutralisation data for single antibodies and pairs of antibodies Fig 60 shows the amino acid sequence for Tch Fig 61 shows the amino acid sequence for Tch Fig 62 shows TEER assay data for Tch in a histogram format Fig 62A shows TEER assay data for Tch in line graph format Fig 63 shows a meier-kaplan curve for the combination of antibodies 997, 1125 and 1151, high concentration is 50mg/Kg and low concentration is 5mg/Kg 50mg/kg’ dose gave 100% protection to day 11, ~82% protection to day 28. 5mg/kg’ dose resulted in non—durable and incomplete protection.

Fig 64 shows bodyweight changes for vancomycin and vehicle treated hamsters Fig 65 shows the bodyweight for low dose dies 5mg/Kg and high dose antibodies 50mg/Kg Fig 66 shows photographs of a colon where the animal received treatment with antibodies ing to the present disclosure vs a l Fig 67-68 show s ofvortexing on antibody stability Fig 69 shows a comparison of aggregation stability for various antibodies Fig 70-73 show neutralisation ofTch for various ribotypes Antibody Generation A range of different immunogens and screening reagents were either purchased or produced by conventional E. 0012' expression techniques in order to provide a e and broad immune response and to tate identification and characterisation of monoclonal antibodies (listed in Table 1). In cases where recombinant proteins or peptides were ted, sequences were based on ribotype 027. The sequence for Tch from ribotype 027 is given in SEQ ID NO: 171 (Uniprot accession number C9YJ37) and the sequence for Tch from ribotype 027 is given is SEQ ID NO: 172 ot accession number C9YJ35).

Sprague Dawley rats and half-lop rabbits were immunised with either synthetic peptides mapping to regions common to both Tch and Tdch full-length toxin, formaldehyde— inactivated toxoid A, binding domain fragments of Toxin A (CROPS l ,2,3 or CROPS4,5,6) or binding domain fragment ofToxin B (CROPS 1 ,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 ted. 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 figure 11.

UCB SLAM was used as a means to generate monoclonal antibodies. B cells were ed directly fiom immunised animals (Zubler et al., 1985). This step enabled sampling of a large percentage ofthe B cell repertoire. By incorporating the ed lymphocyte antibody method (SLAM) (Babcook et al., 1996) it was possible to deconvolute positive culture wells and W0 2013l038156 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 n— specific B cells from culture wells. B cell cultures were set up and atants were first screened for their ability to bind a relevant purified toxin domain (binding, ocation 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 ve for g to Tch or Tch components from this assay were selected for use in cell-based functional assays to identify neutralisers oftoxin-induced cytotoxicity. l0 Approximately 12,000 toxin-specific positives were identified in the primary binding screen from a total of40 x ISO-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 e transcription (RT)—PCR. These V—region genes were then cloned as mouse IgG l/kappa full-length antibodies for rat variable regions and rabbit IgG/kappa fiill-length antibodies for rabbit variable regions. dies 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 y measurements, lead candidates were selected for humanisation. Unless otherwise stated, all the data herein was generated using the humanised antibodies.

A panel mbinant, E. coli-produced toxin fiagments (Tch), C. icz’le—derived toxin or toxoid (A) and synthetic peptides (B) were generated or purchased fi'om commercial sources.

Table 1. Toxin A (Tch) ce related ts for screening and immunizations. e number MI-E659 K577431350 5182702249 62205-R2608 81827-N1978 GI966—N2133 62100-132249 G22l3-N238l UCB E. coli expression 2012/052222 Table 2. Toxin B (Tch) sequence related reagents for screening and immunizations.

Toxin Domain Amino acid Se uence Translocafion Expression and purification of C. difficile anti-toxin Mabs Separate light chain and heavy chain mammalian expression plasmids were combined in equimolar ratios and used to transfect 3 or CHO~S cells. For small scale expression studies lipofectamine and EEK-293 cells were used whereas for tion oflarger batches of lgG electroporation into CHO-S was preferred.

Culture supematants were loaded onto a MabSelect SuRe column (in PBS pH 7.4). dy was eluted with 100% 0.1M Sodium e pH 3 .4 buffer. Samples were neutralized to pH7.4 with Tris.C1pH8.0. Aggregate was removed by Superdex 200 Gel Filtration column in PBS pH 7.4.

TABLE 3 Cell Volume of sion Amount Ant'b d1 o y _n e SN (L) . 3e "I urified (m_) Example 1 In-vitro neutralization of Tch activity by ed Mabs All neutralisation screening assays were run in 96 well polystyrene plates. The assay uses CACO~2 cells grown, and screened in MEM + 20% FCS, 2mM Q, and NEAA. Any antibody combinations are at equal molar ratios unless stated otherwise. Day 1: Cells were plated @ 3000 per well in 50 ul media, and incubated for 24 hrs; Day 2: d samples of humanised Mab were added to 96 well round bottomed polypropylene sterile plates; Spike PP plates with toxin A at a concentration ent to generated the appropriate lethal dose i.e. LDso or above and incubate for 1 hr, at 370C; Add 50 ul 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 405nm. The results are shown in Fig 12 to 24. ECso and % maximum neutralization ofTch activity shown confirm that the selected antibodies have very high potencies as single agents. Combinations of 2 to 5 ofthese did not improve upon the best ECso or % maximum neutralization. Lack of any synergy when combining Mabs CA922, 923, 995, 997 and 1000 is an ant 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 ofthe Mabs (e.g. CA997) are capable of binding to Tch subdomains many times.

Hence it seems probable that these 5 Mabs represent that the maximum affinity, y and valency that is achievable when targeting the C-terminal cell binding domain ofTch. The antibodies were also effective at neutralising very high toxin concentrations g from LDSO to greater than LD95 (LDmx) but some modest increases in ECso (i.e. decreases in potency) were observed with very high levels of [Tch]. These data are also surprising since others have shown substantial reductions in potency when testing elevated Tch concentrations (20).

The high potency and affinity ofthe Mabs described here, e.g. for CA997; is not due solely to their high valency of binding. Others (20 and WOO6/071877) describe ch Mabs capable ofbinding up to 14 times. These Mabs only had affinities in the range 0.3 to lOOnM and showed incomplete protection against Tch mediated cell killing, alone (26-63% protection) or in pairs (31-73% protection). Hence it has been strated that high valency of g to Tch does not necessarily invoke either high affinity of binding to or neutralisation ofTch. Neither the affinities nor valency ofbinding to Tch were described for Mab CDA—l (18 and 559). Thus Mabs described herein to have surprising affinity, y and valency.

TABLE 4 Anti Tch 1, 2 & 3 Mab combinations at a single Tch cone. (LDso) Antibodx Final (highest) Mab conc.ng/ml EC50(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 7 500 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 +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 3+997+1000 500 3.06 922+923+995+997+1000 500 4.72 TABLE 5 Anti Tch single, paired, and triplet Mab combinations at various Tch concentrations, where Tch is at its LDso, LDgo, LD95 and LDmax.

Toxin Tch Sample Final Mab conc.n_lml @ 3000 pg/ml (LDMAX) 922 500 1000 500 922+997 500 00 500 m 997+1000 500 922+997+1000 500 922+997+1000+995 922+997+1000+995+923 @ 1000 pg/ml (LD95) 97—_M- 922+997 997+1000 922+997+1000 922+997+1000+995 922+997+1000+995+923 @ 700 pg/ml (LDgo) — 922+997 922+1000 997+1000 922+997+1000 922+997+1000+995+923 ’ 350 p° m1(LDgo) 922+997 922+1000 997+1000 922+997+1000 7+1000+995 922+997+1000+995+923 500 Example 2 Anti Tch in-vitro neutralization by purified Mab.

Assay methods description: All neutralisation screening assays were run in 96 well polystyrene .

The assay uses CACO-2 cells grown, and screened in MEM + 20% FCS, 2mM Q, and NEAA.Un1ess stated all Ab combinations are in equal ratios. 0 Day 1: Cells are plated @ 3000 per well in 50 n1 media, and incubated for 24 hrs 0 Day 2: Purified samples nised Mab were added to 96 well round bottomed opylene sterile plates 0 Spike PP plates with toxin B lot # 031 and incubate for 1 hr, at 370C 0 Add 50 ul ofthis e to cell plates 0 Incubate for 96 hrs 0 Day 5: Add Methylene blue (0.5%Methylene Blue 50% ethanol) 0 Incubate for 1 hr at room temperature 0 Lyse the cells with 1% N—Lauryl Sarcosine o Read on the BIOTEK Synergy2 plate reader at 405nm The data in Figures 25 to 33 show that single Mabs alone were relatively ineffective at neutralizing Tch, both in terms of% maximum neutralization and activity (ECso). However, when the antibodies were combined in two’s and three’s considerable improvements in both % maximum neutralization and activity (ECso) were observed. 1125 and 1151 were selected as a best g, although other good pairings were ed: 1 125+1 153, 1 125+1 134.

The most effective pairs of Mabs were ed cally 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 Tch neutralisation potential which could result in a d EC50 whilst the Tch neutralisation potential of both CA1125 and CA1151 were insufficient under these assay conditions to result in a defined ECso. However, CA927 was not found to be the most effective Mab to use within a combination. The best CA927 containing combination had an ECSO of 13.5ng/ml whereas other two Mab combinations had ECso’s as low as 2.59 and 4.71ng/ml. In another example, in Table 8 CA1099 had the lowest Tch neutralisation EC50 under the assay conditions used. r, CA1099 was not found to be the most effective Mab to use within a combination. The best CA1099 containing combination had an EC50 of 6ng/rnl whereas other two Mab combinations had EC50’s as low as 2 and lng/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 6 Anti-Tch Mab combinations and relative Mab ratios at constant toxin concentration.

Sample Final Mab ECso(ng/ml) c0nc.n0/ml 1125.g2 1000 >1000 1134.g5 1000 >1000 927. _2 1000 12.89 1]53.g8 1000 >1000 1102.4 1000 >1000 927+1099 1000 >1000 927+] 102 1000 >1000 927+1114 1000 >1]1.]11 927+] 125 1000 13.55 927+1134 1000 51.58 1099+]114 1000 >1000 1102+] 114 1000 >333.333 1102+] 125 1000 15.51 1114+]134 1000 19.70 1114+1151 1000 25.69 1114+] 153 1000 27.48 1125+1134 1000 2.59 151 1000 4.71 1 125+] 153 1000 21.23 1125+1134+1114 1000 3.77 1125+1134+927 1000 2.63 ]5]+]114 1000 4.90 1125+]]51+927 1000 5.69 1125.__2+]134.5+927._2 1000 5.83 1125.g2+1134.__5+]153.8 1000 1125.-2+1134.5+1102._4 1000 272 e 3 Neutralisation of Tch by combinations of purified Mab.

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

The assay uses CACO-2 cells grown, and screened in MEM + 20% FCS, 2mM Q, and NEAA. 0 Day 1: Cells are plated @ 3000 per well in 50 ul media, and incubated for 24 hrs 0 Day 2: Purified samples ofhumanised Mab were added to 96 well round bottomed polypropylene sterile plates 0 Spike PP plates with toxin B (VPI 10463) and incubate for 1 hr, at 370C 0 Add 50 ul ofthis e to cell plates 0 Incubate for 72 hrs 0 Day 5: Add Methylene blue (0.5%Methylene Blue 50% ETOH) 0 te for 1 hr at room temperature 0 Lyse the cells with 1% N—Lauryl Sarcosine o Read on the BIOTEK Synergy2 plate reader at 405nm The s are shown in Figures 34 to 45.

These data show that the best pair of Mabs for neutralizing Tch at a range of toxin concentrations was CA1 125 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 7 Anti-Tch Mab combinations and relative Mab ratios at 3 different toxin COHCS. _-_-_ 1125.2+1134.5 25:75 .2+1151_4 25:75 1125,..2+1153.8 25:75 .2+1134.5 75:25W“ 1125.92 + 1153.98 (75:25) The data show that even the most active specific paired combinations have surprisingly and non- predictably different properties relative to each other. The ECso of the preferred combination of CA1125 and CA1151 in equimolar ratios is largely unaffected by an increasing [Tch]. The three relative molar ratios of Mabs tested (i.e. 25:75 vs 50:50 vs 75:25) have very similar ECso’s to each other, suggesting that CA1125 and CA1151 have especially complementary modes of . This is in contrast to the combination of CA1 125 with CA1134 Where the increase in ECSO (i.e. reduction ofpotency) with higher [Tch] 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 ts that the combined potency of CA1125+CA1134 is more ent upon the CA1125 component. The ECSO of all three molar combinations of CA1125 and CA1153 is substantially affected by increasing [Tch] suggesting that CA1153 is a less suitable partner for combination with CA1125. In rota, these data show that CA1125 and CA1151 are a particularly favourable combination since the highest potency is maintained across a range ofMab and Tch molar ratios.

