U.S. patent application number 16/919576 was filed with the patent office on 2020-12-03 for neutralising antibodies to the major exotoxins tcda and tcdb of clostridium difficile.
This patent application is currently assigned to UCB Biopharma SPRL. The applicant listed for this patent is UCB Biopharma SPRL. Invention is credited to Joanne Elizabeth Compson, Matthew Cox, Nicola Louise Fisher, Karine Jeannine Madeleine Herve, David Paul Humphreys, David Edward Ormonde Knight, Daniel John Lightwood, Brendon MacKenzie, Matthew Jon Timothy Page, Andrew Charles Payne, Kerry Louise Tyson.
Application Number | 20200377578 16/919576 |
Document ID | / |
Family ID | 1000005030693 |
Filed Date | 2020-12-03 |
View All Diagrams
United States Patent
Application |
20200377578 |
Kind Code |
A1 |
Humphreys; David Paul ; et
al. |
December 3, 2020 |
NEUTRALISING ANTIBODIES TO THE MAJOR EXOTOXINS TCDA AND TCDB OF
CLOSTRIDIUM DIFFICILE
Abstract
This present invention describes the derivation and selection of
antibodies capable of neutralising the major exotoxins; TcdA and
TcdB of Clostridium difficile. The invention also describes novel
neutralisation and antigen binding properties of individual Mabs
and mixtures thereof.
Inventors: |
Humphreys; David Paul;
(Slough, GB) ; Lightwood; Daniel John; (Slough,
GB) ; Tyson; Kerry Louise; (Slough, GB) ;
Knight; David Edward Ormonde; (Slough, GB) ; Herve;
Karine Jeannine Madeleine; (Slough, GB) ; Compson;
Joanne Elizabeth; (Slough, GB) ; Page; Matthew Jon
Timothy; (Slough, GB) ; Payne; Andrew Charles;
(Slough, GB) ; Fisher; Nicola Louise; (Slough,
GB) ; MacKenzie; Brendon; (Slough, GB) ; Cox;
Matthew; (Slough, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UCB Biopharma SPRL |
Brussels |
|
BE |
|
|
Assignee: |
UCB Biopharma SPRL
Brussels
BE
|
Family ID: |
1000005030693 |
Appl. No.: |
16/919576 |
Filed: |
July 2, 2020 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14344637 |
Jan 20, 2015 |
10752676 |
|
|
PCT/GB2012/052222 |
Sep 10, 2012 |
|
|
|
16919576 |
|
|
|
|
61535532 |
Sep 16, 2011 |
|
|
|
61638731 |
Apr 26, 2012 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 45/06 20130101;
G01N 33/6854 20130101; A61K 2039/507 20130101; C07K 2317/92
20130101; G01N 2333/91097 20130101; A61K 2039/545 20130101; C07K
2317/94 20130101; G01N 33/56911 20130101; C07K 2317/76 20130101;
A61K 2039/505 20130101; G01N 33/573 20130101; A61K 39/40 20130101;
C07K 16/1282 20130101 |
International
Class: |
C07K 16/12 20060101
C07K016/12; A61K 39/40 20060101 A61K039/40; A61K 45/06 20060101
A61K045/06; G01N 33/569 20060101 G01N033/569; G01N 33/573 20060101
G01N033/573; G01N 33/68 20060101 G01N033/68 |
Claims
1-41. (canceled)
42. A pharmaceutical composition comprising one or more monoclonal
antibodies specific to antigen TcdB1234 wherein the antibody has
high affinity of 600 pM or less for the target antigen TcdB1234 and
said one or more monoclonal antibodies are independently selected
from: i) a heavy chain wherein the variable domain of the heavy
chain comprises a CDR having the sequence given in SEQ ID NO:124
for CDR-H1, a CDR having the sequence given in SEQ ID NO: 125 for
CDR-H2 and a CDR having the sequence given in SEQ ID NO: 126 for
CDR-H3, and a light chain wherein the variable domain of the light
chain comprises a CDR having the sequence given in SEQ ID NO:121
for CDR-L1, a CDR having the sequence given in in SEQ ID NO:122 for
CDR-L2 and a CDR having the sequence given in SEQ ID NO:123 for
CDR-L3; ii) a heavy chain wherein the variable domain of the heavy
chain comprises a CDR having the sequence given in SEQ ID NO:154
for CDR-H1, a CDR having the sequence given in SEQ ID NO:155 for
CDR-H2 and a CDR having the sequence given in SEQ ID NO:156 for
CDR-H3, and a light chain wherein the variable domain of the light
chain comprises a CDR having the sequence given in SEQ ID NO:151
for CDR-L1, a CDR having the sequence given in in SEQ ID NO:152 for
CDR-L2 and a CDR having the sequence given in SEQ ID NO:153 for
CDR-L3; iii) a heavy chain wherein the variable domain of the heavy
chain comprises a CDR having the sequence given in SEQ ID NO:144
for CDR-H1, a CDR having the sequence given in SEQ ID NO:145 for
CDR-H2 and a CDR having the sequence given in SEQ ID NO:146 for
CDR-H3, and a light chain wherein the variable domain of the light
chain comprises a CDR having the sequence given in SEQ ID NO:141
for CDR-L1, a CDR having the sequence given in in SEQ ID NO:142 for
CDR-L2 and a CDR having the sequence given in SEQ ID NO:143 for
CDR-L3; iv) a heavy chain wherein the variable domain of the heavy
chain comprises a CDR having the sequence given in SEQ ID NO:164
for CDR-H1, a CDR having the sequence given in SEQ ID NO:165 for
CDR-H2 and a CDR having the sequence given in SEQ ID NO:166 for
CDR-H3, and a light chain wherein the variable domain of the light
chain comprises a CDR having the sequence given in SEQ ID NO:161
for CDR-L1, a CDR having the sequence given in in SEQ ID NO:162 for
CDR-L2 and a CDR having the sequence given in SEQ ID NO:163 for
CDR-L3; v) a heavy chain wherein the variable domain of the heavy
chain comprises a CDR having the sequence given in SEQ ID NO:64 for
CDR-H1, a CDR having the sequence given in SEQ ID NO: 65 for CDR-H2
and a CDR having the sequence given in SEQ ID NO: 66 for CDR-H3,
and a light chain wherein the variable domain of the light chain
comprises a CDR having the sequence given in SEQ ID NO:61 for
CDR-L1, a CDR having the sequence given in in SEQ ID NO:62 for
CDR-L2 and a CDR having the sequence given in SEQ ID NO:63 for
CDR-L3; vi) a heavy chain wherein the variable domain of the heavy
chain comprises a CDR having the sequence given in SEQ ID NO:74 for
CDR-H1, a CDR having the sequence given in SEQ ID NO: 75 for CDR-H2
and a CDR having the sequence given in SEQ ID NO: 76 for CDR-H3,
and a light chain wherein the variable domain of the light chain
comprises a CDR having the sequence given in SEQ ID NO:71 for
CDR-L1, a CDR having the sequence given in in SEQ ID NO:72 for
CDR-L2 and a CDR having the sequence given in SEQ ID NO:73 for
CDR-L3; vii) a heavy chain wherein the variable domain of the heavy
chain comprises a CDR having the sequence given in SEQ ID NO:84 for
CDR-H1, a CDR having the sequence given in SEQ ID NO: 85 for CDR-H2
and a CDR having the sequence given in SEQ ID NO: 86 for CDR-H3,
and a light chain wherein the variable domain of the light chain
comprises a CDR having the sequence given in SEQ ID NO:81 for
CDR-L1, a CDR having the sequence given in in SEQ ID NO:82 for
CDR-L2 and a CDR having the sequence given in SEQ ID NO:83 for
CDR-L3; viii) a heavy chain wherein the variable domain of the
heavy chain comprises a CDR having the sequence given in SEQ ID
NO:134 for CDR-H1, a CDR having the sequence given in SEQ ID NO:
135 for CDR-H2 and a CDR having the sequence given in SEQ ID NO:
136 for CDR-H3, and a light chain wherein the variable domain of
the light chain comprises a CDR having the sequence given in SEQ ID
NO:131 for CDR-L1, a CDR having the sequence given in in SEQ ID
NO:132 for CDR-L2 and a CDR having the sequence given in SEQ ID
NO:133 for CDR-L3.
43. A pharmaceutical composition according to claim 42, wherein
said one or more monoclonal antibodies are independently selected
from: i) a heavy chain wherein the variable domain of the heavy
chain comprises a CDR having the sequence given in SEQ ID NO:124
for CDR-H1, a CDR having the sequence given in SEQ ID NO: 125 for
CDR-H2 and a CDR having the sequence given in SEQ ID NO: 126 for
CDR-H3, and a light chain wherein the variable domain of the light
chain comprises a CDR having the sequence given in SEQ ID NO:121
for CDR-L1, a CDR having the sequence given in in SEQ ID NO:122 for
CDR-L2 and a CDR having the sequence given in SEQ ID NO:123 for
CDR-L3; ii) a heavy chain wherein the variable domain of the heavy
chain comprises a CDR having the sequence given in SEQ ID NO:154
for CDR-H1, a CDR having the sequence given in SEQ ID NO:155 for
CDR-H2 and a CDR having the sequence given in SEQ ID NO:156 for
CDR-H3, and a light chain wherein the variable domain of the light
chain comprises a CDR having the sequence given in SEQ ID NO:151
for CDR-L1, a CDR having the sequence given in in SEQ ID NO:152 for
CDR-L2 and a CDR having the sequence given in SEQ ID NO:153 for
CDR-L3; iii) a heavy chain wherein the variable domain of the heavy
chain comprises a CDR having the sequence given in SEQ ID NO:144
for CDR-H1, a CDR having the sequence given in SEQ ID NO:145 for
CDR-H2 and a CDR having the sequence given in SEQ ID NO:146 for
CDR-H3, and a light chain wherein the variable domain of the light
chain comprises a CDR having the sequence given in SEQ ID NO:141
for CDR-L1, a CDR having the sequence given in in SEQ ID NO:142 for
CDR-L2 and a CDR having the sequence given in SEQ ID NO:143 for
CDR-L3; and iv) a heavy chain wherein the variable domain of the
heavy chain comprises a CDR having the sequence given in SEQ ID
NO:164 for CDR-H1, a CDR having the sequence given in SEQ ID NO:165
for CDR-H2 and a CDR having the sequence given in SEQ ID NO:166 for
CDR-H3, and a light chain wherein the variable domain of the light
chain comprises a CDR having the sequence given in SEQ ID NO:161
for CDR-L1, a CDR having the sequence given in in SEQ ID NO:162 for
CDR-L2 and a CDR having the sequence given in SEQ ID NO:163 for
CDR-L3.
44. A pharmaceutical composition according to claim 42, wherein a
monoclonal antibody which specifically binds TcdB1234 comprises a
heavy chain wherein the variable domain of the heavy chain
comprises a CDR having the sequence given in SEQ ID NO:124 for
CDR-H1, a CDR having the sequence given in SEQ ID NO: 125 for
CDR-H2 and a CDR having the sequence given in SEQ ID NO: 126 for
CDR-H3, and a light chain wherein the variable domain of the light
chain comprises a CDR having the sequence given in SEQ ID NO:121
for CDR-L1, a CDR having the sequence given in in SEQ ID NO:122 for
CDR-L2 and a CDR having the sequence given in SEQ ID NO:123 for
CDR-L3.
45. A pharmaceutical composition according to claim 44, wherein the
monoclonal antibody has a heavy chain comprising the sequence given
in SEQ ID NO:129 and a light chain comprising the sequence given in
SEQ ID NO:127.
46. A pharmaceutical composition according to claim 42, wherein a
monoclonal antibody which specifically binds TcdB1234 comprising a
heavy chain wherein the variable domain of the heavy chain
comprises a CDR having the sequence given in SEQ ID NO:154 for
CDR-H1, a CDR having the sequence given in SEQ ID NO:155 for CDR-H2
and a CDR having the sequence given in SEQ ID NO:156 for CDR-H3,
and a light chain wherein the variable domain of the light chain
comprises a CDR having the sequence given in SEQ ID NO:151 for
CDR-L1, a CDR having the sequence given in in SEQ ID NO:152 for
CDR-L2 and a CDR having the sequence given in SEQ ID NO:153 for
CDR-L3.
47. A pharmaceutical composition according to claim 46, wherein the
monoclonal antibody has a heavy chain comprising the sequence given
in SEQ ID NO:159 and a light chain comprising the sequence given in
SEQ ID NO:157.
48. A pharmaceutical composition according to claim 42, wherein the
monoclonal antibody has a heavy chain comprising the sequence given
in SEQ ID NO:149 and a light chain comprising the sequence given in
SEQ ID NO:147.
49. A pharmaceutical composition according to claim 42, wherein the
monoclonal antibody has a heavy chain comprising the sequence given
in SEQ ID NO:169 and a light chain comprising the sequence given in
SEQ ID NO:167.
50. A pharmaceutical composition according to claim 42, wherein at
least one of said monoclonal antibodies is a neutralizing antibody
which is effective against ribotypes 003.
51. A pharmaceutical composition according to claim 42, further
comprising two or more antibodies specific to TcdB.
52. A pharmaceutical composition according to claim 42, further
comprising one or more antibodies specific to TcdA.
53. A pharmaceutical composition according to claim 52, wherein the
antibody which specifically binds TcdA comprises a heavy chain
sequence given in SEQ ID NO: 19 and a light chain sequence given in
SEQ ID NO: 17.
54. A pharmaceutical composition according to claim 52, wherein the
antibody which specifically binds TcdA comprises a heavy chain
sequence given in SEQ ID NO: 29 and a light chain sequence given in
SEQ ID NO: 27.
55. A pharmaceutical composition according to claim 52, wherein the
antibody which specifically binds TcdA comprises a heavy chain
sequence given in SEQ ID NO: 49 and a light chain sequence given in
SEQ ID NO: 47.
56. A pharmaceutical composition according to claim 52, wherein the
antibody which specifically binds TcdA comprises a heavy chain
sequence given in SEQ ID NO: 59 and a light chain sequence given in
SEQ ID NO: 57.
57. A pharmaceutical composition according to claim 52, wherein the
antibody which specifically binds TcdA comprises a heavy chain
sequence given in SEQ ID NO: 39 and a light chain sequence given in
SEQ ID NO: 37.
58. A pharmaceutical composition according to claim 52, wherein the
antibody which specifically binds TcdA comprises a heavy chain
sequence given in SEQ ID NO: 9 and a light chain sequence given in
SEQ ID NO: 7.
59. A pharmaceutical composition according to claim 52, which
further comprises a pharmaceutically acceptable excipient.
60. A method of treatment or prophylaxis of Clostridium difficile
infection or complications therefrom comprising administering a
pharmaceutical composition as defined in claim 42 to a patient in
need thereof.
61. A method according to claim 60, wherein the composition is
co-administrated with a compound selected the group comprising
metronidazole, vancomycin, clindamycin, fidaxomicin and
combinations thereof.
Description
[0001] The present invention relates to antibodies to exotoxins of
Clostridium difficile, for example TcdA and TcdB, pharmaceutical
compositions comprising the same, processes of producing said
antibodies and compositions and use of the antibodies and
compositions in treatment and/or prophylaxis, in particular
treatment or prophylaxis of Clostridium difficile infection,
pseudomembranous colitis, fulminant colitis and/or toxic mega
colon.
[0002] The two major exotoxins TcdA and TcdB have been established
as the major pathogenicity determinants of Clostridium difficile in
a large number of in vitro and in vivo studies. Non-toxigenic
strains are not pathogenic to animals and man (1, 2). To date a
clear understanding of the role of binary toxin has yet to be
established (3).
[0003] Both toxins are entero- and cyto-toxic, but the balance of
evidence suggests that TcdA is a more powerful enterotoxin than
TcdB, whilst TcdB is typically observed to be .about.1000.times.
more cytotoxic than TcdA (4). Whilst both toxins are capable of
inducing an inflammatory response, TcdA appears to aid the
migration of the more inflammatory TcdB deeper into the gut mucosa
(5).
[0004] In toto, a large collection of data generated for over 30
years support a model where both toxins are likely to be important
in the human disease process. It is probable that TcdA initiates
early (i.e. before TcdB) and rapid (i.e. 1-3 hours) gut damage
through loss of tight junctions and destruction of villi tips and
hence diarrhoea, probably through albumin driven fluid loss. This
damage to the integrity of the gut lining enables TcdB to exert its
superior molar potency (TcdB is typically cited as being
1000.times. more cytotoxic than TcdA) more rapidly and effectively
(i.e. deeper into tissue, alternative cellular targets and damaging
systemically accessed organs). Either toxin can be effective alone
in vitro on human or animals cells and tissues. Either toxin can be
effective alone in vivo in animals depending upon other eliciting
factors such as mechanical damage, barrier overload and host
specific sensitivities. It is now clear that in hamsters at least
either TcdA or TcdB alone delivered by a Clostridium difficile gut
infection can cause death (1). It is well established that A-B+
strains are capable of causing symptoms and death in humans (6,7).
However, the majority (.about.95%) of clinical strains are A+B+
hence drugs aimed at treating Clostridium difficile infections
(CDI) must be capable of neutralising the activities of and
clearing both toxins effectively.
[0005] CDI is most typically a nosocomial infection of older
patients or those with complicating co-morbidities. However, an
increase in community acquired infections has been noted. Infection
is almost always associated with or induced by use of broad
spectrum antibiotics. Healthcare associated costs are estimated to
be in excess of $1 bn per annum in the US alone. These costs are
primarily due to patients having longer hospitals stays. Current
therapies involve the use of antibiotics such as clindamycin,
vancomycin or fidaxomicin which kill the Clostridium difficile
cells within the gut. Current therapies address the bacterial
infection but do not deal with or prevent directly the significant
pathogenesis caused by TcdA and TcdB which are major contributors
to CDI symptoms and mortality.
[0006] CDI symptoms in humans include mild to severe diarrhoea,
pseudomembranous colitis (PMC) and fulminant colitis or so called
toxic mega colon. Death results in 5-15% of patients receiving
current best care. Thus at the present time there is no specific
therapy available to patients to prevent the damage and injury
caused by C. difficile toxins after infection.
[0007] Raising an antibody response through vaccination and
parenteral administration of polyclonal and monoclonal antibodies
have all been shown to be capable of protecting animals from
symptoms of diarrhoea and death (8-15). Early studies in hamsters
suggested that antibodies against TcdA alone were all that was
necessary for protection. However, use of strains functionally
deleted for TcdA or TcdB demonstrate that either toxin is capable
of causing disease in hamsters, but that both toxins together are
more effective (1).
[0008] For therapeutic applications, monoclonal antibodies (Mabs)
can offer efficacy, safety, manufacturing and regulatory advantages
over serum derived polyclonal antibodies or serum derived
hyper-immune sera. For these reasons Mabs are usually the preferred
option for therapeutic products.
[0009] There have been a number of attempts to generate protective
Mabs against TcdA and TcdB. The most advanced of these in the
clinic is a mixture of 2 IgG1 Mabs, one against each TcdA and TcdB
originally called CDA1 and MDX1388 developed by MBL and Medarex.
They were demonstrated to be unable to fully protect hamsters in
models of acute or relapse infections (15). This Mab combination is
now being developed as MK3415A by Merck Inc. In a human phase II
trial MK3415A resulted in a statistically significant reduction in
disease recurrence (p=0.006) (see also Lowy et al., NEJM (2010)
362: 197-205) but did not affect the duration/severity of diarrhoea
or death rates (16). This may mean that these antibodies may only
be useful for preventing recurrence of infection. Recurrence of
infection results in approximately 25% of patients. Thus there
likely to be a significant patient population in which these
antibodies are not effective.
[0010] In order to be able to have a positive influence upon
diarrhoea (for example as a result of acute damage to gut tight
junctions due to TcdA) and death (for example resulting from
prolonged poor nutritional status, dehydration stress and
initiation of an inflammatory cascade, widespread anatomical damage
to the gut lining and possibly damage to distant organs due to
systemic toxin TcdB more so than TcdA) Mabs are required with
superior affinity, toxin neutralisation, superior prevention of
loss of TEER (trans-epithelial electrical resistance), antigen
decoration and antigen immune clearance.
SUMMARY OF THE PRESENT INVENTION
[0011] The present invention provide a Mab(s) with a very high
level of potency in vitro and in vivo which have the potential to
have an impact upon duration and severity of diarrhoea and death
rate in humans suffering from Clostridium difficile infection
(CDI).
[0012] In one embodiment there is provided a monoclonal antibody
specific to antigen TcdA or TcdB, wherein the antibody has high
affinity for the target antigen and is suitable for reducing the
duration and/or severity of diarrhoea and morbidity in a patient
with Clostridium difficile infection or at risk of said
infection.
[0013] In one embodiment there is provided a Mab specific to TcdA
or TcdB, or a population of at least two Mabs at least one of which
is specific to TcdA and at least one of which is specific to TcdB,
wherein the EC.sub.50 of the or each antibody or the combination of
antibodies is 200 ng/ml or less, for example 150 ng/ml or less such
as 100 ng/ml.
[0014] The antibodies of the present disclosure are useful because
they are likely to provide a means of treating the severity and
duration of symptoms of a primary infection such as diarrhoea in a
patient or preventing death and not just prevent the reoccurrence
of disease symptoms.
[0015] In at least some embodiments the antibodies according to the
present disclosure show no reduction in potency in the presence of
high concentrations of toxin.
DETAILED DESCRIPTION OF THE PRESENT INVENTION
[0016] Specific as employed herein is intended to refer to an
antibody that only recognises the antigen to which it is specific
or an antibody that has significantly higher binding affinity to
the antigen to which is specific compared to binding to antigens to
which it is non-specific, for example 5, 6, 7, 8, 9, 10 times
higher binding affinity.
[0017] Binding affinity may be measured by standard assays such as
surface plasmon resonance, such as BIAcore.
[0018] In one embodiment the EC.sub.50 is less than 75, 70, 60, 65,
55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.5
ng/ml Clostridium difficile infection in cell culture assays and
the patient. This is significantly lower (more potent) than known
antibodies and is thought to be a major factor as to why the
antibodies of the present disclosure have a significant and
positive impact on survival of subjects receiving treatment.
[0019] As employed herein potency is the ability of the antibody to
elicit an appropriate biological response, for example
neutralisation of the deleterious toxin effects, at a given dose or
concentration. Examples of potency include the percent maximal
neutralisation of toxin activity (extent of protection), the lowest
relative concentration of Mab to antigen (e.g. EC.sub.50), the
speed and durability of neutralisation activity.
[0020] In cell culture assays neutralisation might be observed as
one or more of the following: prevention of binding of toxin to
cells, immunoprecipitation of toxin from solution, prevention of
loss of cell form and shape, prevention of loss of cytoskeletal
structures, prevention of loss of cell monolayer tight junctions
and trans-epithelial electrical resistance, prevention of cell
death, apoptosis and production of pro-inflammatory cytokines such
as TNF.alpha., IL-1.beta., IL-6 and MIP1.alpha..
[0021] In tissue section and explant assays neutralisation may, for
example be observed as prevention of necrosis and/or oedematous
fluid accumulation.
[0022] In in vivo assays neutralisation may be observed as one or
more of the following: prevention of fluid accumulation in ligated
ileal loops and prevention of gut tissue necrosis, diarrhoea,
pseudo-membrane formation of death of animals,
[0023] Thus in one embodiment there is provided an antibody (for
example an anti-toxin A antibody) comprising a CDR, such as 1, 2,
3, 4, 5 or 6 CDRs, selected from:
TABLE-US-00001 SEQ ID NO: 1 QASQSISNALA SEQ ID NO: 2 SASSLAS SEQ ID
NO: 3 QYTHYSHTSKNP SEQ ID NO: 4 GFTISSYYMS SEQ ID NO: 5
IISSGGHFTWYANWAKG SEQ ID NO: 6 AYVSGSSFNGYAL
[0024] In one embodiment sequences 1 to 3 are in a light chain of
the antibody.