TABLE 8 Tch neutralisation — 1 or 2 ch Mabs at constant toxin dose (LDso).

Antibody IC5O (nglml) 1099 1102 N/A 1114 103 1125 N A\ 1134 1151 182 1153 260 926 N/A 927 N/A 1099 + 112 1114 + 112 1151 + 112 1134 + 112 1102 + 112 1125 + 115 926 + 1125 42 927 + 1125 A TABLE 9 Tch neutralisation — 1 or 2 anti—Tch Mabs at various Tch doses. 11514 112521111545 50:50 1125.2+1153.e 5050 112502 + 1151-0417525) These data show that combination of Mabs, especially CA1125 and CA1151 improve both the potency as measured by ECso but also as ed by % maximum protection. The % maximum tion is of particular relevance in this assay method since the Maszch mixture is incubated with cells for a long time (72h). Since Tch is toxic to Caco-2 cells in the range ofpg/ml in 2-4h this measure may be considered to be a very difficult test of Mab neutralisation y and may reflect the ability ofMab mixture with regard to their binding kinetics or modalities. In turn this may reflect the ability ofMab mixtures to protect against the effects ofTch during an established infection when there may be substantial quantities ofTch within tissues for many hours, Selected data from Tables 6—9 are further illustrated in Figures 46—59.

Example 4 Valency of binding of Mabs to Tch sub-domains.

The number of moles of binding events of anti~C. difficz'le Tch dies to Tch1234 was determined by Surface Plasmon Resonance (SPR) on a Biacore 3000 (GE Healthcare). avidin was immobilized on a CM5 sensor chip (GE Healthcare) to a level 0RU via amine coupling and biotinylated Tch1234 was bound at 0RU. Two 20ul injections of the same anti-Tch antibody mixtures (final concentration of each antibody was 500nM) were injected over this surface at 10ul/min and the saturating binding response recorded. The surface was regenerated after every cycle using HCl. All the data was corrected for background g using the response to the streptavidin only nce flowcell.

Table 10: Surface plasmon resonance is of the number of IgG binding sites on Tchlm Antibody combination No. of binding Binding Binding relative to cycle repeats Response (RU) CA927 average res | 01188 CA1 125.g2 CA1151.g4 CA1125_CA1151 CA1 125_CA927 CA1151_CA927 CA927 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 Tch1234 once only.

Immobilized CA1125, when bound to Tch1234, does not allow CA1125 to bind further supporting the idea that CA1 125 has one binding site on Tch1234 and that after this has been saturated that no other binding site for CA1125 can be found. However, when Tch1234 has been ted by CA1125, CA1151 can still bind. This demonstrates that CA1151 binds at alternative sites to that occupied by CA1 125. Together these data show that CA1125 is a single binder ofTch1234 whereas 1151 IgG binds to Tchlm more than once, most likely twice. Hence a mixture of CA1125 and CA1151 can bind to Tch1234 approximately 3 times.

All antibodies combinations have an additive binding response showing that there are 2 or more non-competitive sites on Tch1234 bound by these ations.

Example 5 Valency of binding of Mabs to Tch sub-domains.

The number of moles of binding events of anti-Cdifiicile Tch antibodies to Tch123 and A455 were determined by Surface n Resonance (SPR) on a e 3000 (GE Healthcare).

Streptavidin was immobilized on a CMS sensor chip (GE care) Via amine ng to a level of~4000RU and biotinylated Tchm was bound to one flowcell and Tch456 was bound to a ent flowcell to a response of ~500RU. Two 30ul injections of the same anti-Tch antibody at 1 nM were injected over both flowcells at lord/min and the saturating binding response recorded.

The e was regenerated after every cycle using HCl. All the data was corrected for background binding using the response to the streptavidin only reference flowcell.

WO 38156 Antibodies CA997 and CA1000 bind to Tchm in a ratio of six CA997’S to one CA1000 s they bind to Tch456 in a ratio ofthree CA997’s to one CAlOOO (Table 2).

The maximum antibody response for CA997, ted for molecular weight and immobilized toxin level is similar for Tch123 and Tch456. This suggests that CA997 binds Tch456 six times and CAIOOO binds twice to Tch455. Hence antibody CA997 likely binds to Tch whole toxin (Tch) approximately 12 times.

Overall CA997 binds six times or more to A123 and six times or more to A456, Whereas CAlOOO binds at least once to A123 and twice to A456.

Increased valency of binding to Tch and Tch may have two important effects in vivo. The first is that any Mab or Mab mixture which is capable of binding Tch more than once will have increased potential to form inter-toxin binding events and hence immunoprecipitation.

Immunoprecipitation can contribute to y by reducing the solubility of toxin and forming very large macromolecular complexes which hence reduce the ive 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 ho st immune respone. Antigenzantibody complexes bearing Fc nts have been specifically shown to be capable ofpriming a host immune respone t a gut pathogen (21). Also, soluble antigen2antibody complexes have been successfully used as a vaccine ed against the antigen in human clinical trials (22). In addition, immune decoration of toxin with Fe bearing IgG may bute to immune clearance using normal mechanisms through the liver and spleen. In general, higher levels of PC decoration of n 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 ofch required for very rapid and substantial clearance oftoxin (24). The anti-Tch Mab CA997 is likely capable of binding to Tch up to 12 times and the combination of CA1 125 and CA1151 is likely capable of binding to Tch 3 times. Hence the 3 Mab e 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 Tch.

C. a’z'flicz‘le monolayer integrity assay is performed using the Becton—Dickinson (BD) Caco-2 BioCoat HTS plate .

Day 1 — Caco-2 cells seeded @ 2x105/ml per well of the plate insert in 500ul Basal seeding medium (provided by BD). 35ml of Basal seeding medium added to the feeder tray. Cells incubated for 24 hours at 37°C. Day 2 — Basal seeding medium removed from inserts and feeder tray, and replaced with Entero-STIM differentiation medium (supplied by BD). 500ul added per well insert and 35ml to the feeder tray. Cells incubate for a further 72hrs at 37°C. Day 5 — dies prepared at 2x concentration ve 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 125ng/m1 and plate incubated for 1hr at 37°C. lml of Caco-2 growth medium (MEM + 20% FCS, 2mM 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 ed with 400pl of toxin:Ab mixture.

Loss of tight junctions between gut cells is the key early effect ofTch 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 ance in differentiated cultured cells which have formed a monolayer is a useful surrogate for the protection against the acute effects of Tch. Three antibodies shown have good levels of tion against TEER loss, Figure 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 (ECso = 1.21 ng/ml) and yet this is considerably rformed in the TEER assay by an dy (CA1000) which has >le lower potency in a cell proliferation assay (ECso = 19.73ng/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 EC50 of approximately 80ng/ml at 4h. These ations are unexpected since the Mabs in question all had high ies for Tch domains (CA922 ~4pM, CA997 ~132pM, CA1000 ~73pM). These data suggest that CA997 and CA1000 recognise epitopes important in TEER loss or neutralize Tch by different mechanism to other Mabs. Furthermore, since CA1000 is estimated to bind to holotoxin twice (once in Tch123 and once in Tch456) CA1000 may define ‘TEER al’ epitopes within the Tch cell binding regions which might have particular value for defining vaccine immunogens. Results are shown in Figures 62.

Example 7 Affinity of anti—C. difficile toxin dies for mains of Tch and Tch: , Tch456 and 4.

Kinetic constants for the interactions of anti-C. difficile Tch and Tch antibodies were determined by surface plasmon resonance conducted on a BIAcore 3000 using CMS sensor chips.

All experiments were performed at 25°C. Affinipure F(ab’)2 fiagment goat anti—human IgG, Fc nt specific (Jackson ImmunoResearch) was immobilised on a CMS Sensor Chip (GE) Via amine coupling chemistry to a capture level of z7000 response units (RUs). HBS-EP buffer (10mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005 % Surfactant P20, e AB) was used as the running buffer with a flow rate of 10 uL/min. A 10 uL injection of each antibody at lug/ml or lower was used for capture by the immobilised anti-human IgG, Fc. Tch123, Tch456 or Tch1234 were titrated over captured purified antibodies at doubling dilutions from 12.5nM at a flow rate of 30 uL/min. For antibodies present in e supernatants, a single concentration of 12.5nM ofTch123 or Tch456 and 50nM of Tch1234 was passed over the antibodies at 30ul/min. Kinetics were calculated on n=2 The surface was regenerated at a flowrate of lOuL/min by two 10 uL ions of40 mM HCl, and a 5 uL 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 thm.

TABLE 12 Anti-Tch Mab affinities and bindin kinetics Antibod 1D kaonvls) kda/s) KD<M> KD(M) CA164WOO922gl»»»»»» 1.09E+06 06 4.06E~l 2 CA164_00923.g1 5.36E+05 3.47E-05 6.47E-ll Purified Mab 5 point CA164_00995.g1 No binding titration ”SiéléfLQWRZ-gl,,,,,, 7.84E+05 1.03E—04 1.32E—10 CA164_01000gl_____ 1.33E+05 9.78E—06 7.33E-1 l atant 2x lpoint CA164_00993.g1 9.00E+05 5.00E—06 5.5 6E—12 titration __Qél.§fi,99922:gl...... 1.29E+06 3.33E—06 2.59E-12 .QAI.6$QQ92§:.g,1., 6.16E+05 1.92E-04 3.12E-10 d Mab 5 point .Qélfifllwfizgl,,,,, 2.87E+05 3.42E-05 1-1 913-10 titration CA164_00997.g1 05 6.15E—05 6.68E-11 W0 2013l038156 CA164_01000.g1 3.55E+05 2.98E-05 8.41E—11 Supernatant 2x 1point CA164_00993.g1 1.25E+06 5.00E—06 4.00E—12 . titration TABLE 13 Anti-Tch Mab affinities and _ kinetics Antibod ID kaa/Ms) kda/s) KD<M> d Mab 3 point CA164_1 125.g2 05 titration Purified Mab 3 point CA164 l 151. 7 .49E+05 titration Supernatant 2x 1point CA164_926.g1 1.3 8E+05 titration Purified Mab 3 point CA164_927.g2 3.97E+05 titration Purified Mab 3 point CA164_1099.g2 5.24E+05 ion Supernatant 2x 1point CA164_1102.g4 1.17E+05 titration Supernatant 2x 1point CA164_11 l4.g2 2.87E+05 titration Supernatant 2x 1point CA164_1114.g8 2.55E+05 titration Supernatant 2x 1point CA164_1129.g1 1.89E+05 titration d Mab 3 point CA1 64_1 1 34.g5 5 .09E+05 ion TchlZ34 Purified Mab 3 point CA164_1 1 53.g8 1 .43E+05 titration The anti-Tch affinities are particularly high compared to the published affinities of other Mabs.

We demonstrate that affinities as low as 4pM are achievable. The preferred CA997 has an y of l32pM, CA1125 122pM and CA115 551pM. CA995 clearly shows that it does not bind to CROPS A123 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 ies for the binding of these Mabs to holotoxin due to technical constraints. However, given the high affinities and valencies demonstrated for the anti-Tch Mabs it is possible to speculate that the functional affinities against ho lotoxin may be even er than those illustrated for binding to toxin sub-domains.

The anti-Tch Mabs also demonstrated strong ies reaching as low as 31pM. In ular CA1125, 1151, 927, 1099, 1134 and 1153 show ies which surpass those demonstrated by Example 8 Biophysical characteristion of C. difficile anti-toxin humanised IgGl Molecules.

Molecules analysed Anti-Tch IgGl: CA164_00922.g1 CA1 64_0923.g1 CA164_0995.g1 CA164_0997.g1 CA164_01000.g1 Anti-Tch IgGl CA164_01125.gl CA164_01125.g2 CA164_01134.g4 CA164_01134.g5 CA164_01134.g6 CA164_01102.g1 CA164_01102.g4 CA164_01151.g4 Antibody combinations need to be made up of Mabs having high levels of ity 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 al stress such as agitation or shaking. Aggregates are undesirable components of drug W0 2013I’038156 itions 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-Tch Mabs have high Tm stability, whilst three , 923 and 997) have very high Tm’s in the range of 79-81°C. Of the anti-Tch 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°C, 793°C and 80.80C respectively) which makes them suitable for use in a Mab mixture.

In the shaking aggregation assay, CA997 and 922 had the lowest propensity to aggregate of the 5 anti—Tch Mabs. Similarly, CA115 and 1151 had the lowest aggregation propensities of the anti- Tch 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 n concentrations.

Estimation of isoelectric point (p1) by capillary IEF Samples were prepared by mixing the ing: 30u1 Protein sample at l, 0.35% cellulose, 4% pH3-10 ampholytes (Pharmalyte), synthetic pl markers (4.65 and 9.77), lul of each stock solution, and HPLC grade water to make up the final volume to 200ul. The mixture was then analysed using iCE2801EF analyzer(pre-focusing at 1500V for 1 min followed by focusing at 3000V for 6mins). The calibrated electropherograms were then integrated using Empower software (from Waters) Thermal stability (Tm) measured via Thermofluor assay.

This method uses Sypro orange fluorescent dye to monitor the unfolding process of protein domains. The dye binds to exposed hobic regions that become exposed as a consequence of ing which s in a change to the emission spectrum.

The sample (5u1 at 1mg/ml) is mixed with a 5ul of a stock solution of Sypro orange (30x) and the volume made up to 50ul with PBS,pH 7.40. 10u1 aliquots ofthis solution is applied towells in a 384 well plate (n=4).

The plate is placed in a 7900HT fast real-time PCR system containing a heating device for accurate temperature control. The ature is increased from 20°C to 990 C (Ramp rate of 1.1°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.

During manufacture antibody samples are ted 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 ation stability.

Both anti-Tch and anti-Tch IgGl les were subjected to stress by agitation, by vortexing using an Eppendorf Thermomixer Comfort at 25 °C, l400rpm. Sample size was 250uL, (X3 per sample) in a 1.5 mL conical orf—style capped tube (plastic), in PBS pH 7.4. Each sample was brought to a concentration of lmg/ml (using tion ient calculated from sequence) and aggregation was monitored by absorbance at 340nm and/or 595nm, by use of a Varian Cary SO-Bio spectrophotometer, measured at intervals for up to 24 hours.

Results Table 14 provides a summary ofthe measured pl and Tm data for both anti—Tch and ch IgGl molecules.

Table 14 : Compilation of p1 and Tm Data Anti-TCdA I_ G1 CA164 00922-1"—8 l CA164 0923.—_79 CA1640995.1"—71 CA164 0997-1 —-_— *denotes that it was not possible to discern the Fab and CH2 domains.

Measured pI The measured pI of the molecules were high (except for CA164_01000.gl_P3) and away from the pH of formulation buffers such as PBS, pH 7.4 and 50m sodium acetate/125mM sodium chloride, W0 20131038156 pH 5. This may mean that s with pH’s suitable for co-formulation oftwo or more Mabs can be selected.

Thermal Stability (Tm) Measured via Thermofluor assay Since all of the les are IgGl, 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 ofthe Fab’ domain (Tm(Fab)).

For the anti-Tch molecules, the rank order (most stable first) was CA9222997>923>995>1000 and for the anti-Tch les (most stable first) was CA115l.g4>1125.g1,g4>1134.g4>1134.g521134.g6>1102.g1=1102.g4.

Stressing of samples by agitation It was possible to determine ent aggregation stability between the different antibodies, Figure 67 shows the effect of agitation via vortexing on different anti-Tch IgGl les in PBS,pH 7.4.

It was possible to determine a ranking order (most aggregation stable first) : CA9222997>9232995>1000 Figure 68 shows the effect of agitation via vortexing on different anti-Tch molecules.

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.

A further study was performed to compare directly the aggregation ity ofthe ch molecule (CA1151.g4) with the more stable le CA1 125.g2 (see Figure 2) and more aggregation stable anti—Tch molecules (CA922.gl and CA997.gl). The results can be seen in Figure 69.

Further results for these 4 Mabs are also shown in Figures 67 and 68.

For the anti-Tch molecules, CA922.g1 and g1, CA922 were preferable based on the analyses above, although apart from CA1000) all molecules could be considered suitable candidates for use as therapeutic IgGl.

For the anti- '1"ch molecules, the biophysical characteristics could be grouped within the family of grafts based on the ation stability and Tm, such that the CA1 125 grafts potentially proved more stable. The CA1102 grafts showed poorest Tm data and also showed the greatest tendancy to aggregate via stress by agitation.

A study using CA1151.g4 showed that this molecule exhibited slightly increased aggregation stability relative to CA1 1 125.g2 and seemed equivalent to the Tch molecules (CA922.gl 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 ion.

Example 9 Anti-C. difficile toxin Mab r infection study.

The hamster ion study was performed by Ricerca Biosciences LLC, Cleveland, Ohio, USA.

The study protocol was approved by the Ricerca IACUC committee. Active and l components (composition and dose) were blinded to Ricerca staff until after completion of the planned 28 day study period.

Golden Syrian male hamsters (weight 82—103g, 54 days old) were individually housed in HEPA d able 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 l) once a day for each of 4 days: days -3, -2, -1 and 0. Two doses of Mab were investigated: high dose = 50mg/kg each of anti-Tch and anti-Tch components and low dose 5mg/kg each ofanti-Tch and anti-Tch components.

The drug combination tested was composed of one anti-Tch antibody .gl) which constituted 50% of the injected protein and two anti-Tch antibodies (CA1125.g2 and CA1 15 1 . g4) which together constituted 50% ofthe injected protein but which alone constituted % ofthe injected protein. Hamsters were sensitised (day -1) with 50mg/kg of Clindarnycin ate in PBS (3.0.) before being challenged 1 day later (day 0) with 3.4 X 106 c.f.u. of vegetative cells from strain ATCC43596. Vancomycin was dosed at 5mg/kg twice a day for 5 days (p.o.) on days 1, 2, 3, 4, 5.

Viability checks were performed on animals twice a day, animals found to be in extremis were euthanised and d as dead. Body weights were determined on each day of dosing, then twice weekly and before euthanising ors. 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 ycin 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.

Survival curves can be seen in Figure 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 +1 1. rs receiving the high dose ofUCB 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 ofUCB Mab PCT/G32012/052222 mixture all survived until day +3, thereafter animals were lost fairly ly until day +16 when all had died. The data show ional 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 ofvery high level performance for neutralization and stability.

There is no apparent link between death and body weight during the acute phase (days 1-5) of the ion, Figures 64-65. Hence it may be supposed that hamsters die of overwhelming direct and indirect effects ofTch and Tch. 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 ofthe 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 or protective effects of the UCB Mabs enabling the gut to function as normal.

Table 15. Gross atholo_ scores roup 1: lack Dark red aecum caecum It is clear that UCB Mabs were able to protect the large and small intestines from the bloody effusions caused by Tch and Tch.

The results are shown in Figures 63 to 66 The photographs in Figure 66 show l gross pathologies for the swelling and bloody effusions of caeca caused by Tch and Tch (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 ofUCB Mabs the large intestine can return to normal morphology and function.

Example 10 Neutralisation of Tch from different ribotyped strains by purified Mab.

Clinical ions are caused by a variety of different strains. Strain ences are characterized using a number of different methods of which ribotyping is a key one. Different ribotype strains are observed to have different enicity, ion and sporulation properties. All of the Tch neutralization shown above used Tch purified from strain known as VPIlO463. However, the predominant aggressively pathogenic strain ated with out-breaks is called ribotype 027.

Other key pes include 078, 001 , 106. Amino acid sequence difference have been observed between toxins produced by ent ribotypes and hence it is important that Mabs are e of neutralizing toxin from a diverse set of clinical isolates. CA922, 997 and 1000 were tested for their ability to neutralize Tch from strains 027 and 078 and compared to their abilities against Tch from VP110463. Mabs were tested at 4 [Tch] and found to be capable of neutralizing all toxins without significant difference at LDso, LD90 and LD95 Table 16 EC50 values (nglml) - Tch strain VPl 10463 Table 17 EC50 values (nglml) - Tch ribotype 027 Table 18 EC50 values (nglml) - Tch ribotype 078 Example 11 PK data A PK study of a human IgGl (20mg/kg) in y hamsters. The hamster PK was found a half— life similar to Mabs in mice or rats. (tl/z 6-8 days). i.p. and s.c. dosing were essentially the same.

The pharmacokinetics and bution to the gut of a hlgGl Mab was studied in ‘normal’ (non- infected) golden Syrian hamsters. Purified Mab was stered to male hamsters (l20—l35g) by CARE Research LLC, Fort Collins, Colorado, USA and samples were assayed by UCB Pharma.

The study was approved by the CARE IACUC committee. Eight s each received a single dose of 20 mg/kg ofIgG1, four were closed i.p., four were dosed s.c. Blood was collected at l, 3, 8, 24, 48, 72, 103 and 168 hours post-dose, serum was separated before storage at -80°C. Blood was also taken from two untreated hamsters in order to provide assay controls. Following euthanasia, a 20m length of colon was cut from the caeca junction onwards from each r. The colon section was flushed with wash buffer (50% (v/v) PBS ning 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 0.51111 of wash buffer homogenized until ly uniform and stored at 4°C before ate shipping on wet ice. For the anti-human IgGl ELISA Nunc maxisorp 96 well plates were coated overnight in 0.1M NaHCO3 pH 8.3 with Goat F(ab’)2 anti-human IgG-Fcy fragment (Jackson 109098), 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 alter washing were revealed with goat uman kappa—HRP (Cambridge Bioscience 2060-05) in sample—conjugate buffer and TMB with a 2.5M H2804 stop solution.

Gut, Mucosa and Serum Levels: Serum samples collected from blood taken at 168 hour time point and colon samples were removed after this. 20mg/kg IP at 168 hour —ng/mL per cm mucosa 1001 20mg/kg SC at 168 hour 76.6 163.7 153-3 WO 38156 Serum Data 22626 1378 22371 2258 43287 7169 61290 17637 The data is also shown1n Figure 70 and 71 It was also shown that hlgGl could be found in ‘scrapings’ of the gut i.e that hIgGl gets into the vasculature thy gut —- and so could be protective in ylactic 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 This study was to determine the serum concentration of CA725.0, CA726.0, CA997.g1 CA1125.g2, and CAO] 151.g4 following i.p. administration us doses detailed below) in the Golden Syrian Hamster.

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.

A unique (“proteotypic”) peptide common to all of the humanised Mabs investigated was selected (DTLMISR, a CH2 region peptide) and both s and calibration samples were tryptically digested as ed. Tryptic digest of 5 ul serum samples was performed overnight using sequencing grade modified Trypsin ga, Southampton, UK) following ration/ reduction with acetonitrile / Tris (2-carboxyethyl) phosphine and carbamido-methylation with iodoacetamide (Sigma-Aldrich, Poole, UK).

The LC—MS/MS system consisted of a CTC HTS-x Autosampler (CTC ics, 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 (100x4.6 mm, enex, 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 ion volume was 10 uL; all of the eluent was introduced into the mass spectrometer source. The source temperature of the mass spectrometer was maintained at 600 °C and other source parameters (e.g. collision energy, declustering potential, n 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 es of interest; samples were analysed following tryptic digestion.

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

For CA164_00997 and CAl64~01151, interfering peaks were ed in the MRM traces. For this reason, these two analytes could not be quantified in the samples.

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 ofthis approach is demonstrated by the fact that the sum of the concentrations ed for CA164_00725 and CA164_00726 are in good agreement (within experimental error) of the concentration observed for total h—IgG.

Using this approach, the total concentration G in the samples of animals dosed with CAl64_00997, CA164_01125 and CA164_01151 was ined.