[0025] In one embodiment sequences 4 to 6 are in a heavy chain of
the antibody.
[0026] In one embodiment SEQ ID NO: 1 is CDR L1, SEQ ID NO: 2 is
CDR L2 and SEQ ID NO; 3 is CDR L3.
[0027] In one embodiment SEQ ID NO: 4 is CDR H1, SEQ ID NO: 5 is
CDR H2 and SEQ ID NO; 6 is CDR H3.
[0028] In one embodiment SEQ ID NO: 1 is CDR L1, SEQ ID NO: 2 is
CDR L2, SEQ ID NO; 3 is CDR L3, SEQ ID NO: 4 is CDR H1, SEQ ID NO:
5 is CDR H2 and SEQ ID NO; 6 is CDR H3.
[0029] In one embodiment there is provided a variable region, such
as a light chain variable region with the following sequence
(Antibody 922 anti-toxin A antibody; Light chain Variable region
sequence) SEQ ID NO: 7:
TABLE-US-00002 DPVMTQSPSTLSASVGDRVTITCQASQSISNALAWYQQKPGKAPKLLIYS
ASSLASGVPSRFKGSGSGTEFTLTISSLQPDDFATYYCQYTHYSHTSKNP FGGGTKVEIK
wherein the CDRs are underlined and construct is referred to herein
as 922.g1 VK (gL1).
[0030] The polynucleotide sequence encoding SEQ ID NO: 7 is shown
in FIG. 1 and SEQ ID NO: 8 therein.
[0031] In one embodiment there is provided a variable region, such
as a heavy chain variable region with the following sequence
(Antibody 922 anti-toxin A antibody heavy chain variable region
sequence) SEQ ID NO: 9:
TABLE-US-00003 EVQLVESGGGLVQPGGSLRLSCAASGFTISSYYMSWVRQAPGKGLEWIGI
ISSGGHFTWYANWAKGRFTISSDSTTVYLQMNSLRDEDTATYFCARAYVS
GSSFNGYALWGQGTLVTVS
wherein the CDRs are underlined and construct is referred to herein
as 922.g1 VH (gH1)
[0032] The polynucleotide sequence encoding SEQ ID NO: 9 is shown
in FIG. 1 and SEQ ID NO: 10 therein.
[0033] In one embodiment the antibody comprises the variable
regions shown in SEQ ID NO: 7 and 9.
[0034] Thus in one embodiment there is provided an antibody (for
example an anti-toxin A antibody) comprising a CDR, such as 1, 2,
3, 4, 5 or 6 CDRs, selected from:
TABLE-US-00004 SEQ ID NO: 11 QASQSISNYLA SEQ ID NO: 12 SASTLAS SEQ
ID NO: 13 QYSHYGTGVFGA SEQ ID NO: 14 AFSLSNYYMS SEQ ID NO: 15
IISSGSNALKWYASWPKG SEQ ID NO: 16 NYVGSGSYYGMDL
[0035] In one embodiment sequences 11 to 13 are in a light chain of
the antibody.
[0036] In one embodiment sequences 14 to 16 are in a heavy chain of
the antibody.
[0037] In one embodiment SEQ ID NO: 11 is CDR L1, SEQ ID NO: 12 is
CDR L2 and SEQ ID NO: 13 is CDR L3.
[0038] In one embodiment SEQ ID NO: 14 is CDR H1, SEQ ID NO: 15 is
CDR H2 and SEQ ID NO; 16 is CDR H3.
[0039] In one embodiment SEQ ID NO: 11 is CDR L1, SEQ ID NO: 12 is
CDR L2, SEQ ID NO: 13 is CDR L3, SEQ ID NO: 14 is CDR H1, SEQ ID
NO: 15 is CDR H2 and SEQ ID NO; 16 is CDR H3.
[0040] In one embodiment there is provided a variable region, such
as a light chain variable region with the following sequence
(Antibody 923 anti-toxin A antibody; Light chain Variable region
sequence) SEQ ID NO: 17:
TABLE-US-00005 DVVMTQSPSSLSASVGDRVTITCQASQSISNYLAWYQQKPGKVPKLLIYS
ASTLASGVPSRFKGSGSGTQFTLTISSLQPEDVATYYCQYSHYGTGVFGA FGGGTKVEIK
wherein the CDRs are underlined and construct is referred to herein
as CA923.g1 gL1
[0041] The polynucleotide sequence encoding SEQ ID NO: 17 is shown
in FIG. 1 and SEQ ID NO: 18 therein.
[0042] In one embodiment there is provided a variable region, such
as a heavy chain variable region with the following sequence
(Antibody 923 anti-toxin A antibody heavy chain variable region
sequence) SEQ ID NO: 19:
TABLE-US-00006 EVQLVESGGGLVQPGGSLRLSCAASAFSLSNYYMSWVRQAPGKGLEWIGI
ISSGSNALKWYASWPKGRFTISKDSTTVYLQMNSLRAEDTATYFCARNYV
GSGSYYGMDLWGQGTLVTVS
wherein the CDRs are underlined and construct is referred to herein
as CA923.g1 gH1
[0043] The polynucleotide sequence encoding SEQ ID NO: 19 is shown
in FIG. 2 and SEQ ID NO: 20 therein.
[0044] In one embodiment an antibody according to the invention
comprises variable regions shown in SEQ ID NO 17: and SEQ ID NO:
19.
[0045] In one embodiment there is provided an antibody (for example
an anti-toxin A antibody) comprising a CDR, such as 1, 2, 3, 4, 5
or 6 CDRs, selected from:
TABLE-US-00007 SEQ ID NO: 21 QASQSISSYFS SEQ ID NO: 22 GASTLAS SEQ
ID NO: 23 QCTDYSGIYFGG SEQ ID NO: 24 GFSLSSYYMS SEQ ID NO: 25
IISSGSSTTFTWYASWAKG SEQ ID NO: 26 AYVGSSSYYGFDP
[0046] In one embodiment sequences 21 to 23 are in a light chain of
the antibody.
[0047] In one embodiment sequences 24 to 26 are in a heavy chain of
the antibody.
[0048] In one embodiment SEQ ID NO: 21 is CDR L1, SEQ ID NO: 22 is
CDR L2 and SEQ ID NO; 23 is CDR L3.
[0049] In one embodiment SEQ ID NO: 24 is CDR H1, SEQ ID NO: 25 is
CDR H2 and SEQ ID NO; 26 is CDR H3.
[0050] In one embodiment SEQ ID NO: 21 is CDR L1, SEQ ID NO: 22 is
CDR L2, SEQ ID NO; 23 is CDR L3, SEQ ID NO: 24 is CDR H1, SEQ ID
NO: 25 is CDR H2 and SEQ ID NO; 26 is CDR H3.
[0051] In one embodiment there is provided a variable region, such
as a light chain variable region with the following sequence
(Antibody 993 anti-toxin A antibody; Light chain Variable region
sequence) SEQ ID NO: 27:
TABLE-US-00008 DVVMTQSPSTLSASVGDRVTITCQASQSISSYFSWYQQKPGKAPQLLIYG
ASTLASGVPSRFKGSGSGTELTLTISSLQPDDFATYYCQCTDYSGIYFGG FGGGTKVEIK
wherein the CDRs are underlined and construct is referred to herein
as CA993.g1 gL1
[0052] The polynucleotide sequence encoding SEQ ID NO: 27 is shown
in FIG. 2 and SEQ ID NO: 28 therein.
[0053] In one embodiment there is provided a variable region, such
as a heavy chain variable region with the following sequence
(Antibody 993 anti-toxin A antibody heavy chain variable region
sequence) SEQ ID NO: 29:
TABLE-US-00009 EVQLVESGGGLVQPGGSLKLSCTASGFSLSSYYMSWVRQAPGKGLEWIGI
ISSGSSTTFTWYASWAKGRFTISKTSTTVYLQMNSLKTEDTATYFCARAY
VGSSSYYGFDPWGQGTLVTVS
wherein the CDRs are underlined and construct is referred to herein
as CA993.g1 gH1
[0054] The polynucleotide sequence encoding SEQ ID NO: 29 is shown
in FIG. 2 and SEQ ID NO: 30 therein.
[0055] In one embodiment an antibody according to the invention
comprises variable regions shown in SEQ ID NO: 27 and SEQ ID NO:
29.
[0056] In one embodiment there is provided an antibody (for example
an anti-toxin A antibody) comprising a CDR, such as 1, 2, 3, 4, 5
or 6 CDRs, selected from:
TABLE-US-00010 SEQ ID NO: 31 QASQSINNYFS SEQ ID NO: 32 GAANLAS SEQ
ID NO: 33 QNNYGVHIYGAA SEQ ID NO: 34 GFSLSNYDMI SEQ ID NO: 35
FINTGGITYYASWAKG SEQ ID NO: 36 VDDYIGAWGAGL
[0057] In one embodiment sequences 31 to 33 are in a light chain of
the antibody.
[0058] In one embodiment sequences 34 to 36 are in a heavy chain of
the antibody.
[0059] In one embodiment SEQ ID NO: 31 is CDR L1, SEQ ID NO: 32 is
CDR L2 and SEQ ID NO; 33 is CDR L3.
[0060] In one embodiment SEQ ID NO: 34 is CDR H1, SEQ ID NO: 35 is
CDR H2 and SEQ ID NO: 36 is CDR H3.
[0061] In one embodiment SEQ ID NO: 31 is CDR L1, SEQ ID NO: 32 is
CDR L2, SEQ ID NO; 33 is CDR L3, SEQ ID NO: 34 is CDR H1, SEQ ID
NO: 35 is CDR H2 and SEQ ID NO; 36 is CDR H3.
[0062] In one embodiment there is provided a variable region, such
as a light chain variable region with the following sequence
(Antibody 995 anti-toxin A antibody; Light chain Variable region
sequence) SEQ ID NO: 37:
TABLE-US-00011 DVVMTQSPSTLSASVGDRVTITCQASQSINNYFSWYQQKPGKAPKLLIYG
AANLASGVPSRFKGSGSGTEYTLTISSLQPDDFATYSCQNNYGVHIYGAA FGGGTKVEIK
wherein the CDRs are underlined
[0063] The polynucleotide sequence encoding SEQ ID NO: 37 is shown
in FIG. 3 and SEQ ID NO: 38 therein.
[0064] In one embodiment there is provided a variable region, such
as a heavy chain variable region with the following sequence
(Antibody 995 anti-toxin A antibody heavy chain variable region
sequence) SEQ ID NO: 39
TABLE-US-00012 EVQLVESGGGLVQPGGSLRLSCTASGFSLSNYDMIWVRQAPGKGLEYIGF
INTGGITYYASWAKGRFTISRDSSTVYLQMNSLRAEDTATYFCARVDDYI
GAWGAGLWGQGTLVTVS
wherein the CDRs are underlined
[0065] The polynucleotide sequence encoding SEQ ID NO: 39 is shown
in FIG. 3 and SEQ ID NO: 40 therein.
[0066] In one embodiment an antibody according to the invention
comprises variable regions shown in SEQ ID NO: 37 and SEQ ID NO:
39.
[0067] In one embodiment there is provided an antibody (for example
an anti-toxin A antibody) comprising a CDR, such as 1, 2, 3, 4, 5
or 6 CDRs, selected from:
TABLE-US-00013 SEQ ID NO: 41 QASQSISSYLS SEQ ID NO: 42 RASTLAS SEQ
ID NO: 43 LGVYGYSNDDGIA SEQ ID NO: 44 GIDLSSHHMC SEQ ID NO: 45
VIYHFGSTYYANWATG SEQ ID NO: 46 ASIAGYSAFDP
[0068] In one embodiment sequences 41 to 43 are in a light chain of
the antibody.
[0069] In one embodiment sequences 44 to 46 are in a heavy chain of
the antibody.
[0070] In one embodiment SEQ ID NO: 41 is CDR L1, SEQ ID NO: 42 is
CDR L2 and SEQ ID NO; 43 is CDR L3.
[0071] In one embodiment SEQ ID NO: 44 is CDR H1, SEQ ID NO: 45 is
CDR H2 and SEQ ID NO: 46 is CDR H3.