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

Study groups Blinded labels ent components Actual Treatments Dose days Grp Treatment Anti-toxin A Anti-toxin B 4 Treatment 3 Vehicle PBS SmL/kg i.p. 3, —2, —1, 0 2 Vancomycin Vancomycin Smg/kg b.i.d. p.0. 1, 2, 3, 4, 5 1 Treatment 1 UCB LD* 3, -2, ~1, 0 g1_P3 CA1125.g2_P3 CA1151.g4_P3 5mg/kg A 5mg/kg i.p. Smg/kg 2.5mg/kg 2.5mg/kg Treatment 4 UCB HD* 3, -2, -1, 0 CA997.g1_P3 CA1125.g2_P3 CA1151.g4_P3 50mg/kg A 50mg/kg i.p. g g 25mg/kg 6 Treatment 5 Competitor LD“ 3, —2, -1, O CA726__P3 CA725_P3 5mg/kg A 5mg/kg i.p. Smg/kg Smg/kg 3 Treatment 2 Competitor HD‘ 3, -2, -1, O CA726_P3 CA725_P3 SOmg/kg A 50mg/kg i.p. 50mg/kg SOmg/kg W0 2013;1038156 2012/052222 Group/time-m Serum conc ug/mLtotal h-lgG mg/kg 997, 2.5 mg/kg 4345-8}- LONG 1125, 2.5 n mg/kg 1151 II CDC) N_\ Illllllll 0‘)N 50 mg/kg 725, O)co 50 mg/kg 726 0').h N O’) 4:.

N O) 01 \l —x \1co I 00 (.0 I (X) 4:. 0)(A) 50 mg/kg 997, co 25 mg/kg i00 00N 1125, 25 N00 00 (A) mg/kg 1151 004:. 03 01 mg/kg 725, mg/kg 726 nd - not detected (LOQ = 2.5 pg/mL for all analytes na - not analysed: interference in the sample was observed for 997 and 1151 Table 20 Antibody CA725 is prior art antibody MDX1388. Antibody CA726 is prior art antibody CDAl as described (15) A summary of this data is ted in Figure 72. - atholo 3 ----“Red Red _-____-control References 1. Kuehne, S et al., “The role of toxin A and toxin B in Closlridz'um diflicile Infection” Nature (2010) 467: 711-713. 2. Davies AH et a1, “Super toxins from a super bug: structure and function of Closirz‘dz'um le toxins” Biochem. J (2011) 436: 517—526. 3. n, S et a1., “Differential xic Effects of Toxins A and B Isolated from Clostrz'dz‘um diflicz‘le” Infect. 1m. (1984) 46: 324—331. 4. Du, T and Alfa, MJ “Translocation of Clostrz'dz'um diflicz‘le toxin B across polarized Caco—Z cell monolayers is enhanced by toxin A” Can J Infect Dis. (2004) 15: 83-88.

Kim, Iaconis and Rolfe. “Immunization of Adult Hamsters against Clostrz'dz‘um diffcz'le- ated Ileocecitis and Transfer of Protection to Infant Hamsters” Infect. 1mm. (1987) 55:2984-2992 Rupnik JCM (2003) 41:1118-1125 Chaves-Olarte IBC (1999) 274:11046—11052.

Lylerly, DM et al., “Passive Immunization of Hamsters against Disease Caused by Closlrz'dium le by Use ofBovine Immunoglobulin G Concentrate” Infection and ty (1991) 59:2215—2218.

Lylerly, DM et al., “Vaccination against Lethal Clostridium difificile Enterocolitis With a Nontoxic Recombinant Peptide ofToxin A” Current Microbiology (1990) 21:29-32.

. Lylerly, DM et al., “Characterization ofToxins A and B of Clostrz’dium diflicz'le with Monoclonal dies” Infect. 11m. (1986) 54:70-76. 11. Corthier et al., “Protection against Experimental Pseudomembranous s in Gnotobiotic Mice by Use clonal Antibodies against Clostridium diflicile Toxin A” . Imm. (1991) 59: 1192-1195. 12. Kink JA and Williams JA, odies to Recombinant Clostridz'um diflicz‘le Toxins A and B Are an Effective Treatment and Prevent Relapse of C. diflicz‘le-Associated e in a Hamster Model of ion” Infect. 11mm. (1998) 66:2018-2025. 13. Ma D, et al., Progenics inc. ASM Poster 27th May 2010 14. Hansen, G and Demarest, SJ. A2 . Babcock GJ, et al., “Human monoclonal antibodies directed against toxins A and B prevent Closzridz'um dijj‘icile-induced mortality in hamster” . Imm.(2006) 74:6339—6347. 16. Lowy I et al., “Treatment with Monoclonal Antibodies against Clostrz'dz'um diflicile Toxins” NEJM (2010) 362: 197~205. 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 te murine and human B cells via direct cell interaction. J. Iimnunol. 134, 3662-3668 18. Babcook, J. 8., Leslie, K. B., Olsen, 0. A., Salmon, R. A. & Schrader, J. W. (1996). A novel strategy for generating monoclonal antibodies from , isolated lymphocytes producing antibodies of defined specificities. Proc. Natl. Acad. Sci. U. S. A 93, 848 19. Tickle, S., Adams, R., Brown, D., Griffiths, M., Lightwood, D. & Lawson, A. (2010). High- Throughput Screening for High Affinity Antibodies ., pp. 303-307.

. Demarest et al., mAbs (2010) 2:190-198 21. a et al., J. Clin. Invest. (2006) 116: 2142-2151 22. Xu et a1., Vaccine (2005) 23:2658~2664. 23. Yousaf et a1., Clin. Exp. Immunol. (1986) 66:654-660 24. Mannik et a1., J. Exp. Med. (1971) 133: 713-739 . Nusrat et a1., Infection and Immunity (2001) 69: 1329-1336 26. Lima et a1., Infect Immun (1988) 56:582—588 27. Ravichandran et al J of Pharmacology and Experimental Therapeutics. (2006) 318: 1343—1351 28. shi et a1., (2009) e 27:2616-2619 29. Cohen et a1., Infect. Cont. and Hosp. Epidem. (2010) 31: 431-455 . Barbut et al., J. Clin. Microbiol. (2000) 38: 2386-2388 31. Wilcox et al., J. Hospital Infection (1998) 38: 93-100.

What is claimed is: 1. A pharmaceutical composition comprising a monoclonal antibody suitable for reducing the duration and/or ty of diarrhoea or morbidity and/or mortality in a patient with Clostridium ile ion or at risk of said infection n the antibody is specific to: antigen Clostrz'dium diflicz'le toxin A (Tch) and binds 3 or more times in the range $1827-D2249 or G2205-R2608 of SEQ ID NO: 171 with an EC50 in the range 0.1 to 10 ng/ml when toxin is used at an LDgo or higher.

A pharmaceutical composition according to claim 1, wherein the anti-Tch antibody binds 4, 5, 6, 7, 8, 9,10,11,12,13,14 or 15 times or more.

A pharmaceutical ition according to claim 2, wherein the antibody EC50 is between 1 and l when toxin is at an LDgo or higher.

A pharmaceutical composition according to claim 2 or claim 3 wherein the maximal inhibition of toxin is n 50 and 100% when toxin is used at an LD30 or higher.

A pharmaceutical composition according to any one of claims 1 to 4, further comprising a monoclonal antibody specific to antigen Clostridium difiz‘cile toxin B (Tch).

A pharmaceutical ition according to any one of claims 1 to 5, wherein the anti— Tch and/or anti—Tch antibody has an affinity in the range 50 to 600pM.

A pharmaceutical composition according to any one of claims 1 to 6, wherein one or more dies is a neutralizing antibody effective against ribotypes 003, 012, 027 and 078.

A pharmaceutical composition according to any one of claims 1 to 7 comprising a monoclonal antibody which specifically binds Tch comprising a heavy chain wherein the variable domain of the heavy chain comprises at least one of a CDR having the sequence given in SEQ ID NO:44 for , a CDR having the sequence given in SEQ ID NO:45 for CDR—H2 and a CDR having the sequence given in SEQ ID NO:46 for CDR— H3 and a light chain wherein the variable domain of the light chain comprises at least one of a CDR having the sequence given in SEQ ID NO:41 for CDR—L1, a CDR having the sequence given in in SEQ ID NO:42 for CDR—L2 and a CDR having the sequence given in SEQ ID NO:43 for CDR—L3.

A pharmaceutical composition according to claim 8 comprising a monoclonal antibody having a heavy chain sing the sequence given in SEQ ID NO:49 and a light chain comprising the sequence given in SEQ ID NO:47.

. A pharmaceutical composition according to claim 1 comprising a onal antibody which specifically binds Tch comprising a heavy chain wherein the variable domain of the heavy chain comprises at least one of a CDR having the sequence given in SEQ ID NO:54 for CDR—H1 a CDR having the sequence given in SEQ ID NOISS for CDR—H2 and a CDR having the ce given in SEQ ID NO:56 for CDR-H3 and a light chain wherein the variable domain of the light chain comprises at least one of a CDR having the sequence given in SEQ ID NO:51 for CDR-L1, a CDR having the sequence given in in SEQ ID NO:52 for CDR-L2 and a CDR having the sequence given in SEQ ID NO:53 for CDR-L3. 11. A pharmaceutical composition according to claim 10 comprising a monoclonal dy having a heavy chain sing the sequence given in SEQ ID NO:59 and a light chain comprising the sequence given in SEQ ID NO:57. 12. A pharmaceutical composition according to claim 1 comprising a monoclonal antibody which specifically binds Tch comprising a heavy chain n the variable domain of the heavy chain comprises at least one of a CDR having the sequence given in SEQ ID NO: 124 for CDR-H1, a CDR having the sequence given in 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 le domain of the light chain comprises at least one of a CDR having the sequence given in SEQ ID NO: 121 for , a CDR having the ce 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. 13. A pharmaceutical composition according to claim 12 comprising a monoclonal antibody having a heavy chain comprising the sequence given in SEQ ID NO: 129 and a light chain comprising the sequence given in SEQ ID . 14. A pharmaceutical composition according to claim 1 comprising a onal antibody which specifically binds Tch comprising a heavy chain wherein the variable dOmain of the heavy chain comprises at least one of a CDR having the sequence given in SEQ ID NO:154 for CDR—H1, a CDR having the sequence given in 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 at least one of a CDR having the sequence given in SEQ ID NO:151 for CDR-Ll, 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.

. A pharmaceutical composition according to claim 14 comprising a monoclonal antibody having 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. 16. A pharmaceutical composition according to claim 1 sing a monoclonal antibody which specifically binds Tch having a heavy chain and a light chain wherein the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID N029, SEQ ID NO:19, SEQ ID NO:29 and SEQ ID N039 and the light chain variable region comprises a sequence ed from the group consisting of SEQ ID NO:7, SEQ ID NO:17, SEQ ID N027 and SEQ ID NO:37. 17. A ceutical composition ing to claim 5 comprising a monoclonal antibody which specifically binds Tch having a heavy chain and a light chain wherein the heavy chain variable region comprises a ce selected from the group consisting of SEQ ID N0269, SEQ ID NO:79, SEQ ID NO:89, SEQ ID NO:99, SEQ ID NO:109, SEQ ID NO:119, SEQ ID NO:139, SEQ ID NO:149 and SEQ ID NO:159 and the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NO:67, SEQ ID NO:77, SEQ ID NO:87, SEQ ID NO:97, SEQ ID NO:107, SEQ ID NO:117, SEQ ID NO:137, SEQ ID NO:147 and SEQ ID NO:157. 18. A pharmaceutical composition according to any one of claims 1 to 17, wherein at least one antibody in the composition is c to Tch and at least one antibody in the composition is specific to Tch. 19. A pharmaceutical composition according to claim 18, wherein the composition further comprises at least a second antibody specific to Tch.

. Use of the pharmaceutical composition according to any one of claims 1 to 19 in the manufacture of a medicament for the treatment or prophylaxis of Clostridium difi’zcz’le ion or cations therefrom. 21. A pharmaceutical composition according to any one of claims 1 to 19, substantially as herein described with reference to any one of the Examples and/or Figures thereof. 1/69 Figure 1 SEQ ID NO: 8 polynucleotide sequence encoding anti-toxin A antibody 922.g1 VK (ng) GACCCTGTGA TGACCCAGAG TCCGAGCACT CTTTCTGCCT CCGTGGGAGA CCGCGTGACC ATTACATGTC AGGCTTCACA AAGTATCTCC AATGCTCTGG CCTGGTATCA GCAGAAACCC GGCAAAGCCC CTAAGCTGCT CATCTACTCT GCATCAAGCC TGGCTAGCGG CGTGCCAAGC CGATTCAAGG GGAGCGGTTC TGGCACTGAG TTTACGCTGA CCATCAGTAG CTTGCAGCCT GACGATTTTG CAACCTATTA CTGCCAGTAC ACACACTACT CCCATACATC TAAAAACCCA TTCGGAGGGG GTACTAAGGT CGAAATAAAG SEQ ID NO: 10 polynucleotide sequence encoding anti-toxin A antibody 922.g1 VH(gH1) GAAGTGCAAT TGGTGGAAAG TGGCGGAGGA CTGGTGCAAC CCGGGGGTAG TCTGCGACTG AGCTGTGCTG CCTCCGGCTT TACCATTAGC TATA TGAGCTGGGT TCGACAGGCC CCTGGAAAAG GACTCGAATG GATCGGCATC ATATCTTCCG GTGGGCATTT CACCTGGTAC GCAAACTGGG CTAAGGGGAG ATTCACGATT AGCAGCGACT CCACAACCGT GTACCTGCAA ATGAACAGCC TGAGGGATGA GGACACTGCC ACATATTTCT GCGCACGCGC TTACGTGAGC GGAAGCTCAT TTAATGGCTA TGCACTGTGG GGGCAAGGAA TGAC TGTCTCG SEQ ID NO: 18 polynucleotide ce ng anti-toxin A antibody g1 ng GACGTCGTGATGACTCAGAGCCCATCTAGTCTGAGCGCTAGCGTCGGAGACCGAGTCACAATTACC TGTCAAGCCTCCCAGAGCATCTCCAACTACCTGGCCTGGTACCAACAGAAACCTGGCAAGGTGCCC AAGCTGCTGATCTATAGTGCTTCCACACTCGCAAGCGGCGTTCCGTCACGCTTTAAGGGATCTGGC TCTGGCACTCAGTTCACCTTGACGATCTCAAGCCTGCAGCCAGAAGATGTGGCCACCTATTACTGC CAGTATTCCCACTACGGGACTGGGGTGTTCGGTGCCTTTGGAGGTGGGACCAAAGTGGAGATAAAG Figure 2 SEQ ID NO: 20 polynucleotide sequence encoding anti-toxin A antibody CA923.g1 ng GAAGTTCAACTTGTGGAATCTGGAGGCGGGCTCGTGCAGCCTGGTGGAAGCCTTAGACTGAGCTGC GCTGCATCCGCATTTTCCCTGTCCAACTACTACATGAGCTGGGTGCGACAAGCACCAGGCAAGGGA CTGGAATGGATTGGCATCATAAGCTCCGGTTCCAATGCCCTGAAATGGTACGCATCATGGCCGAAA GGCCGCTTTACCATAAGCAAGGACTCCACCACCGTCTATCTGCAGATGAACTCATTGCGTGCCGAG GACACTGCAACGTACTTCTGTGCTCGCAACTACGTGGGAAGCGGATCTTATTATGGCATGGATCTG TGGGGACAAGGTACACTCGTGACCGTCTCG SEQ ID NO: 28 polynucleotide ce encoding anti-toxin A antibody CA993.g1 ng GATGTCGTGA TGACTCAGTC CCCCTCTACA TTGAGTGCCT CTGTCGGTGA TACC ATCACCTGTC AAGCAAGCCA CAGC TCCTACTTCT CTTGGTACCA GCAAAAGCCG GGAAAAGCCC TGCT GATTTATGGG GCCTCAACAC TGGCTTCTGG CGTGCCATCA AGATTCAAGG GATCTGGCTC CGGCACTGAG CTTACACTGA CCATTAGCTC CCTGCAACCT GACGATTTTG CTACCTACTA GTGC ACCGACTATA GTGGGATATA CGGA TTTGGGGGAG GGACGAAAGT GGAAATCAAG SEQ ID NO: 30 polynucleotide sequence encoding oxin A antibody CA993.g1 ng GAAGTTCAGC TGGTCGAGAG CGGAGGCGGA CTGGTGCAAC CTGGTGGTAG CCTGAAACTC TCTTGTACTG CCTCCGGGTT TTCCCTGAGC TCTTACTATA TGTCATGGGT GAGACAGGCT CCCGGGAAAG AATG GATCGGGATT ATCTCCTCCG GCTCTTCCAC CACA TGGTACGCCT CATGGGCAAA GGGGAGGTTT ACCATAAGCA AGACAAGCAC GACCGTGTAT CTTCAGATGA ACTCCCTGAA GACGGAGGAT ACTGCCACCT ACTTTTGCGC TCGGGCCTAT GTGGGCTCAA GCTCTTACTA TGGCTTCGAC CCATGGGGAC CACT TGTGACCGTC SUBSTITUTE SHEET (RULE 26) 2/69 Figure 3 SEQ ID NO: 38 polynucleotide sequence encoding anti-toxin A antibody 995.g1 VL region GACGTCGTGA TGACACAGAG CCCTTCAACA CTGTCTGCAA GCGTGGGCGA TAGGGTCACC ATAACGTGCC AGGCCTCTCA ATCCATCAAC AACTATTTTA GCTGGTACCA GCAGAAGCCA GCTC CGAAACTTCT GATCTACGGA GCTGCCAACC TGGCAAGTGG CGTGCCATCA CGGTTCAAGG GATCCGGGAG TGAG TATACCCTGA CCATTTCATC TCTCCAACCC TTCG CCACCTACTC CTGCCAGAAT GGCG TGCACATCTA TGGAGCTGCC TTTGGCGGTG GGACAAAAGT GGAAATTAAG SEQ ID NO: 40 polynucleotide sequence encoding anti-toxin A antibody 995.gl VH region GAAGTTCAGC TGGTCGAGAG AGGG CTTGTGCAAC CTGGTGGCTC CCTCCGTCTG AGCTGTACTG CTTCTGGATT CTCACTGAGC AATTACGACA TGATCTGGGT GCGACAGGCA CCCGGCAAAG GACTGGAGTA CATTGGCTTC ATCAACACCG GGGGTATAAC GTACTATGCC GCTA AGGGGCGCTT TACAATTAGT AGGGATTCCT CTACCGTGTA CCTGCAGATG AACTCACTGA GAGCCGAGGA CACTGCCACA TATTTCTGCG CTCGGGTGGA TATC GGGGCCTGGG GATT GTGGGGCCAA GGAACACTGG TCACCGTCTC G SEQ ID NO: 48 polynucleotide sequence encoding anti-toxin A antibody 997.g1 VL region GCACTCGTGATGACACAGAGCCCGAGTAGCTTTAGTGCTTCAACCGGTGATAGGGTCACTATTACT TGCCAAGCCTCTCAGAGTATATCTAGCTATCTGAGCTGGTACCAGCAAAAGCCCGGGAAGGCTCCT AAACTGCTGATCTACCGGGCTTCCACATTGGCCTCCGGCGTTCCCTCACGCTTTAGCGGCTCCGGA TCCGGAACCGAGTACACCCTGACTATCTCTTGCCTGCAATCTGAGGACTTCGCAACCTACTATTGT CTGGGCGTCTACGGATATAGCAACGATGACGGGATCGCCTTCGGCGGCGGTACCAAAGTGGAAATT Figure 4 SEQ ID NO: 50 polynucleotide sequence encoding anti-toxin A dy 997.g1 VH region GAGGTGCAACTTGTGGAAAGCGGGGGAGGACTGGTGCAGCCTGGGGGCTCATTGAGACTGAGCTGC ACCGTTTCTGGTATTGACCTGAGCTCCCATCATATGTGCTGGGTGCGCCAGGCACCCGGAAAAGGA CTGGAATACATCGGCGTCATATACCACTTTGGCTCTACATACTATGCCAACTGGGCAACTGGGCGA TTCACAATTAGCAAGGACTCAACTACCGTTTACCTGCAAATGAATAGCCTGAGGGCTGAGGATACT GCCACCTATTTCTGTGCCCGGGCTTCAATCGCCGGCTATTCTGCCTTTGATCCATGGGGGCAAGGA ACACTCGTGACCGTCTCG SEQ ID NO: 58 polynucleotide sequence encoding anti-toxin A antibody 1000.g1 VL region GAAATCGTGA TGACGCAGTC ACCAAGCACA CTGAGCGCTT CTGTGGGAGA TCGGGTCACA ATAACCTGTC AGGCCTCCCA GAGCATCTAC TCTTATCTGG CATGGTACCA GCAGAAGCCA GGGAAAGCTC CCAAGCTGCT GATTTATGAC ACTT TGGCTTCCGG TGTTCCTAGT AGGTTCAAAG GCTCCGGAAG CGAG TTTACCCTGA CCATCTCATC TCTGCAACCC GATGACTTTG CCACATACTA TTGCCAGGGG AATGCCTACA ACTC ACACGACAAC GCATTCGGGG GAGGCACCAA AGTCGAAATT AAG SEQ ID NO: 60 polynucleotide sequence encoding anti-toxin A antibody 1000.gl VH region GAAGTTCAGC TGGTCGAGAG GGGT TTGATTCAGC CCGGTGGCTC ACTTAGATTG AGCTGCACCG TGTCCGGAAT CGATCTGTCA TCTGATGCCG TGGGCTGGGT GGCA CCTGGGAAAG GACTGGAGTA TATAGGGATC ATCGCCACCT TCGACTCCAC ATACTACGCT AGCTGGGCAA AAGGGCGCTT TACGATTAGC TCCT CTACTACCGT GTACCTCCAA ATGAACTCAC CCGA TGCC ACTTATTTCT GTGCTCGGAC CGGTAGCTGG TACTACATCT CTGGCTGGGG CTCCTACTAT TATGGCATGG ACCTGTGGGG GACA CTCGTGACCG TCTCG SUBSTITUTE SHEET (RU LE 26) 3/69 Figure 5 SEQ ID NO: 68 polynucleotide sequence encoding oxin B antibody 926.gl VL region GATACCGTGCTGACCCAGAGCCCTGCTACATTGTCACTGAGCCCCGGGGAGAGGGCCACATTGAGC TGCCGGGCTTCAAAATCCGTGTCCACCCTCATGCACTGGTTTCAGCAAAAGCCCGGGCAGGCCCCA AAACTGCTGATCTACCTCGCATCTAACCTTGAATCTGGCGTGCCGGCCCGCTTTAGTGGCTCCGGA AGCGGAACCGACTTCACACTGACGATTAGCTCCCTGGAGCCTGAGGATTTCGCCGTGTACTATTGC CAGCAAACTTGGAATGACCCTTGGACTTTCGGGGGCGGTACTAAGGTCGAAATAAAG SEQ ID NO: 70 polynucleotide sequence encoding anti-toxin B antibody 926.gl VH region GAGGTGGAACTGCTCGAATCTGGTGGTGGGCTGGTGCAGCCCGGTGGATCTCTGAGATTGTCATGC GAGGCATCCGGCTTTACCTTTTCCAACTACGGAATGGCCTGGGTGAGACAGGCCCCAACGAAGGGG CTCGAATGGGTTACAAGCATCAGCTCTTCTGGGGGATCTACTTACTATCGCGATAGCGTCAAAGGC CGGTTTACCATTAGCCGAGATAATGCCAAATCAAGCCTGTATCTGCAAATGAACAGCCTGAGGGCT GAGGACACCGCCACATACTATTGTACAACCGTGATAAGGGGCTACGTGATGGACGCATGGGGACAG GGGACATTGGTTACCGTCTCG SEQ ID NO: 78 polynucleotide sequence ng anti-toxin B antibody 927.g2 VL region GACACACAGA TGACCCAGAG CCCATCCACT TTGTCTGCAT CCGTGGGCGA CCGAGTGACA ATCACCTGTA GAGCAAGCGG TTCCGTGAGC ACACTGATGC ATTGGTACCA GCAGAAGCCT GGGAAGGCTC TGCT CAAA AACC TTGCCTCCGG CGTTCCAAGC CGGTTTAGCG GTTCCGGATC TGGAACCGAG TTCACCCTGA CAAG CCTGCAACCC GACGACTTCG CCACCTACTA TTGCCACCAG AGCTGGAATA GCGACACGTT CGGGCAAGGC ACAAGGCTGG AAATCAAA SEQ ID NO: 80 polynucleotide sequence encoding oxin B antibody 927.g2 VH region GAGGTGCAAC TTGTGGAAAG CGGAGGGGGC GTGGTCCAAC CCGGAAGAAG TCTCCGTCTT TCTTGCGCCG CAAGTGGCTT CACCTTTTCC AACTACGGAA TGGCCTGGGT TCGACAAGCT CCTGGGAAAG GATTGGAGTG GGTGGCCACT ATCAACTATG GCAC GACACACTAC CGAGACTCTG TTAAGGGGCG CTTTACGATT TCCCGCGACA ATAGCAAGAG CACCCTCTAC CTGCAAATGA ATAGCCTCCG GGCCGAGGAT ACTGCTGTGT ACTATTGTAC CTCCATCTCA CGGAGCCACT ATTG CTGGGGACAA GGCACACTCG TGACTGTCTCG SUBSTITUTE SHEET (RULE 26) 2012/052222 4/69 Figure 6 SEQ ID NO: 88 polynucleotide sequence ng anti-toxin B antibody 1099.g2 VL region GACGTCCAGC TCACTCAATC TCCCTCCTTT CTGTCTGCTT GCGA TCGCGTGACA ATAACCTGCA CCAA ATCAATTAGC AACCATCTGG CATGGTATCA GGAGAAGCCT GGCAAAGCCA ATAAGCTGCT CTCC GGCTCAACTC CCGG AAGC CGATTTAGCG GATCTGGGAG CGGAACCGAG TTCACACTTA CCATTAGCTC CCTGCAACCG GAGGACTTCG CCACCTATTA CTGCCAGCAA TACGACGAAT ACCCCTATAC GTTCGGCCAA GGGACAAGAT TGGAAATCAA GCGTACG SEQ ID NO: 90 polynucleotide sequence encoding anti-toxin B antibody 1099.g2 VH region GAAGTTCAGC TGCAGGAATC TGGACCTGGC TTGGTGAAAC CAAGCGAGAC ACTTAGTCTC ACTTGCACCG GCTT CTCCCTTCAA TCCTACACGA TCTCTTGGGT GCGGCAACCA CCCGGGAAAG GACTGGAATG GATCGCAGCC GGGG GAGGGAGCAC CTATTACAAC TTGCCTCTCA AGAGCCGCGT GACCATATCC CGTGACACAA GCAAGAGCCA GGTTTCCCTG AAGCTGAGCT CCGTGACTGC TGCCGATACG GCTGTTTACT ATTGCACCCG CTGG TATCCCCGTT CCTATTTCGA CTACTGGGGA AGAGGCACAC TGGTTACCGT CTCG SEQ ID NO: 98 polynucleotide sequence encoding anti-toxin B antibody 1102.g4 VL region AACATCGTGC TGACACAGTC TCCTGCAACC CTTTCACTGT CTCCAGGTGA ACGAGCAACC CTGAGTTGTA GAGCCAGTCA GAGGATCTCC ACGAGCATTC ACTGGTATCA GCAAAAGCCT GGGCAAGCTC CCAGACTCTT GATCAAGTAC GCCTCTCAGA GCATAAGTGG AGCT AGGTTTAGCG GCTCAGGCTC AGGAACAGAC TTCACTCTGA GCTC CCTGGAACCG GAGGACTTTG ATTA CTGCCAGCAA TCCTACTCCA GTCTGTACAC CTTCGGGCAG GGTACTAAAC TGGAGATAAA G Figure 7 SEQ ID NO: 100 polynucleotide sequence encoding anti-toxin B antibody 1102.g4 VH region GAAGTGCAGC AATC CGGGGGAGGT TTGGTGCAAC CAGGTGGCTC ACTGAGACTG AGCTGTGCCG TTTCCGGCTT TACGTTCTCA GACAGTTATA TGGCCTGGGT GCGTCAAGCA CCTGGAAAAG GGCTGGAGTG GATTGCCAGT ATCAGCTATG GTGGGACCAT AATCCAGTAC GGCGATAGCG TCAAGGGCAG GTTTACTATC TCCAGGGACA ACGCCAAGTC AAGCCTTTAC CTGCAGATGA ATTCTCTCCG CGCAGAGGAT ACCGCTGTGT ATTACTGCGC TAGACGGCAG GGAACCTACG CTCGATACCT GGACTTCTGG GGTCAGGGAA CACTCGTTAC AGTCTCG SEQ ID NO: 108 polynucleotide sequence encoding anti-toxin B antibody 1114.g2 VL region GCGACGCAAA TGACTCAGTC GCCCTCATCG CTTAGCGCGT CCGTCGGAGA GACG ATCACCTGCC GCGCATCAGA GTCC ACACTCCTCC ACTGGTATCA GCAGAAACCG GGGAAGGCAC CAAAACTCTT GATCTACAAA GCCAGCAACC CCGG TGTCCCGTCA AGGTTCTCCG GGAGCGGTTC GGGGACAGAC TTTACTTTGA CCATTTCGTC GCCG GAGGACTTCG CCACCTATTA CTGTCATCAG TCATGGAACT CACCTCCCAC ATTTGGCCAG GGAACGAAAC TCGAAATCAA G SEQ ID NO: 110 polynucleotide sequence encoding anti—toxin B antibody 1114.g2 VH region GAAGTACAAC TCGTAGAGTC AGGGGGTGGG CTGGTCCAAC GCTC CCTTCGGCTT TCGTGTGCCG CCTCGGGATT CACGTTTAGC GGTA TGGCCTGGGT GAGGCAGGCA AAGG GTCTTGAGTG GGTAGCGATC ATCAACTATG ATGCAAGCAC CACCCACTAC AGGGATAGCG TCAAGGGACG CTTTACTATC AGCCGGGATA ATGCGAAATC CTCGCTCTAT CTGCAGATGA ACTCCCTCAG AGCCGAGGAC ACCGCAGTGT GCAC ACGATACGGA CGCTCGCACT ATTTCGACTA ACAG GGGACGCTCG TAACTGTCTC G SUBSTITUTE SHEET (RULE 26) /69 Figure 8 SEQ ID NO: 118 polynucleotide sequence encoding anti-toxin B antibody 1114.g8 VL region GACACGGTCC TGACTCAGTC GCCCTCATCG CTTAGCGCGT GAGA TAGAGTGACG ATCACCTGCC GCGCATCAGA GTCGGTGTCC ACACTCCTCC ACTGGTATCA GCAGAAACCG GCAC CAAAACTCTT GATCTACAAA GCCAGCAACC TTGCGTCCGG TGTCCCGTCA AGGTTCTCCG GTTC GGGGACAGAC TTGA CCATTTCGTC GCTTCAGCCG GAGGACTTCG CCACCTATTA CTGTCATCAG TCATGGAACT CACCTCCCAC ATTTGGCCAG GGAACGAAAC TCGAAATCAA G SEQ ID NO: 120 polynucleotide sequence ng anti-toxin B antibody 1114.g8 VH region GAAGTACAAC TCGTAGAGTC AGGGGGTGGG CTGGTCCAAC GCTC CCTTCGGCTT TCGTGTGCCG CCTCGGGATT CACGTTTAGC AATTACGGTA TGGCCTGGGT GAGGCAGGCA AAGG GTCTTGAGTG GGTAGCGATC ATCAACTATG ATGCAAGCAC CACCCACTAC AGGGATAGCG TCAAGGGACG CTTTACTATC AGCCGGGATA ATGCGAAATC CTCGCTCTAT ATGA ACTCCCTCAG AGCCGAGGAC ACCGCAGTGT ACTATTGCAC ACGATACGGA CGCTCGCACT ATTTCGACTA TTGGGGACAG GGGACGCTCG TAACTGTCTC G SEQ ID NO: 128 polynucleotide sequence ng anti-toxin B antibody 1125.g2 VL region GATATACAAA TGACTCAGAG CCCTAGCTCA CTGAGCGCTT CTGTGGGCGA TCGTGTGACA ATCACTTGCA AAGCAAGCCA GAACATCTAT ATGTACCTGA ATTGGTACCA GCAAAAACCG GGAAAAGCTC CCAAGCGCCT GATTTACAAC ACCAATAAGC TGCATACCGG CGTGCCAAGC CGTTTTAGCG GATCTGGCTC TGGAACCGAA CTGA CCATAAGCTC CCTGCAACCG GAAGACTTTG ACTA TTGCCTCCAG CACAAATCCT TCCCCTATAC GTTCGGACAA GGGACCAAAC TGGAAATCAA A SEQ ID NO: 130 polynucleotide sequence encoding anti-toxin B antibody 1125.g2 VH region CAGC AAAG CGGCGGAGGA