[0072] In one embodiment SEQ ID NO: 41 is CDR L1, SEQ ID NO: 42 is
CDR L2, SEQ ID NO; 43 is CDR L3, SEQ ID NO: 44 is CDR H1, SEQ ID
NO: 45 is CDR H2 and SEQ ID NO; 46 is CDR H3.
[0073] In one embodiment there is provided a variable region, such
as a light chain variable region with the following sequence
(Antibody 997 anti-toxin A antibody; Light chain Variable region
sequence) SEQ ID NO: 47:
TABLE-US-00014 ALVMTQSPSSFSASTGDRVTITCQASQSISSYLSWYQQKPGKAPKLLIYR
ASTLASGVPSRFSGSGSGTEYTLTISCLQSEDFATYYCLGVYGYSNDDGI AFGGGTKVEIK
wherein the CDRs are underlined
[0074] The polynucleotide sequence encoding SEQ ID NO: 47 is shown
in FIG. 3 and SEQ ID NO: 48 therein.
[0075] In one embodiment there is provided a variable region, such
as a heavy chain variable region with the following sequence
(Antibody 997 anti-toxin A antibody heavy chain variable region
sequence) SEQ ID NO: 49:
TABLE-US-00015 EVQLVESGGGLVQPGGSLRLSCTVSGIDLSSHHMCWVRQAPGKGLEYIGV
IYHFGSTYYANWATGRFTISKDSTTVYLQMNSLRAEDTATYFCARASIAG
YSAFDPWGQGTLVTVS
wherein the CDRs are underlined
[0076] The polynucleotide sequence encoding SEQ ID NO: 49 is shown
in FIG. 4 and SEQ ID NO: 50 therein.
[0077] In one embodiment an antibody according to the invention
comprises variable regions shown in SEQ ID NO: 47 and SEQ ID NO:
49.
[0078] In one embodiment there is provided an antibody (for example
an anti-toxin A antibody) comprising a CDR, such as 1, 2, 3, 4, 5
or 6 CDRs, selected from:
TABLE-US-00016 SEQ ID NO: 51 QASQSIYSYLA SEQ ID NO: 52 DASTLAS SEQ
ID NO: 53 QGNAYTSNSHDNA SEQ ID NO: 54 GIDLSSDAVG SEQ ID NO: 55
IIATFDSTYYASWAKG SEQ ID NO: 56 TGSWYYISGWGSYYYGMDL
[0079] In one embodiment sequences 51 to 53 are in a light chain of
the antibody.
[0080] In one embodiment sequences 54 to 56 are in a heavy chain of
the antibody.
[0081] In one embodiment SEQ ID NO: 51 is CDR L1, SEQ ID NO: 52 is
CDR L2 and SEQ ID NO: 53 is CDR L3.
[0082] In one embodiment SEQ ID NO: 54 is CDR H1, SEQ ID NO: 55 is
CDR H2 and SEQ ID NO; 56 is CDR H3.
[0083] In one embodiment SEQ ID NO: 51 is CDR L1, SEQ ID NO: 52 is
CDR L2, SEQ ID NO; 53 is CDR L3, SEQ ID NO: 54 is CDR H1, SEQ ID
NO: 55 is CDR H2 and SEQ ID NO: 56 is CDR H3.
[0084] In one embodiment there is provided a variable region, such
as a light chain variable region with the following sequence
(Antibody 1000 anti-toxin A antibody; Light chain Variable region
sequence) SEQ ID NO: 57:
TABLE-US-00017 EIVMTQSPSTLSASVGDRVTITCQASQSIYSYLAWYQQKPGKAPKLLIYD
ASTLASGVPSRFKGSGSGTEFTLTISSLQPDDFATYYCQGNAYTSNSHDN AFGGGTKVEIK
wherein the CDRs are underlined.
[0085] The polynucleotide sequence encoding SEQ ID NO: 57 is shown
in FIG. 4 and SEQ ID NO: 58 therein.
[0086] In one embodiment there is provided a variable region, such
as a heavy chain variable region with the following sequence
(Antibody 1000 anti-toxin A antibody heavy chain variable region
sequence) SEQ ID NO: 59:
TABLE-US-00018 EVQLVESGGGLIQPGGSLRLSCTVSGIDLSSDAVGWVRQAPGKGLEYIGI
IATFDSTYYASWAKGRFTISKASSTTVYLQMNSLRAEDTATYFCARTGSW
YYISGWGSYYYGMDLWGQGTLVTVS
wherein the CDRs are underlined.
[0087] The polynucleotide sequence encoding SEQ ID NO: 59 is shown
in FIG. 4 and SEQ ID NO: 60 therein.
[0088] In one embodiment an antibody according to the invention
comprises variable regions shown in SEQ ID NO: 57 and SEQ ID NO:
59.
[0089] In one embodiment there is provided an antibody (for example
an anti-toxin B antibody) comprising a CDR, such as 1, 2, 3, 4, 5
or 6 CDRs, selected from:
TABLE-US-00019 SEQ ID NO: 61 RASKSVSTLMH SEQ ID NO: 62 LASNLES SEQ
ID NO: 63 QQTWNDPWT SEQ ID NO: 64 GFTFSNYGMA SEQ ID NO: 65
SISSSGGSTYYRDSVKG SEQ ID NO: 66 VIRGYVMDA
[0090] In one embodiment sequences 61 to 63 are in a light chain of
the antibody.
[0091] In one embodiment sequences 64 to 66 are in a heavy chain of
the antibody.
[0092] In one embodiment SEQ ID NO: 61 is CDR L1, SEQ ID NO: 62 is
CDR L2 and SEQ ID NO: 63 is CDR L3.
[0093] In one embodiment SEQ ID NO: 64 is CDR H1, SEQ ID NO: 65 is
CDR H2 and SEQ ID NO: 66 is CDR H3.
[0094] In one embodiment SEQ ID NO: 61 is CDR L1, SEQ ID NO: 62 is
CDR L2, SEQ ID NO; 63 is CDR L3, SEQ ID NO: 64 is CDR H1, SEQ ID
NO: 65 is CDR H2 and SEQ ID NO: 66 is CDR H3.
[0095] In one embodiment there is provided a variable region, such
as a light chain variable region with the following sequence
(Antibody 926 anti-toxin B antibody; Light chain Variable region
sequence) SEQ ID NO: 67:
TABLE-US-00020 DTVLTQSPATLSLSPGERATLSCRASKSVSTLMHWFQQKPGQAPKLLIYL
ASNLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQTWNDPWTFGG GTKVEIK
wherein the CDRs are underlined.
[0096] The polynucleotide sequence encoding SEQ ID NO: 67 is shown
in FIG. 5 and SEQ ID NO: 68 therein.
[0097] In one embodiment there is provided a variable region, such
as a heavy chain variable region with the following sequence
(Antibody 926 anti-toxin B antibody heavy chain variable region
sequence) SEQ ID NO: 69:
TABLE-US-00021 EVELLESGGGLVQPGGSLRLSCEASGFTFSNYGMAWVRQAPTKGLEWVTS
ISSSGGSTYYRDSVKGRFTISRDNAKSSLYLQMNSLRAEDTATYYCTTVI
RGYVMDAWGQGTLVTVS
wherein the CDRs are underlined.
[0098] The polynucleotide sequence encoding SEQ ID NO: 69 is shown
in FIG. 5 and SEQ ID NO: 70 therein.
[0099] In one embodiment there is provided an antibody (for example
an anti-toxin B antibody) comprising a CDR, such as 1, 2, 3, 4, 5
or 6 CDRs, selected from:
TABLE-US-00022 SEQ ID NO: 71 RASGSVSTLMH SEQ ID NO: 72 KASNLAS SEQ
ID NO: 73 HQSWNSDT SEQ ID NO: 74 GFTFSNYGMA SEQ ID NO: 75
TINYDGRTTHYRDSVKG SEQ ID NO: 76 ISRSHYFDC
[0100] In one embodiment sequences 71 to 73 are in a light chain of
the antibody.
[0101] In one embodiment sequences 74 to 76 are in a heavy chain of
the antibody.
[0102] In one embodiment SEQ ID NO: 71 is CDR L1, SEQ ID NO: 72 is
CDR L2 and SEQ ID NO: 73 is CDR L3.
[0103] In one embodiment SEQ ID NO: 74 is CDR H1, SEQ ID NO: 75 is
CDR H2 and SEQ ID NO: 76 is CDR H3.
[0104] In one embodiment SEQ ID NO: 71 is CDR L1, SEQ ID NO: 72 is
CDR L2, SEQ ID NO; 73 is CDR L3, SEQ ID NO: 74 is CDR H1, SEQ ID
NO: 75 is CDR H2 and SEQ ID NO: 76 is CDR H3.
[0105] In one embodiment there is provided a variable region, such
as a light chain variable region with the following sequence
(Antibody 927 anti-toxin B antibody; Light chain Variable region
sequence) SEQ ID NO: 77:
TABLE-US-00023 DTQMTQSPSTLSASVGDRVTITCRASGSVSTLMHWYQQKPGKAPKLLIYK
ASNLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCHQSWNSDTFGQG TRLEIK
wherein the CDRs are underlined
[0106] The polynucleotide sequence encoding SEQ ID NO: 77 is shown
in FIG. 5 and SEQ ID NO: 78 therein.
[0107] In one embodiment there is provided a variable region, such
as a heavy chain variable region with the following sequence
(Antibody 927 anti-toxin B antibody heavy chain variable region
sequence) SEQ ID NO: 79:
TABLE-US-00024 EVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMAWVRQAPGKGLEWVAT
INYDGRTTHYRDSVKGRFTISRDNSKSTLYLQMNSLRAEDTAVYYCTSIS
RSHYFDCWGQGTLVTVS
wherein the CDRs are underlined.
[0108] The polynucleotide sequence encoding SEQ ID NO: 79 is shown
in FIG. 5 and SEQ ID NO: 80 therein.
[0109] In one embodiment an antibody according to the invention
comprises variable regions shown in SEQ ID NO: 77 and SEQ ID NO:
79.
[0110] In one embodiment there is provided an antibody (for example
an anti-toxin B antibody) comprising a CDR, such as 1, 2, 3, 4, 5
or 6 CDRs, selected from:
TABLE-US-00025 SEQ ID NO: 81 KASKSISNHLA SEQ ID NO: 82 SGSTLQS SEQ
ID NO: 83 QQYDEYPYT SEQ ID NO: 84 GFSLQSYTIS SEQ ID NO: 85
AISGGGSTYYNLPLKS SEQ ID NO: 86 PRWYPRSYFDY
[0111] In one embodiment sequences 81 to 83 are in a light chain of
the antibody.
[0112] In one embodiment sequences 84 to 86 are in a heavy chain of
the antibody.
[0113] In one embodiment SEQ ID NO: 81 is CDR L1, SEQ ID NO: 82 is
CDR L2 and SEQ ID NO: 83 is CDR L3.
[0114] In one embodiment SEQ ID NO: 84 is CDR H1, SEQ ID NO: 85 is
CDR H2 and SEQ ID NO: 86 is CDR H3.
[0115] In one embodiment SEQ ID NO: 81 is CDR L1, SEQ ID NO: 82 is
CDR L2, SEQ ID NO; 83 is CDR L3, SEQ ID NO: 84 is CDR H1, SEQ ID
NO: 85 is CDR H2 and SEQ ID NO: 86 is CDR H3.
[0116] In one embodiment there is provided a variable region, such
as a light chain variable region with the following sequence
(Antibody 1099 anti-toxin B antibody; Light chain Variable region
sequence) SEQ ID NO: 87:
TABLE-US-00026 DVQLTQSPSFLSASVGDRVTITCKASKSISNHLAWYQEKPGKANKLLIHS
GSTLQSGTPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYDEYPYTFGQ GTRLEIKRT
wherein the CDRs are underlined.
[0117] In one embodiment the last two amino acids (RT) of SEQ ID
NO: 87 are omitted.