TTGGTGCAAC CTGGTGGCTC TCTTCGCCTG TCTTGCGCTG CAAGCGGCTT TACGTTCCGC GATAGCTTTA TGGCTTGGGT GCGACAAGCT CCTGGGAAAG GGCTGGAATG GGTCGCTAGC TACG AAGGCGACAA GACTTACTAT GGGGACTCTG TGAAAGGCCG ATTCACCATT AGCCGAGACA ACGCAAAGAA GTAC CTGCAGATGA TGCG TGCCGAAGAT ACCGCCGTGT ACTATTGCGC TAGGCTGACG ATCACTACAA GCGGAGATAG CTGGGGACAA GGGACAATGG TGACCGTCTC GAGC SEQ ID NO: 138 polynucleotide sequence encoding anti-toxin B antibody 1129.g1 VL region GACACCCAGA AGTC TCCGTCAAGC CTTTCTGCCT CTGTTGGAGA TCGAGTCACA ATTACGTGCA GCCA ACACGTGGGT ACCAACGTGG ACTGGTATCA ACAGAAGCCA GGGAAGGTCC CCAAACTGCT GATCTACGGT GCCAGTATTC GCTATACCGG CGTGCCTGAT CGCTTCACCG GAAGCGGGTC CGAT TTCACACTGA CAATCAGCTC CCTGCAACCT GAAGACGTGG CTACTTACTA CTGCCTGCAG TACAACTATA ATCCCTACAC CTTTGGCCAG GGCACCAAAC TGGAGATAAA G SEQ ID NO: 140 polynucleotide sequence ng anti-toxin B antibody 1129.g1 VH region GAGGTGCAAC AATC AGGAGGTGGC GTGGTTCAGC CCGGTAGATC ACTTCGTCTG AGTTGTGCAA CAAGCGGCTT CTCC AACTTCGGGA TGTCTTGGGT TAGACAGGCT AAGG GCCTCGAATG GGTGGCTAGT ATTAGCCCAA GCGGGGGAAA CGCCTACTAT AGGGACAGCG TGAAAGGACG CTTCACTATC AGCCGAGATA ACTCCAAGAC CACGCTGTAT CTGCAGATGA ATAGTCTGAG GGCCGAGGAT ACCGCAGTGT ACTACTGCAC TCGACGGGCC TATTCTTCCC CTTTTGCCTT TTGGGGACAG GGGACTCTGG TGACAGTCTC GAGC SUBSTITUTE SHEET (RULE 26) 6/69 Figure 9 SEQ ID NO: 148 polynucleotide sequence encoding anti-toxin B antibody 1134.g5 VL region GACGTCCAGC AATC TCCCTCCTTT CTGTCTGCTT CTGTGGGCGA TCGCGTGACA ATAACCTGCA AGGCCTCCAA ATCAATTAGC AACCATCTGG CATGGTATCA GGAGAAGCCT GGCAAAGCCA ATAAGCTGCT GATCCACTCC GGCTCAACTC TGCAACCCGG TACCCCAAGC CGATTTAGCG GATCTGGGAG CGGAACCGAG CTTA CCATTAGCTC CCTGCAACCG GAGGACTTCG CCACCTATTA CTGCCAGCAA TACGACGAAT ACCCCTATAC GTTCGGCCAA GGGACAAGAT TGGAAATCAA G SEQ ID NO: 150 polynucleotide sequence encoding anti-toxin B antibody 1134.g5 VH region GAAGTTCAGC TGCAGGAATC TGGACCTGGC TTGGTGAAAC AGAC ACTTAGTCTC ACTTGCACCG TTTCCGGCTT CTCCCTTAAT TCCTACACGA TCACTTGGGT GCGGCAACCA CCCGGGAAAG GACTGGAATG AGCC ATTAGCGGGG GAGGGAGCAC CTATTTCAAC TCGGCTCTCA AGAGCCGCGT GACCATATCC CGTGACACAA GCAAGAGCCA GGTTTCCCTG AAGCTGAGCT CCGTGACTGC TGCCGATACG GCTGTTTACT ATTGCACCCG ACCTCGCTGG TATCCCCGTT CCTATTTCGA CTACTGGGGA AGAGGCACAC CCGT CTCG SEQ ID NO: 158 polynucleotide sequence encoding oxin B antibody 1151.g4 VL region CAAA TGACTCAGTC GCCCTCATCG CTTAGCGCGT CCGTCGGAGA TAGAGTGACG ATCACGTGCA AAGCATCACA AAATGTCGGG AACAATGTGG CATGGTATCA ACCG GGGAAGGCAC CAAAACTCTT GATCTACTAC GCCAGCAACA CTGG TGTCCCGTCA AGGTTCACGG GAGGGGGTTA CGGGACAGAC TTTACTTTGA CCATTTCGTC GCCG GAGGACTTCG CCACCTATTA GAGG GTCTACCAGT GGAC CCAG GGAACGAAAG TGGAAATCAA G Figure 10 SEQ ID NO: 160 poiynucleotide sequence encoding anti-toxin B antibody 1151.g4 VH region GAAGTACAAC TCCAAGAGTC GGGGCCTGGT CTGGTCAAGC CGTCCGAAAC ACTTTCGCTG ACGTGTACGG TATCAGGATT CTCACTTACA TCATACTACG TCCACTGGGT GAGGCAGCCA CCCGGGAAGG GTCTTGAGTG GATGGGCTGC ATTAGAACCG GAGGGAATAC CGAGTACCAG AGCGAATTTA AGAGCCGCGT CACTATCAGC CGGGATACGT CCAAAAACCA GGTGTCGCTC AAATTGTCCT CCGTGACGGC CGCTGACACC TACT ATTGCGCGCG AGGAAACTAT GGCTTTGCGT ATTGGGGACA GGGGACGCTC GTAACTGTCT CG SEQ ID NO: 168 polynucleotide sequence encoding anti-toxin B antibody 1153.g8 VL region GATATACAGA TGACTCAGTC CCCTTCTAGC CTTTCAGCTT GCGA TAGAGTGACT ATCACGTGTA AGGCTAGTCA TAAC AAGTATCTGG ACTGGTACCA GCAGAAACCC GGGAAGGTTC CCAAGCTGCT GATCTACAAC ATCCAGTCCC TGCATACAGG CATTCCTAGC CGGTTTAGCG GATCTGGTTC CGAC TTCACCCTGA CAATCAGCTC TCTGCAACCA GAAGACGTGG CCACCTATTA CTGCTTCCAG CACAATAGTG GCTGGACTTT TGGACAAGGT ACCAGGCTGG AGATCAAA SEQ ID N0: 170 polynucleotide ce encoding anti-toxin B dy 1153.g8 VH region GAGGTTCAGC TGGTGGAATC AGGAGGGGGT CTGGTGCAAC CAGGAGGCTC CCTGAAACTG TCTTGCGCCG CAAGCGGCTT TACGTTTACC CAGGCCGCTA TGTTCTGGGT TAGGCAGGCC AAGG AAGG AAGA ATCAGCACCA AGAGCAACAA TTTCGCTACG TACTATCCGG ACTCCGTGAA AGGCCGGTTT ACCATTTCTC GCGATGACAG CAAGAACACC CTGC AGATGAACAG TCTCAAGACC GAGGACACAG CCGTGTACTA TTGTACTGCT CCCGCCTATT ATTACGATGG CACAGTGCCT TTCGCATACT GGGGACAGGG TACTTTGGTG ACTGTCTCG SUBSTITUTE SH EET (RU LE 26) 7/69 Figure 11 Sera titres from 4 rabbits immunised with Tch toxoid and 5 rats immunised with Tch binding domain (TchIZ34). ELISA data generated using Tch toxin or Tch binding domain coated on an ELISA plate Rabbit Terminal sera titre against Tch «mm-- Oontroi sera Dilution factor Rat al sera titre against Tch1234 ~0— Oontrol sera Dilution factor SUBSTITUTE SHEET (RULE 26) 8/69 Figure 12 Anti Tch (Ribotype 003) in-vitro neutralization data for single Mabs (X axis cone. (ng/ml) and Y axis % Neutralization) lisation conc. n / ml Neutralisafion SU BSTITUTE SH EET (RU LE 26) 9/69 Figure 13 Anti Tch (Ribotype 003) in-vitro neutralization data for single Mabs (X axis cone. (ng/ml) and Y axis % lization) Neutralisation Neutralisafion 922+923 Neutralisafion SUBSTITUTE SHEET (RULE 26) /69 Figure 14 Anti Tch (Ribotype 003) in-vitro neutralization data for paired Mabs (X axis conc. (ng/ml) and Y axis % Neutralization) 922+925 Neutralisation Neutralisafion SUBSTITUTE SHEET (RULE 26) 11/69 Figure 15 Anti Tch (Ribotype 003) in-vitro neutralization data for paired Mabs (X axis conc. ) and Y axis % Neutralization) Neutralisation 923+1000 Neutralisation SUBSTITUTE SHEET (RULE 26) 2012/052222 12/69 Figure 16 Anti Tch (Ribotype 003) in-vitro neutralization data for three Mab mixtures (X axis conc. (ng/ml) and Y axis % Neutralization) 922+923+995 Neutralisafion 922+923+997 Neutralisafion SUBSTITUTE SHEET (RULE 26) 13/69 Figure 17 Anti Tch (Ribotype 003) in-vitro neutralization data for three Mab mixtures (X axis conc. (ng/ml) and Y axis % Neutralization) +997 Neutralisafion 922+997+1000 Neutralisafion SUBSTITUTE SH EET (RULE 26) 2012/052222 14/69 Figure 18 Anti Tch (Ribotype 003) in—vitro neutralization data for three Mab mixtures (X axis conc. (ng/ml) and Y axis % Neutralization) 923+995+997 Neutralisation Neutralisation SUBSTITUTE SHEET (RULE 26) /69 Figure 19 Anti '1‘ch (Ribotype 003) in-vitro neutralization data for four and five Mab mixtures (X axis cone. (ng/ml) and Y axis % Neutralization) 922+923+995+997 lisafion Neutralisation SUBSTITUTE SHEET (RULE 26) WO 38156 16/69 Figure 20 Anti Tch (Ribotype 003) in-vitro neutralization data for four and five Mab mixtures (X axis conc. (ng/ml) and Y axis % Neutralization) 923+995+997+1000 Neutralisa’don Neutralisafion SU BSTITUTE SH EET (RU LE 26) 17/69 Figure 21 Anti Tch (Ribotype 003) ro neutralization data for single and paired Mabs at different Tch concentrations (X axis is conc ng/ml) Neutraiization LD max (circle) LD 95 (triangle) LD 90 (square) % LD 80 (diamond) w'fi ‘tw$55 Mr)» 9’;«a Neutralization z s:é; at; ’1’} LD max (Circle) LD 95 (triangle) 0/0 LD 90 (square) LD 80 (diamond) Neutralization ‘3." egg}! WWWW 2“ ; 'W‘XFIVAbIOOO > 3 LD max (circle) f 1 LD 95 (triangle) wt??? LD 90 (square) €33) E LD 80 (diamond) SUBSTITUTE SHEET (RULE 26) 18/69 Figure 22 Anti '1‘ch (Ribotype 003) in—vitro neutralization data for single and paired Mabs at different Tch concentrations (X axis is cone. ng/ml) M 6% c”; {5 Ab 922 ~12»,~22; LD max (circle) 4..) LD 95 (triangle) (D LD 90 (square) O LD 80 (diamond) Neutralization Ab 922+997 LD max (circle) LD 95 (triangle) LD 90 (square) 0/0 LD 80 nd) Neutralization Ab 922+1000 LD max e) LD 95 (triangle) LD 90 (square) LD 80 (diamond) SUBSTlTUTE SHEET (RULE 26) 19/69 Figure 23 Anti '1‘ch (Ribotype 003) ro neutralization data for single and to five Mab mixtures at different Tch concentrations (X axis cone. ng/ml) Neutraiization 4.3";a; .3145. €272 fl 7.? LD max (circle) 3 ‘ ’6 l ' g f l; ”f, 3 LD 95 (triangle) LD 90 (square) 837’ % LD 80 nd) Neutralization f1}? if: Ab 997+1009 WW.___. ’ ” jfle' LD max (circie) :5 3; LD 95 (triangle) w" LD 90 (square) LD 80 (diamond) Neutralization _/ Ab 922+ 997+1000 mM...... 77777 if...“ ..... é”; If E? LD max (circle) iii LD 95 (triangle) , ! f LD 90 (square) 0/0 LD 80 (diamond) SUBSTITUTE SHEET (RULE 26) /69 Figure 24 Anti Tch (Ribotype 003) in-vitro neutralization data for single and to five Mab mixtures at different Tch concentrations (X axis is cone. ng/ml yst ‘ x’ LD max (circle) 4:" l 3‘ } 3:" l LD 95 (triangle) k} 455* LD 90 (square) ‘33) LD 80 (diamond) Neutralization Wmmmqunmq Ab922+997+1000+995 fl iii (I 3 LD max (circle) a E LD 95 (triangle) ' V 0/0 { LD 90 e) LD 80 (diamond) w}. R}(if! ”Jo at? ‘65 a: 1—3- Ab922+997+1000+995 +923 g ‘3? LD max (circle) a 33 LB 95 gle) 2 LD 90 (square) a" 3 LD 80 (diamond) SUBSTITUTE SHEET (RULE 26) 21/69 Figure 25 Anti Tch (Ribotype 003) in—vitro neutralization data for single Mabs (Y axis neutralization X axis conc ng/ml for 1125.g2, 1134.g5 and 927.g2 respectively) Neutralisation .._. lisafion SUBSTITUTE SHEET (RU LE 26) 22/69 Figure 26 Anti Tch (Ribotype 003) in-vitro neutralization data for single Mabs Y axis lization X axis cone ng/ml for1153.g8 and 1102.g4 respectively) .‘12 Neutralisafion SUBSTITUTE SHEET (RULE 26) 23/69 Figure 27 Anti Tch (Ribotype 003) in-vitro neutralization data for paired Mabs Y axis neutralization X axis conc ng/ml for combinations of 927+1099, 927+1102, 927+1114 respectively) 927+1099 Neutralisafion 927+‘l 102 Neutralisa’u’on SUBSTITUTE SHEET (RULE 26) 24/69 Figure 28 Anti Tch (Ribotype 003) in—vitro neutralization data for paired Mabs (Y axis neutralization X axis conc ng/ml for combinations of 25, 927+1134, 1099+1114 respectively) 927+1125 Neutralisafion 927+1 134 1099+1114 Neutralisafion SUBSTITUTE SHEET (RULE 26) /69 Figure 29 Anti Tch (Ribotype 003) in-vitro neutralization data for paired Mabs (Y axis neutralization X axis conc ng/ml for combinations of 1102+1114, 125, 1114+1134 respectively) 1102+1114 Neutralisatjon Neutralisafion SU BSTITUTE SHEET (RULE 26) 26/69 Figure 30 Anti Tch ype 003) in—vitro neutralization data for paired Mabs (Y axis neutralization X axis conc ng/ml for combinations of 1114+1151, 1114+1153, 1125+1134 respectively) 1114+1151 Neutralisafi 1114+1153 Neutralisation SUBSTITUTE SHEET (RULE 26) 27/69 Figure 31 Anti Tch (Ribotype 003) in-vitro neutralization data for three Mab mixtures (Y axis neutralization X axis conc ng/ml for combinations of 1125+1134+1114, l34+927, 1125+1151+1114 respectively) 1125+1134+1114 Neutralisafion Neutralisation "('6 SUBSTITUTE SHEET (RULE 26) 28/69 Figure 32 Anti Tch (Ribotype 003) in-vitro neutralization data for three Mab mixtures (Y axis neutralization X axis cone [1ng for 1125.+1151+927, 1125.g2+1134.g5+927.g2 respectively) 151+927 Neutralisafion 1125.gz+1134.gs+927.gz 1125.g2+1134.95+1153.g8 Neutralisafion SU BSTITUTE SHEET (RULE 26) 29/69 Figure 33 Anti Tch (Ribotype 003) ro neutralization data for three Mab mixtures 1125.gz+1134.gs+11o2.g4 SU BSTITUTE SHEET (RULE 26) /69 Figure 34 Anti Tch (Ribotype 003) in-vitro neutralization data for two Mab mixtures at ent toxin concentrations CA164_1125 + CA164_927 - TCdB strain VP! 10463 (LD60) percentage neutralisation 1 00 (LD77) percentage neutralisation CA164_1125 + CA164_927 - Tch strain VP! 10463 (LD85) percentage neutralisation SUBSTITUTE SHEET (RULE 26) 31/69 Figure 35 Anti Tch (Ribotype 003) in—vitro neutralization data for two Mab mixtures at different toxin concentrations 1125 + CA164_1102 - Tch strain VPI 10463 (LD60) percentage neutralisation CA164__1125 + CA164_1102 — Tch strain VPI 10463 (LD77) percentage neutralisation 1125 + CA164_1102 — Tch strain VPI 10463 (LD85) percentage neutralisation SU BSTITUTE SH EET (RU LE 26) WO 38156 32/69 Figure 36 Anti 1‘ch (Ribotype 003) in-vitro neutralization data for two Mab es at different toxin concentrations CA164_1125 + CA164_1114 - Tch strain VPI 10463 (LD60) percentage neutralisation CA164_1125 + CA164_1114 - Tch strain VPI 10463 (LD77) percentage neutralisation 1125.2 + 1114.