[0118] The polynucleotide sequence encoding SEQ ID NO: 87 is shown
in FIG. 6 and SEQ ID NO: 88 therein. In one embodiment the codons
encoding the last two amino acids (RT) are omitted.
[0119] In one embodiment there is provided a variable region, such
as a heavy chain variable region with the following sequence
(Antibody 1099 anti-toxin B antibody heavy chain variable region
sequence) SEQ ID NO: 89:
TABLE-US-00027 EVQLQESGPGLVKPSETLSLTCTVSGFSLQSYTISWVRQPPGKGLEWIAA
ISGGGSTYYNLPLKSRVTISRDTSKSQVSLKLSSVTAADTAVYYCTRPRW
YPRSYFDYWGRGTLVTVS
wherein the CDRs are underlined
[0120] The polynucleotide sequence encoding SEQ ID NO: 89 is shown
in FIG. 6 and SEQ ID NO: 90 therein.
[0121] In one embodiment an antibody according to the invention
comprises variable regions shown in SEQ ID NO 87: and SEQ ID NO:
89.
[0122] In one embodiment there is provided an antibody (for example
an anti-toxin B antibody) comprising a CDR, such as 1, 2, 3, 4, 5
or 6 CDRs, selected from:
TABLE-US-00028 SEQ ID NO: 91 RASQRISTSIH SEQ ID NO: 92 YASQSIS SEQ
ID NO: 93 QQSYSSLYT SEQ ID NO: 94 GFTFSDSYMA SEQ ID NO: 95
SISYGGTIIQYGDSVKG SEQ ID NO: 96 RQGTYARYLDF
[0123] In one embodiment sequences 91 to 93 are in a light chain of
the antibody.
[0124] In one embodiment sequences 94 to 96 are in a heavy chain of
the antibody.
[0125] In one embodiment SEQ ID NO: 91 is CDR L1, SEQ ID NO: 92 is
CDR L2 and SEQ ID NO; 93 is CDR L3.
[0126] In one embodiment SEQ ID NO: 94 is CDR H1, SEQ ID NO: 95 is
CDR H2 and SEQ ID NO: 96 is CDR H3.
[0127] In one embodiment SEQ ID NO: 91 is CDR L1, SEQ ID NO: 92 is
CDR L2, SEQ ID NO; 93 is CDR L3, SEQ ID NO: 94 is CDR H1, SEQ ID
NO: 95 is CDR H2 and SEQ ID NO: 96 is CDR H3.
[0128] In one embodiment there is provided a variable region, such
as a light chain variable region with the following sequence
(Antibody 1102 anti-toxin B antibody; Light chain Variable region
sequence) SEQ ID NO: 97:
TABLE-US-00029 NIVLTQSPATLSLSPGERATLSCRASQRISTSIHWYQQKPGQAPRLLIKY
ASQSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSYSSLYTFGQ GTKLEIK
wherein the CDRs are underlined
[0129] The polynucleotide sequence encoding SEQ ID NO: 97 is shown
in FIG. 6 and SEQ ID NO: 98 therein.
[0130] In one embodiment there is provided a variable region, such
as a heavy chain variable region with the following sequence
(Antibody 1102 anti-toxin B antibody heavy chain variable region
sequence) SEQ ID NO: 99:
TABLE-US-00030 EVQLVESGGGLVQPGGSLRLSCAVSGFTFSDSYMAWVRQAPGKGLEWIAS
ISYGGTIIQYGDSVKGRFTISRDNAKSSLYLQMNSLRAEDTAVYYCARRQ
GTYARYLDFWGQGTLVTVS
wherein the CDRs are underlined.
[0131] The polynucleotide sequence encoding SEQ ID NO: 99 is shown
in FIG. 7 and SEQ ID NO: 100 therein.
[0132] In one embodiment an antibody according to the invention
comprises variable regions shown in SEQ ID NO 97: and SEQ ID NO:
99.
[0133] In one embodiment there is provided an antibody (for example
an anti-toxin B antibody) comprising a CDR, such as 1, 2, 3, 4, 5
or 6 CDRs, selected from:
TABLE-US-00031 SEQ ID NO: 101 RASESVSTLLH SEQ ID NO: 102 KASNLAS
SEQ ID NO: 103 HQSWNSPPT SEQ ID NO: 104 GFTFSNYGMA SEQ ID NO: 105
IINYDASTTHYRDSVKG SEQ ID NO: 106 YGRSHYFDY
[0134] In one embodiment sequences 101 to 103 are in a light chain
of the antibody.
[0135] In one embodiment sequences 104 to 106 are in a heavy chain
of the antibody.
[0136] In one embodiment SEQ ID NO: 101 is CDR L1, SEQ ID NO: 102
is CDR L2 and SEQ ID NO: 103 is CDR L3.
[0137] In one embodiment SEQ ID NO: 104 is CDR H1, SEQ ID NO: 105
is CDR H2 and SEQ ID NO: 106 is CDR H3.
[0138] In one embodiment SEQ ID NO: 101 is CDR L1, SEQ ID NO: 102
is CDR L2, SEQ ID NO; 103 is CDR L3, SEQ ID NO: 104 is CDR H1, SEQ
ID NO: 105 is CDR H2 and SEQ ID NO; 106 is CDR H3.
[0139] In one embodiment there is provided a variable region, such
as a light chain variable region with the following sequence
(Antibody 1114 anti-toxin B antibody; Light chain Variable region
sequence) SEQ ID NO: 107:
TABLE-US-00032 ATQMTQSPSSLSASVGDRVTITCRASESVSTLLHWYQQKPGKAPKLLIYK
ASNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQSWNSPPTFGQ GTKLEIK
wherein the CDRs are underlined.
[0140] The polynucleotide sequence encoding SEQ ID NO: 107 is shown
in FIG. 7 and SEQ ID NO: 108 therein.
[0141] In one embodiment there is provided a variable region, such
as a heavy chain variable region with the following sequence
(Antibody 1114 anti-toxin B antibody heavy chain variable region
sequence) SEQ ID NO: 109:
TABLE-US-00033 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMAWVRQAPGKGLEWVAI
INYDASTTHYRDSVKGRFTISRDNAKSSLYLQMNSLRAEDTAVYYCTRYG
RSHYFDYWGQGTLVTVS
wherein the CDRs are underlined.
[0142] The polynucleotide sequence encoding SEQ ID NO: 109 is shown
in FIG. 7 and SEQ ID NO: 110 therein.
[0143] In one embodiment an antibody according to the invention
comprises variable regions shown in SEQ ID NO: 107 and SEQ ID NO:
109.
[0144] In one embodiment there is provided an antibody (for example
an anti-toxin B antibody) comprising a CDR, such as 1, 2, 3, 4, 5
or 6 CDRs, selected from:
TABLE-US-00034 SEQ ID NO: 111 RASESVSTLLH SEQ ID NO: 112 KASNLAS
SEQ ID NO: 113 HQSWNSPPT SEQ ID NO: 114 GFTFSNYGMA SEQ ID NO: 115
IINYDASTTHYRDSVK SEQ ID NO: 116 YGRSHYFDY
[0145] In one embodiment sequences 111 to 113 are in a light chain
of the antibody.
[0146] In one embodiment sequences 114 to 116 are in a heavy chain
of the antibody.
[0147] In one embodiment SEQ ID NO: 111 is CDR L1, SEQ ID NO: 112
is CDR L2 and SEQ ID NO: 113 is CDR L3.
[0148] In one embodiment SEQ ID NO: 114 is CDR H1, SEQ ID NO: 115
is CDR H2 and SEQ ID NO: 116 is CDR H3.
[0149] In one embodiment SEQ ID NO: 111 is CDR L1, SEQ ID NO: 112
is CDR L2, SEQ ID NO; 113 is CDR L3, SEQ ID NO: 114 is CDR H1, SEQ
ID NO: 115 is CDR H2 and SEQ ID NO: 116 is CDR H3.
[0150] In one embodiment there is provided a variable region, such
as a light chain variable region with the following sequence
(Antibody 1114 graft 8 anti-toxin B antibody; Light chain Variable
region sequence) SEQ ID NO: 117:
TABLE-US-00035 DTVLTQSPSSLSASVGDRVTITCRASESVSTLLHWYQQKPGKAPKLLIYK
ASNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQSWNSPPTFGQ GTKLEIK
wherein the CDRs are underlined.
[0151] The polynucleotide sequence encoding SEQ ID NO: 117 is shown
in FIG. 8 and SEQ ID NO: 118 therein.
[0152] In one embodiment there is provided a variable region, such
as a heavy chain variable region with the following sequence
(Antibody 1114 graft 8 anti-toxin B antibody heavy chain variable
region sequence) SEQ ID NO: 119:
TABLE-US-00036 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMAWVRQAPGKGLEWVAI
INYDASTTHYRDSVKGRFTISRDNAKSSLYLQMNSLRAEDTAVYYCTRYG
RSHYFDYWGQGTLVTVS
wherein the CDRs are underlined.
[0153] The polynucleotide sequence encoding SEQ ID NO: 119 is shown
in FIG. 8 and SEQ ID NO: 120 therein.
[0154] In one embodiment an antibody according to the invention
comprises variable regions shown in SEQ ID NO: 117 and SEQ ID NO:
119.
[0155] In one embodiment there is provided an antibody (for example
an anti-toxin B antibody) comprising a CDR, such as 1, 2, 3, 4, 5
or 6 CDRs, selected from:
TABLE-US-00037 SEQ ID NO: 121 KASQNIYMYLN SEQ ID NO: 122 NTNKLHT
SEQ ID NO: 123 LQHKSFPYT SEQ ID NO: 124 GFTFRDSFMA SEQ ID NO: 125
SISYEGDKTYYGDSVKG SEQ ID NO: 126 LTITTSGDS
[0156] In one embodiment sequences 121 to 123 are in a light chain
of the antibody.
[0157] In one embodiment sequences 124 to 126 are in a heavy chain
of the antibody.
[0158] In one embodiment SEQ ID NO: 121 is CDR L1, SEQ ID NO: 122
is CDR L2 and SEQ ID NO: 123 is CDR L3.
[0159] In one embodiment SEQ ID NO: 124 is CDR H1, SEQ ID NO: 125
is CDR H2 and SEQ ID NO: 126 is CDR H3.
[0160] In one embodiment SEQ ID NO: 121 is CDR L1, SEQ ID NO: 122
is CDR L2, SEQ ID NO: 123 is CDR L3, SEQ ID NO: 124 is CDR H1, SEQ
ID NO: 125 is CDR H2 and SEQ ID NO: 126 is CDR H3.
[0161] In one embodiment there is provided a variable region, such
as a light chain variable region with the following sequence
(Antibody 1125 anti-toxin B antibody; Light chain Variable region
sequence) SEQ ID NO: 127:
TABLE-US-00038 DIQMTQSPSSLSASVGDRVTITCKASQNIYMYLNWYQQKPGKAPKRLIYN
TNKLHTGVPSRFSGSGSGTEYTLTISSLQPEDFATYYCLQHKSFPYTFGQ GTKLEIK
wherein the CDRs are underlined.
[0162] The polynucleotide sequence encoding SEQ ID NO: 127 is shown
in FIG. 8 and SEQ ID NO: 128 therein.
[0163] In one embodiment there is provided a variable region, such
as a heavy chain variable region with the following sequence
(Antibody 1125 anti-toxin B antibody heavy chain variable region
sequence) SEQ ID NO: 129:
TABLE-US-00039 EVQLVESGGGLVQPGGSLRLSCAASGFTFRDSFMAWVRQAPGKGLEWVAS
ISYEGDKTYYGDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARLT
ITTSGDSWGQGTMVTVSS
wherein the CDRs are underlined.
[0164] In one embodiment the last amino acid (S) of SEQ ID NO: 129
is omitted.
[0165] The polynucleotide sequence encoding SEQ ID NO: 129 is shown
in FIG. 9 and SEQ ID NO: 130 therein. In one embodiment the codon
AGC encoding the last amino acid S is omitted.
[0166] In one embodiment an antibody according to the invention
comprises variable regions shown in SEQ ID NO: 127 and SEQ ID NO:
129.
[0167] In one embodiment there is provided antibody (for example an
anti-toxin B antibody) comprising a CDR, such as 1, 2, 3, 4, 5 or 6
CDRs, selected from:
TABLE-US-00040 SEQ ID NO: 131 KASQHVGTNVD SEQ ID NO: 132 GASIRYT
SEQ ID NO: 133 LQYNYNPYT SEQ ID NO: 134 GFIFSNFGMS SEQ ID NO: 135
SISPSGGNAYYRDSVKG SEQ ID NO: 136 RAYSSPFAF
[0168] In one embodiment sequences 131 to 133 are in a light chain
of the antibody.
[0169] In one embodiment sequences 134 to 136 are in a heavy chain
of the antibody.
[0170] In one embodiment SEQ ID NO: 131 is CDR L1, SEQ ID NO: 132
is CDR L2 and SEQ ID NO: 133 is CDR L3.
[0171] In one embodiment SEQ ID NO: 134 is CDR H1, SEQ ID NO: 135
is CDR H2 and SEQ ID NO: 136 is CDR H3.
[0172] In one embodiment SEQ ID NO: 131 is CDR L1, SEQ ID NO: 132
is CDR L2, SEQ ID NO: 133 is CDR L3, SEQ ID NO: 134 is CDR H1, SEQ
ID NO: 135 is CDR H2 and SEQ ID NO: 136 is CDR H3.
[0173] In one embodiment there is provided a variable region, such
as a light chain variable region with the following sequence
(Antibody 1129 anti-toxin B antibody; Light chain Variable region
sequence) SEQ ID NO: 137:
TABLE-US-00041 DTQMTQSPSSLSASVGDRVTITCKASQHVGTNVDWYQQKPGKVPKLLIYG
ASIRYTGVPDRFTGSGSGTDFTLTISSLQPEDVATYYCLQYNYNPYTFGQ GTKLEIK
wherein the CDRs are underlined.
[0174] The polynucleotide sequence encoding SEQ ID NO: 137 is shown
in FIG. 8 and SEQ ID NO: 138 therein.
[0175] In one embodiment there is provided a variable region, such
as a heavy chain variable region with the following sequence
(Antibody 1129 anti-toxin B antibody heavy chain variable region
sequence) SEQ ID NO: 139:
TABLE-US-00042 EVQLVESGGGVVQPGRSLRLSCATSGFIFSNFGMSWVRQAPGKGLEWVAS
ISPSGGNAYYRDSVKGRFTISRDNSKTTLYLQMNSLRAEDTAVYYCTRRA
YSSPFAFWGQGTLVTVSS
wherein the CDRs are underlined.
[0176] In one embodiment the last amino acid (S) of SEQ ID NO: 139
is omitted.
[0177] The polynucleotide sequence encoding SEQ ID NO: 139 is shown
in FIG. 8 and SEQ ID NO: 140 therein. In one embodiment the codon
AGC encoding the last amino acid S is omitted.
[0178] In one embodiment an antibody according to the invention
comprises variable regions shown in SEQ ID NO: 137 and SEQ ID NO:
139.
[0179] In one embodiment there is provided an antibody (for example
an anti-toxin B antibody) comprising a CDR, such as 1, 2, 3, 4, 5
or 6 CDRs, selected from:
TABLE-US-00043 SEQ ID NO: 141 KASKSISNHLA SEQ ID NO: 142 SGSTLQP
SEQ ID NO: 143 QQYDEYPYT SEQ ID NO: 144 GFSLNSYTIT SEQ ID NO: 145
AISGGGSTYFNSALKS SEQ ID NO: 146 PRWYPRSYFDY
[0180] In one embodiment sequences 141 to 143 are in a light chain
of the antibody.
[0181] In one embodiment sequences 144 to 146 are in a heavy chain
of the antibody.
[0182] In one embodiment SEQ ID NO: 141 is CDR L1, SEQ ID NO: 142
is CDR L2 and SEQ ID NO: 143 is CDR L3.
[0183] In one embodiment SEQ ID NO: 144 is CDR H1, SEQ ID NO: 145
is CDR H2 and SEQ ID NO: 146 is CDR H3.
[0184] In one embodiment SEQ ID NO: 141 is CDR L1, SEQ ID NO: 142
is CDR L2, SEQ ID NO: 143 is CDR L3, SEQ ID NO: 144 is CDR H1, SEQ
ID NO: 145 is CDR H2 and SEQ ID NO: 146 is CDR H3.
[0185] In one embodiment there is provided a variable region, such
as a light chain variable region with the following sequence
(Antibody 1134 anti-toxin B antibody; Light chain Variable region
sequence):
TABLE-US-00044 SEQ ID NO: 147
DVQLTQSPSFLSASVGDRVTITCKASKSISNHLAWYQEKPGKANKLLIH
SGSTLQPGTPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYDEYPYTF GQGTRLEIK
wherein the CDRs are underlined.
[0186] The polynucleotide sequence encoding SEQ ID NO: 147 is shown
in FIG. 9 and SEQ ID NO: 148 therein.
[0187] In one embodiment there is provided a variable region, such
as a heavy chain variable region with the following sequence
(Antibody 1134 anti-toxin B antibody heavy chain variable region
sequence) SEQ ID NO: 149:
TABLE-US-00045 EVQLQESGPGLVKPSETLSLTCTVSGFSLNSYTITWVRQPPGKGLEWIA
AISGGGSTYFNSALKSRVTISRDTSKSQVSLKLSSVTAADTAVYYCTRP
RWYPRSYFDYWGRGTLVTVS
wherein the CDRs are underlined
[0188] The polynucleotide sequence encoding SEQ ID NO: 149 is shown
in FIG. 9 and SEQ ID NO: 150 therein.
[0189] In one embodiment an antibody according to the invention
comprises variable regions shown in SEQ ID NO 147: and SEQ ID NO:
149.
[0190] In one embodiment there is provided antibody (for example an
anti-toxin B antibody) comprising a CDR, such as 1, 2, 3, 4, 5 or 6
CDRs, selected from:
TABLE-US-00046 SEQ ID NO: 151 KASQNVGNNVA SEQ ID NO: 152 YASNRFT
SEQ ID NO: 153 QRVYQSTWT SEQ ID NO: 154 GFSLTSYYVH SEQ ID NO: 155
CIRTGGNTEYQSEFKS SEQ ID NO: 156 GNYGFAY
[0191] In one embodiment sequences 151 to 153 are in a light chain
of the antibody.
[0192] In one embodiment sequences 154 to 156 are in a heavy chain
of the antibody.
[0193] In one embodiment SEQ ID NO: 151 is CDR L1, SEQ ID NO: 152
is CDR L2 and SEQ ID NO: 153 is CDR L3.
[0194] In one embodiment SEQ ID NO: 154 is CDR H1, SEQ ID NO: 155
is CDR H2 and SEQ ID NO: 156 is CDR H3.
[0195] In one embodiment SEQ ID NO: 151 is CDR L1, SEQ ID NO: 152
is CDR L2, SEQ ID NO; 153 is CDR L3, SEQ ID NO: 154 is CDR H1, SEQ
ID NO: 155 is CDR H2 and SEQ ID NO; 156 is CDR H3.
[0196] In one embodiment there is provided a variable region, such
as a light chain variable region with the following sequence
(Antibody 1151 anti-toxin B antibody; Light chain Variable region
sequence) SEQ ID NO: 157:
TABLE-US-00047 AIQMTQSPSSLSASVGDRVTITCKASQNVGNNVAWYQHKPGKAPKLLIY
YASNRFTGVPSRFTGGGYGTDFTLTISSLQPEDFATYYCQRVYQSTWTF GQGTKVEIK
wherein the CDRs are underlined.
[0197] The polynucleotide sequence encoding SEQ ID NO: 157 is shown
in FIG. 9 and SEQ ID NO: 158 therein.
[0198] In one embodiment there is provided a variable region, such
as a heavy chain variable region with the following sequence
(Antibody 1151 anti-toxin B antibody heavy chain variable region
sequence) SEQ ID NO: 159:
TABLE-US-00048 EVQLQESGPGLVKPSETLSLTCTVSGFSLTSYYVHWVRQPPGKGLEWMG
CIRTGGNTEYQSEFKSRVTISRDTSKNQVSLKLSSVTAADTAVYYCARG
NYGFAYWGQGTLVTVS
wherein the CDRs are underlined.
[0199] The polynucleotide sequence encoding SEQ ID NO: 159 is shown
in FIG. 9 and SEQ ID NO: 160 therein.
[0200] In one embodiment an antibody according to the invention
comprises variable regions shown in SEQ ID NO: 157 and SEQ ID NO:
159.
[0201] In one embodiment there is provided an antibody (for example
an anti-toxin B antibody) comprising a CDR, such as 1, 2, 3, 4, 5
or 6 CDRs, selected from:
TABLE-US-00049 SEQ ID NO: 161 KASQNINKYLD SEQ ID NO: 162 NIQSLHT
SEQ ID NO: 163 FQHNSGW SEQ ID NO: 164 GFTFTQAAMF SEQ ID NO: 165
RISTKSNNFATYYPDSVKG SEQ ID NO: 166 PAYYYDGTVPFAY
[0202] In one embodiment sequences 161 to 163 are in a light chain
of the antibody.
[0203] In one embodiment sequences 164 to 166 are in a heavy chain
of the antibody.
[0204] In one embodiment SEQ ID NO: 161 is CDR L1, SEQ ID NO: 162
is CDR L2 and SEQ ID NO: 163 is CDR L3.
[0205] In one embodiment SEQ ID NO: 164 is CDR H1, SEQ ID NO: 165
is CDR H2 and SEQ ID NO: 166 is CDR H3.
[0206] In one embodiment SEQ ID NO: 161 is CDR L1, SEQ ID NO: 162
is CDR L2, SEQ ID NO: 163 is CDR L3, SEQ ID NO: 164 is CDR H1, SEQ
ID NO: 165 is CDR H2 and SEQ ID NO: 166 is CDR H3.
[0207] In one embodiment there is provided a variable region, such
as a light chain variable region with the following sequence
(Antibody 1153 anti-toxin B antibody; Light chain Variable region
sequence) SEQ ID NO: 167:
TABLE-US-00050 DIQMTQSPSSLSASVGDRVTITCKASQNINKYLDWYQQKPGKVPKLLIY
NIQSLHTGIPSRFSGSGSGTDFTLTISSLQPEDVATYYCFQHNSGWTFG QGTRLEIK
wherein the CDRs are underlined.
[0208] The polynucleotide sequence encoding SEQ ID NO: 167 is shown
in FIG. 10 and SEQ ID NO: 168 therein.
[0209] In one embodiment there is provided a variable region, such
as a heavy chain variable region with the following sequence
(Antibody 1153 anti-toxin B antibody heavy chain variable region
sequence) SEQ ID NO: 169:
TABLE-US-00051 EVQLVESGGGLVQPGGSLKLSCAASGFTFTQAAMFWVRQASGKGLEGIA
RISTKSNNFATYYPDSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYC
TAPAYYYDGTVPFAYWGQGTLVTVS
wherein the CDRs are underlined.
[0210] The polynucleotide sequence encoding SEQ ID NO: 169 is shown
in FIG. 10 and SEQ ID NO: 170 therein.
[0211] In one embodiment an antibody according to the invention
comprises variable regions shown in SEQ ID NO: 167 and SEQ ID NO:
169.
[0212] In one embodiment there is provided antibody comprising 6
CDRs independently selected from SEQ ID NOs 1, 2, 3, 4, 5, 6, 11,
12, 13, 14, 15, 16, 21, 22, 23, 24, 25, 26, 31, 32, 33, 34, 35, 36,
41, 42, 43, 44, 45, 46, 51, 52, 53, 54, 55, 56, 61, 62, 63, 64, 65,
66, 71, 72, 73, 74, 75, 76, 81, 82, 83, 84, 85, 86, 91, 92, 93, 94,
95, 96, 101, 102, 103, 104, 105, 106, 111, 112, 113, 114, 115, 116,
121, 122, 123, 124, 125, 126, 131, 132, 133, 134, 135, 136, 141,
142, 143, 144, 145, 146, 151, 152, 153, 154, 155, 156, 161, 162,
163, 164, 165 and 166.