CA164__1125 + CA164__1114 — Tch strain VPI 10463 (LD85) percentage neutralisation SUBSTITUTE SH EET (RULE 26) 33/69 Figure 37 Anti Tch (Ribotype 003) in—vitro neutralization data for two Mab mixtures at different toxin concentrations CA164_1125 + CA164_1134 - Tch strain VPI 10463 (LD60) percentage lisation 1125.2 + 1134.5 n-lml CA164_1125 + CA164_1134 - Tch strain VPI 10463 (LD77) percentage neutralisation percentage neutralisation SUBSTITUTE SH EET (RULE 26) 2012/052222 34/69 Figure 38 Anti 1‘ch (Rihotype 003) in-vitro neutralization data for two Mab mixtures at different toxin concentrations CA164_1125 + CA164_1151 - Tch strain VPI 10463 (L060) percentage neutralisation CA164_1125 + CA164_1151 - Tch strain VPI 10463 (LD77) percentage neutralisation 1125.2 + 1151. 1125 + CA164_1151 — Tch strain VP! 10463 (LD85) percentage neutralisation SUBSTITUTE SH EET (RU LE 26) /69 Figure 39 Anti 1‘ch (Ribotype 003) in-vitro neutralization data for two Mab mixtures at different toxin concentrations CA164_1125 + CA164_1153 - Tch strain VPI 10463 (LD60) percentage neutralisation CA164_1125 + _115-3 Tch 'strain VPI 10463 (LD77) percentage lisation CA164_1125 + CA164_1153 - Tch strain VPI 10463 (LD85) percentage neutralisation SUBSTITUTE SHEET (RULE 26) 36/69 Figure 40 Anti 1‘ch ype 003) in-vitro neutralization data for two Mab mixtures at different toxin concentrations CA164_1125 + CA164_1134 (25:75) - Tch strain VPI 10463 (LD60) percentage neutralisation 1125.2 + 1134.5 25:75 n/ml CA164_1125 + CA164_1134 (25:75) - Tch strain VPI 10463 (LD77) tage neutralisation 1125.2 + 1134.35 25:75 nulmi CA164_1125 + +CA164_1134 (25:75) - Tch strain VPI 10463 (LD85) percentage neutralisation 100 2 + 1134.5 25:75 no/ml SUBSTITUTE SHEET (RULE 26) 37/69 Figure 41 Anti Tch (Ribotype 003) in-vitro neutralization data for two Mab mixtures at different relative Mab ratios and different toxin concentrations CA164_1125 + CA164_1151 (25:75) - Tch strain VPI 10463 (LD60) percentage neutralisation CA164_1125 + CA164__1151 ) - Tch strain VPl 10463 (LD77) percentage neutralisation 1125 + CA164_1151 (25:75) - Tch strain VPI 10463 (LD85) percentage neutralisation SUBSTITUTE SHEET (RULE 26) 38/69 Figure 42 Anti 1‘ch (Ribotype 003) in-vitro lization data for two Mab mixtures at different relative Mab ratios and different toxin concentrations CA164_1125 + CA164_1153 (25:75) - Tch strain VPI 10463 (LDSO) tage neutralisation CA164,1125 + CA164_1153 (25:75) — Tch strain VPI 10463 (LD77) percentage neutralisation 1 10 1125.02 +1153.

CA164_1125 + CA164__1153 (25:75) - Tch strain VPI 10463 (L085) percentage neutralisation SUBSTITUTE SHEET (RULE 26) 39/69 Figure 43 Anti Tch (Ribotype 003) in-vitro neutralization data for two Mab mixtures at different relative Mab ratios and different toxin trations CA164_1125 + CA164_1134 (75:25) - Tch strain VPI 10463 (LD60) percentage neutralisation CA164__1125 + CA164__1134 (75:25) - Tch strain VPI 10463 (LD77) percentage lisation . 10 100 2 + 1134.05 7525 nu/mt CA164_1125 + CA164_1134 (75:25) - Tch strain VPI 10463 (LD85) percentage neutralisation SUBSTITUTE SHEET (RULE 26) 40/69 Figure 44 Anti 1‘ch (Ribotype 003) ro neutralization data for two Mab mixtures at different relative Mab ratios and different toxin concentrations CA164_1125 + CA164_1151 (75:25) - Tch strain VPI 10463 (LDBO) percentage neutralisation CA164__1125 + CA164__1151 (75:25) - Tch strain VPI 10463 (LD77) percentage lisation 0.1 1 10 1125.2 +1151.

CA164_1125 + CA164_1151 (75:25) - Tch strain VPI 10463 (LD85) percentage neutralisation SUBSTITUTE SHEET (RULE 26) 41/69 Figure 45 Anti 1‘ch (Ribotype 003) in-vitro neutralization data for two Mab mixtures at different relative Mab ratios and different toxin concentrations CA164_1125 + CA164_1153 (75:25) - Tch strain VPI 10463 (LD60) tage neutralisation CA164_1125 + 1153 (75:25) - Tch strain VPI 10463 (LD77) percentage neutralisation SUBSTITUTE SH EET (RULE 26) 42/69 Figure 46 Tch strain VPI 10463 neutralisation, Antibody singles and pairs, Constant toxin dose (LD80) Vfii$§e neaéérsasaiéan f .926 (star) 112:5 (circle) 11._34+~~1125 (square). §€fz§a§§8§§§ $39§§§¥3§§2$32§§a 3‘538‘ \ 1134 (star) $33i \ 1125 (circle) 3;} 1134+1125 (square)W $3 flex 3‘.~**"*me gg ‘5;wa ”'23 $3"? 3 $3 ”33%. “KKK?, §n§o§ mm? mama-gel mafisfimfiw 1102 (star) 1125 e) 8»? ’ 1102+1125 (square) \afiiii‘fliiQ mm: SUBSTITUTE SHEET (RULE 26) 43/69 Figure 47 Tch neutralisation, Antibody singles and pairs, Constant toxin dose (LDSO) §€f£€§§i$§£ 22233223283299 1151 (star) “93%. 1125 (circie) 1151+1125 (square) gammmga afim “$29 927 (star) “$99"' 1125 (circle) mrt: 25 (square) {3"V1) 6;: w!!- ”A; .4 ’2»; Ma 2123£72 145. ‘LJ f22ff» 3:32 . 1114 (star) 1125 (circle) 92 1114+1125 (square) SUBSTITUTE SHEET (RULE 26) 44/69 Figure 48 Tch neutralisation, Antibody s and pairs, Constant toxin dose (LDSO) Tch strain VPl 10463 (LD80) percentage neutralisation 100 [antibody] ng/ml fir 1125 O 1153 I 1125+1153 SUBSTITUTE SHEET (RULE 26) 45/69 Figure 49 Tch neutralisation, dy singles and pairs, Varying toxin dose (straddling) Tch strain VPI 10463 (LD7’5) percentage neutralisation Tch strain VPI 10463 (LD86) percentage neutralisation Tch strain VPI 10463 (LDQO) percentage neutralisation SUBSTITUTE SHEET (RULE 26) 46/69 Figure 50 '1‘ch neutralisation, Antibody singles and pairs, Varying toxin dose (straddling) Tch strain VP] 10463 (LD75) percentage lisation percentage neutralisation Tch strain VPI 10463 (LDQO) percentage neutralisation SUBSTITUTE SHEET (RULE 26) 47/69 Figure 51 Tch neutralisation, Antibody singles and pairs, Varying toxin dose (straddling) Tch strain VP! 10463 (L075) percentage neutralisation ‘1'ch strain VPII 10463 (LD86) percentage neutralisation 1134.05 nI/ml TCdB strain VPI 10463 (LDQO) percentage lisation 0.1 10 SU BSTITUTE SHEET (RULE 26) 48/69 Figure 52 Tch neutralisation, Antibody singles and pairs, Varying toxin dose (straddling) TCdB strain VP! 10463 (LD75) percentage neutralisation tage neutralisation percentage neutralisation SUBSTITUTE SHEET (RULE 26) 49/69 Figure 53 Tch neutralisation, Antibody s and pairs, Varying toxin dose (straddling) Tch strain VPl 10463 (LD75) percentage neutralisation Tch strain VPl 10463 (LD86) percentage neutralisation Tch strain VP]. 10463 (LD90) percentage neutralisation SUBSTITUTE SHEET (RULE 26) 50/69 Figure 54 Tch neutralisation, Antibody s and pairs, Varying toxin dose (straddling) Tch strain VPI 10463 (LD75) percentage neutralisation Tch strain VPI 10463 (LD86) percentage neutralisation Tch strain VPI 10463 (LDQO) percentage neutralisation SUBSTITUTE SHEET (RULE 26) 51/69 Figure 55 Tch neutralisation, Antibody singles and pairs, Varying toxin dose (straddling) Tch strain VPI 10463 (LD75) percentage neutralisation tage neutralisation Tch strain VP! 10463 (LD90) percentage neutralisation SUBSTITUTE SHEET (RULE 26) 52/69 Figure 56 Tch neutralisation, Antibody singles and pairs, g toxin dose (straddling) Tch strain VPI 10463 (LD75) percentage neutralisation Tch strain VPI 10463 (LD86) percentage neutralisation 1125.2 + 1153.08 n0/ml Tch strain VPI 10463 (LDQO) percentage neutraiisation SUBSTITUTE SH EET (RULE 26) 53/69 Figure 57 Tch lisation, Antibody singles and pairs, Varying toxin dose (straddling) Tch strain VPI 10463 (LD75) percentage neutralisation Tch strain VPI 10463 (LD86) percentage neutralisation percentage neutralisation SU BSTITUTE SHEET (RULE 26) PCT/G32012/052222 54/69 Figure 58 Tch neutralisation, Antibody singles and pairs, Varying toxin dose (straddling) Tch strain VPI 10463 (LD75) percentage neutralisation .4 25:75 no/ml Tch strain VPI 10463 (L086) percentage lisation 100 02 + 1151.4 25:75 no/mi Tch strain VPI 10463 (LDQO) percentage neutralisation SUBSTITUTE SHEET (RULE 26) WO 38156 55/69 Figure 59 Tch neutralisation, Antibody singles and pairs, Varying toxin dose (straddling) Tch strain VP! 10463 (LD75) percentage neutralisation Tch strain VPI 10463 (LD86) percentage neutralisation Tch strain VPI 10463 (LD90) percentage neutralisation SU BSTITUTE SHEET (RU LE 26) 56/69