[0213] In one embodiment there is provided an anti-TcdA antibody
comprising 6 CDRs independently selected from SEQ ID NOs 1, 2, 3,
4, 5, 6, 11, 12, 13, 14, 15, 16, 21, 22, 23, 24, 25, 26, 31, 32,
33, 34, 35, 36, 41, 42, 43, 44, 45, 46, 51, 52, 53, 54, 55 and
56.
[0214] In one embodiment there is provided an anti-TcdB antibody
comprising 6 CDRs independently selected from SEQ ID NOs 61, 62,
63, 64, 65, 66, 71, 72, 73, 74, 75, 76, 81, 82, 83, 84, 85, 86, 91,
92, 93, 94, 95, 96, 101, 102, 103, 104, 105, 106, 111, 112, 113,
114, 115, 116, 121, 122, 123, 124, 125, 126, 131, 132, 133, 134,
135, 136, 141, 142, 143, 144, 145, 146, 151, 152, 153, 154, 155,
156, 161, 162, 163, 164, 165 and 166.
[0215] In one embodiment there is provided an antibody which
comprises two variable regions independently selected from SEQ ID
NOs: 7, 9, 17, 19, 27, 29, 37, 39, 47, 49, 57, 59, 67, 69, 77, 79,
87, 89, 97, 99, 107, 109, 117, 119, 127, 129, 137, 139, 147, 149,
157 and 159.
[0216] In one embodiment there is provided an antibody which
comprises two variable regions independently selected from SEQ ID
NOs: 7, 9, 17, 19, 27, 29, 37, 39, 47, 49, 57 and 59.
[0217] In one embodiment there is provided an antibody which
comprises two variable regions independently selected from SEQ ID
NOs: 67, 69, 77, 79, 87, 89, 97, 99, 107, 109, 117, 119, 127, 129,
137, 139, 147, 149, 157 and 159.
[0218] In one embodiment the antibodies according to the invention
are humanized.
[0219] In one embodiment the antibody or antibodies are directed to
the C terminal "cell binding" portion of the TcdA and/or TcdB
toxin.
[0220] In one embodiment an antibody according to the invention is
suitable for neutralising toxin A or toxin B.
[0221] Neutralising as employed herein is intended to refer to the
elimination or reduction of harmful/deleterious effects of the
target toxin, for example at least a 50% reduction in the relevant
harmful effect.
[0222] The inventors have established by using internal comparisons
between antibodies discovered in this application and by comparison
against antibodies well described in the art (Babcock et al. 2006;
Lowy et al., 2010) that some antibodies have the desirable
characteristic of maintaining effective neutralization (for example
low EC.sub.50 and high % protection) even at high toxin
concentrations. Other antibodies including those described in the
art do not maintain effective toxin neutralization at high toxin
concentrations.
[0223] Effective toxin concentrations can be defined as a `lethal
dose` (LD) in titration studies in the absence of neutralizing
antibodies. Neutralisation assays are typically conducted at an LD
of 50% of complete cell killing (i.e. an LD.sub.50) but may be more
rigorously conducted at an LD.sub.50.
[0224] Assays may also be performed under considerably more
challenging conditions such as LD.sub.90, LD.sub.95 and/or
LD.sub.max (LD.sub.max is the maximal toxin quantity which can be
included in an assay as constrained by assay volume and maximum
toxin concentration/solubility). Such assays aim to mimic the early
stages of infection of humans when C. difficile growth in the bowel
is rampant and diarrhea and other symptoms lead one to hypothesise
that toxin concentrations are at their highest. Antibodies which
effectively neutralize damaging toxin activities under high toxin
concentration conditions are thought by the present inventors to
have special clinical value for the control of symptoms in human
infections. In one embodiment the antibody or antibodies of the
present disclosure have useful, for example low EC.sub.50 values
and/or high % protection from cell death for one or more the
LD.sub.80, LD.sub.90, LD.sub.95 and/or LD.sub.max. In one
embodiment the EC.sub.50 in the one or more of the latter
situations is 15 ng/ml or less, for example 10 ng/ml or less, such
as 5 ng/ml or less, in particular 1 ng/ml or less. In one
embodiment the % protection from cell death is >90%, or >75%
or >50%.
[0225] Thus in one embodiment the present disclosure provides an
antibody or a combination of antibodies which maintain toxin
neutralization even in the presence of high levels of toxin, for
example as measured in an assay provided herein.
[0226] The harmful effect of toxin may, for example be measured in
a suitable in vitro assay. In one embodiment the neutralization is
measured in an assay given in Example 1 below. Also provided is an
antibody or antibodies identified in a neutralization assay, for
example wherein the potency of the antibody is maintained in the
presence of high levels of toxin.
[0227] Toxin A is used interchangeably with TcdA.
[0228] Toxin B is used interchangeably with TcdB.
[0229] In one embodiment an antibody according to the invention is
a monoclonal antibody or binding fragment thereof.
[0230] In one embodiment a monoclonal antibody according to the
invention is capable of neutralising TcdA with very high potency
and affinity.
[0231] In one embodiment a monoclonal antibody according to the
invention is capable of neutralising TcdA with very high potency
and affinity and high avidity.
[0232] Avidity as employed herein refers to the combined strength
of multiple binding affinities.
[0233] In one embodiment a monoclonal antibody according to the
invention is capable of neutralising TcdA with very high potency
and affinity and high avidity and high valency of binding.
[0234] Valency of binding as employed herein refers to the ability
for a monoclonal antibody to bind to an antigen multiple times.
High valency of binding hence results in high levels of decoration
of antigen with antibodies and/or high levels of cross-linking of
toxin molecules, which is thought to be advantageous.
[0235] Anti-TcdA Mabs according to the present disclosure may be
suitable for neutralising the early effects of TcdA, for example on
cells such as loss of tight junctions.
[0236] Tight junction as employed herein is intended to refer to
impermeable zone of connection between cells within a monolayer or
anatomical tissue structure. Fluid loss does not occur when tight
junctions retain their structural and functional integrity. Loss of
tight junctions is an indication that the cell has been compromised
by toxin and is well documented as being an early step in the toxic
effects of TcdA and TcdB (25) and results in loss of fluid
containing serum, immunoglobulin and ions (26, 3). Loss of tight
junctions is thought to be a first step on the onset of diarrhoea
in humans.
[0237] The TEER assay system, can be used to measure the loss of
tight junction in vitro. TEER is an acronym for trans epithelial
electric resistance assay and it is generally employed to measure
the permeability of a differentiated cell layer representative of a
gut endothelial lining. However, in the context of screening for
antibodies TEER loss can be employed to identify antibodies that
slow or prevent damage to the tight junctions and hence is a
surrogate for protection against tissue damage leading to
diarrhoea.
[0238] Often Caco-2 cells are employed since they are derived from
human colon cells and are known to form differentiated monolayers
with cells connected by tight junctions. A kit is commercially
available from Becton-Dickinson named the Caco-2 BioCoat HTS plate
system (BD Biosciences/354802). The instructions in the kit are
suitable for testing in the present context. The resistance of the
membrane changes when the membrane has been compromised.
[0239] Generally the antibody will be pre-incubated with the toxin
before addition to the TEER system to establish if the antibody can
prevent or slow the damage to the membrane caused by the toxin. The
assay may be performed over a suitable period, for example 24 hours
taking measurements at certain time-points. The present inventors
have established that the 4 hour time point is particularly
discriminating for therapeutically useful antibodies. The
concentration of toxin employed in the TEER assay is generally in
the range 100-200 ng/ml, most preferably 125 ng/ml
[0240] The concentration of antibody (for example IgG1) employed in
the TEER assay is generally in the range of 4 to 2000 ng/ml, for
example 50 to 1000 ng/ml, such as 100 to 500 ng/ml.
[0241] In one embodiment the EC.sub.50 of the antibody in the TEER
assay employed in said condition is at least 200 ng/ml, for example
less than 100 ng/ml, such as about 60-80 ng/ml.
[0242] In one embodiment there is provided an anti-TcdA antibody or
an anti-TcdB antibody suitable for use as a therapeutic agent in
the treatment or prevention of C. difficile infection, wherein said
antibody was screened and selected employing a TEER assay.
[0243] In one aspect there is provided a method of screening an
antibody in a TEER assay for the ability to slow or prevent loss of
tight junctions. In one embodiment the antibody or antibodies
screened are anti-TcdA antibodies. In one embodiment the antibody
or antibodies screened are anti-TcdB antibodies. In one embodiment
the antibody or antibodies screened are a combination of anti-TcdA
and anti-TcdB antibodies. In one embodiment the method comprises
the step of identifying an appropriate antibody or antibodies and
expressing suitable quantities of same. In one embodiment the
method comprises the further step of formulating said antibody or
antibodies in a pharmaceutical formulation. In one embodiment the
method comprises the further step of administering said antibody or
antibodies or said formulation to a patient in need thereof.
[0244] In one embodiment multiple antibodies of the disclosure may
be capable of binding to the target toxin (TcdA or TcdB), which may
aid immune clearance of the toxin.
[0245] Multiple antibodies as employed herein is intended to refer
to multiple copies of an antibody with the same sequence or an
antibody with the same amino acid sequence or an antibody specific
to the same target antigen but with a different amino acid
sequence.
[0246] In one embodiment the antibodies according to the invention
are specific to the target antigen, for example specific to an
epitope in the target antigen.
[0247] In one embodiment the antibodies of the invention are able
to bind to the target antigen in two or more locations, for example
two or three locations, such as four, five, six, seven, eight,
nine, ten or more locations, for example the toxin may comprise
repeating domains and thus an antibody may be specific to an
epitope and in fact that epitope may be present in the antigen
several times i.e. in more than one location. Thus given antibodies
may bind the same epitope multiple times in different locations in
the antigen.
[0248] In one embodiment the antibody binds to the given antigen
multiple times, for example 2 to 20 times such as 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15 or 16 times. In one embodiment the
antibody binds the given antigen at least 3 times. This multiple
binding is thought to be important in neutralisation and/or
clearance of the toxin. Whilst not wishing to be bound by theory it
is thought that multiple binding, for example 3 more times, i.e. by
decoration with 3 or more Fc fragments is important in triggering
rapid clearance of the toxin (24) primarily via the liver and
spleen (27, 28).
[0249] In one embodiment the anti-TcdA antibody binds 3 or more
times, for example 3 to 16 times.
[0250] In one embodiment the anti-TcdA antibody binds 12 times.
[0251] In one embodiment the anti-TcdA antibody binds 2 times.
[0252] In one embodiment an anti-TcdA antibody binds in the
catalytic-terminal cell binding domain of TcdA.
[0253] In one embodiment the anti-TcdB antibody binds 2 or more
times, for example 2 times.
[0254] In one embodiment an anti-TcdB antibody binds in the
catalytic-terminal cell binding domain of TcdB.
[0255] In one embodiment the antibody or antibodies according to
disclosure are capable of cross-linking toxin molecules, for
example one arm of the antibody molecule binds one toxin molecule
and another of the antibody binds a epitope in a different toxin
molecule, thereby forming a sort of immune complex. The formation
of the latter may also facilitate activation of the immune system
to clear the relate toxin and thereby minimise the deleterious in
vivo effects of the same.
[0256] In one embodiment an innate immune response, such as
complement is activated.
[0257] In one embodiment the antibody or antibodies of the
disclosure have high potency against toxins derived from strains of
different ribotypes, for example 003, 027, 078.
[0258] In one embodiment antibodies against TcdA may have an
EC.sub.50 in the range of 0.1-100 ng/ml, such as 1 to 10 ng/ml and
a maximal inhibition in the range of 50-100% at toxin
concentrations of LD.sub.80-95, for example against toxins from
strains of ribotypes 003, 027 and 078.
[0259] In one embodiment antibodies against TcdA may have an
EC.sub.50 in the range of 0.1 100 ng/ml, such as 1 to 10 ng/ml and
a maximal inhibition in the range of 60-100%, 70-100%, 80-100% or
90-100% at toxin concentrations of LD.sub.80-95, for example
against toxins from strains of ribotypes 003, 027 and 078.
[0260] In one embodiment antibodies against TcdB may have EC.sub.50
in the range of 0.1-100 ng/ml, such as 1 to 10 ng/ml and a maximal
inhibition in the range of 50-100% at toxin concentrations of
LD.sub.80-95, for example against toxins from strains of ribotype
003.
[0261] In one embodiment antibodies against TcdB may have EC.sub.50
in the range of 0.1-100 ng/ml, such as 1 to 10 ng/ml and a maximal
inhibition in the range of 60-100%, 70-100%, 80-100% or 90-100% at
toxin concentrations of LD.sub.80-95, for example against toxins
from strains of ribotype 003.
[0262] In one embodiment there are provided combinations of
antibodies according to the invention, for example combinations of
antibodies specific to TcdA, combinations of antibodies specific to
TcdB or combinations of antibodies to specific to TcdA and
antibodies specific to TcdB.
[0263] Combinations of antibodies specific to TcdA will generally
refer to combinations of antibodies directed to different epitopes
on the target antigen TcdA, or at least with different binding
properties.
[0264] Combinations of antibodies specific to TcdB will generally
refer to combinations of antibodies directed to different epitopes
on the target antigen TcdB, or at least with different binding
properties.
[0265] The combinations may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14 or 15 distinct antibodies, for example 2, 3, 4 or 5
antibodies.
[0266] In one embodiment there is provided a combination of one
anti-TcdA antibody and two anti-TcdB, for example wherein the
anti-TcdA antibody is 997 and where the anti-TcdB antibodies are
1125 and 1151
[0267] In particular there is provided a combination of one
anti-TcdA antibody comprising a heavy variable region with a
sequence as shown in SEQ ID NO:49 and a light variable region with
a sequence shown in SEQ ID NO: 47 and two anti-TcdB antibodies the
first with a heavy variable region shown in SEQ ID NO: 129 and a
light variable region shown in SEQ ID NO: 127, and the second with
a heavy variable region shown in SEQ ID NO: 159 and light variable
region shown in SEQ ID NO: 157.
[0268] Distinct antibodies as employed herein is intended to refer
to antibodies with different amino acid sequences, which may bind
the same epitope or different epitopes on the target antigen.
[0269] Also provided by the present invention is a specific region
or epitope of TcdA which is bound by an antibody provided by the
present invention, in particular an antibody comprising the heavy
chain sequence given in SEQ ID NO:49 and the light chain sequence
given in SEQ ID NO:47.
[0270] Also provided by the present invention is a specific region
or epitope of TcdB which is bound by an antibody provided by the
present invention, in particular an antibody comprising the heavy
chain sequence given in SEQ ID NO:129 and the light chain sequence
given in SEQ ID NO:127 or an antibody comprising the heavy chain
sequence given in SEQ ID NO:159 and the light chain sequence given
in SEQ ID NO:157.
[0271] This specific region or epitope of the TcdA or TcdB toxins
can be identified by any suitable epitope mapping method known in
the art in combination with any one of the antibodies provided by
the present invention. Examples of such methods include screening
peptides of varying lengths derived from the toxins for binding to
the antibody of the present invention with the smallest fragment
that can specifically bind to the antibody containing the sequence
of the epitope recognised by the antibody. The peptides may be
produced synthetically or by proteolytic digestion of the toxin
polypeptide. Peptides that bind the antibody can be identified by,
for example, mass spectrometric analysis. In another example, NMR
spectroscopy or X-ray crystallography can be used to identify the
epitope bound by an antibody of the present invention. Once
identified, the epitopic fragment which binds an antibody of the
present invention can be used, if required, as an immunogen to
obtain additional antagonistic antibodies which bind the same
epitope.
[0272] Antibodies which cross-block the binding of an antibody
according to the present invention may be similarly useful in
neutralizing toxin activity. Accordingly, the present invention
also provides a neutralizing antibody having specificity for TcdA
or TcdB, which cross-blocks the binding of any one of the
antibodies described above to TcdA or TcdB and/or is cross-blocked
from binding these toxins by any one of those antibodies. In one
embodiment, such an antibody binds to the same epitope as an
antibody described herein above. In another embodiment the
cross-blocking neutralising antibody binds to an epitope which
borders and/or overlaps with the epitope bound by an antibody
described herein above. In another embodiment the cross-blocking
neutralising antibody of this aspect of the invention does not bind
to the same epitope as an antibody of the present invention or an
epitope that borders and/or overlaps with said epitope.
[0273] Cross-blocking antibodies can be identified using any
suitable method in the art, for example by using competition ELISA
or BIAcore assays where binding of the cross blocking antibody to
TcdA or TcdB prevents the binding of an antibody of the present
invention or vice versa.
[0274] In one embodiment there is provided a method of generating
an anti-TcdA or anti-TcdB antibody, in particular a neutralizing
antibody and/or an antibody which cross-blocks the binding of an
antibody described herein, said method comprising the steps of
immunizing a host with a suitable antigen, for example an antigen
shown in any one of SEQ ID Nos 173 to 194 or a combination thereof.
The said method may also comprise one or more the following steps,
for example identifying an antibody of interest (in particular
using a functional assay such as TEER assay), expressing the
antibody of interest, and optionally formulating the antibody as a
pharmaceutically acceptable composition.
[0275] Thus in one aspect the present disclosure provides a method
of immunizing a host with an amino acid sequence shown in SEQ ID
Nos 173 to 194 or a combination thereof.
[0276] In one embodiment the antibodies according to the invention
have an affinity to the target antigen of 10 nM or less, for
example 1 nM or less such as 900 pM, in particular 800 pM, 700 pM,
600 pM or 500 pM, such as 60 pM.
[0277] In one embodiment the affinity is for TcdA or TcdB or a
fragment thereof. In one example the fragment is TcdA123
corresponding to residues S1827-D2249 of TcdA. In one example the
fragment is TcdA456 corresponding to residues G2205-R2608. In one
embodiment the fragment is TcdB1234 corresponding to residues
S1833-E2366 of TcdB.
[0278] In one embodiment antibodies according to the invention or a
combination thereof have an EC.sub.50 of 200 ng/ml or less, for
example 150 ng/ml or less such as 100 ng/ml or less, such as in the
range 0.1 to 10 ng/ml.
[0279] The individual component antibodies of mixtures are not
required to have an EC.sub.50 in said range provided that when they
are used in combination with one or more antibodies the combination
has an EC.sub.50 in said range.
[0280] Advantageously, the antibodies of the invention are stable,
for example are thermally stable at temperatures above 50.degree.
C. such as 60 or 70.degree. C.
[0281] The antibodies and combinations according to the present
invention also have one or more of the following advantageous
properties: slow off rate, high affinity, high potency, the ability
to bind multiple times to the target antigen, to neutralise the
toxin by a mechanism which reduces the loss of measurable TEER
activity, to stimulate or assist the hosts natural immune response,
to catalyse or assist in immune clearance of the pathogen (or
toxin) and/or to educate the immune system to respond appropriately
to the pathogen (or toxin).
[0282] The residues in antibody variable domains are conventionally
numbered according to a system devised by Kabat et al. This system
is set forth in Kabat et al., 1987, in Sequences of Proteins of
Immunological Interest, US Department of Health and Human Services,
NIH, USA (hereafter "Kabat et al. (supra)"). This numbering system
is used in the present specification except where otherwise
indicated.
[0283] The Kabat residue designations do not always correspond
directly with the linear numbering of the amino acid residues. The
actual linear amino acid sequence may contain fewer or additional
amino acids than in the strict Kabat numbering corresponding to a
shortening of, or insertion into, a structural component, whether
framework or complementarity determining region (CDR), of the basic
variable domain structure. The correct Kabat numbering of residues
may be determined for a given antibody by alignment of residues of
homology in the sequence of the antibody with a "standard" Kabat
numbered sequence.
[0284] The CDRs of the heavy chain variable domain are located at
residues 31-35 (CDR-H1), residues 50-65 (CDR-H2) and residues
95-102 (CDR-H3) according to the Kabat numbering system. However,
according to Chothia (Chothia, C. and Lesk, A. M. J. Mol. Biol.,
196, 901-917 (1987)), the loop equivalent to CDR-H1 extends from
residue 26 to residue 32. Thus unless indicated otherwise `CDR-H1`
as employed herein is intended to refer to residues 26 to 35, as
described by a combination of the Kabat numbering system and
Chothia's topological loop definition.
[0285] The CDRs of the light chain variable domain are located at
residues 24-34 (CDR-L1), residues 50-56 (CDR-L2) and residues 89-97
(CDR-L3) according to the Kabat numbering system.
[0286] Antibodies for use in the present invention may be obtained
using any suitable method known in the art. The toxin A and/or
toxin B polypeptide/protein including fusion proteins, for example
toxin-Fc fusions proteins or cells (recombinantly or naturally)
expressing the polypeptide (such as activated T cells) can be used
to produce antibodies which specifically recognise the target
toxins. The toxin polypeptide may be the full length polypeptide or
a biologically active fragment or derivative thereof.
[0287] Polypeptides may be prepared by processes well known in the
art from genetically engineered host cells comprising expression
systems or they may be recovered from natural biological sources.
In the present application, the term "polypeptides" includes
peptides, polypeptides and proteins. These are used interchangeably
unless otherwise specified. The sequence for TcdA from ribotype 027
is given in SEQ ID NO: 171 (Uniprot accession number C9YJ37) and
the sequence for TcdB from ribotype 027 is given is SEQ ID NO: 172
(Uniprot accession number C9YJ35).
[0288] The antigen polypeptide may in some instances be part of a
larger protein such as a fusion protein for example fused to an
affinity tag.
[0289] Antibodies generated against the antigen polypeptide may be
obtained, where immunisation of an animal is necessary, by
administering the polypeptides to an animal, preferably a non-human
animal, using well-known and routine protocols, see for example
Handbook of Experimental Immunology, D. M. Weir (ed.), Vol 4,
Blackwell Scientific Publishers, Oxford, England, 1986). Many
warm-blooded animals, such as rabbits, mice, rats, sheep, cows,
camels or pigs may be immunized. However, mice, rabbits, pigs and
rats are generally most suitable.
[0290] Monoclonal antibodies may be prepared by any method known in
the art such as the hybridoma technique (Kohler & Milstein,
1975, Nature, 256:495-497), the trioma technique, the human B-cell
hybridoma technique (Kozbor et al., 1983, Immunology Today, 4:72)
and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies
and Cancer Therapy, pp 77-96, Alan R Liss, Inc., 1985).
[0291] Antibodies for use in the invention may also be generated
using single lymphocyte antibody methods by cloning and expressing
immunoglobulin variable region cDNAs generated from single
lymphocytes selected for the production of specific antibodies by,
for example, the methods described by Babcook, J. et al., 1996,
Proc. Natl. Acad. Sci. USA 93(15):7843-78481; WO92/02551;
WO2004/051268 and International Patent Application number
WO2004/106377.
[0292] Humanised antibodies (which include CDR-grafted antibodies)
are antibody molecules having one or more complementarity
determining regions (CDRs) from a non-human species and a framework
region from a human immunoglobulin molecule (see, e.g. U.S. Pat.
No. 5,585,089; WO91/09967). It will be appreciated that it may only
be necessary to transfer the specificity determining residues of
the CDRs rather than the entire CDR (see for example, Kashmiri et
al., 2005, Methods, 36, 25-34). Humanised antibodies may optionally
further comprise one or more framework residues derived from the
non-human species from which the CDRs were derived.
[0293] As used herein, the term `humanised antibody molecule`
refers to an antibody molecule wherein the heavy and/or light chain
contains one or more CDRs (including, if desired, one or more
modified CDRs) from a donor antibody (e.g. a murine monoclonal
antibody) grafted into a heavy and/or light chain variable region
framework of an acceptor antibody (e.g. a human antibody). For a
review, see Vaughan et al, Nature Biotechnology, 16, 535-539, 1998.
In one embodiment rather than the entire CDR being transferred,
only one or more of the specificity determining residues from any
one of the CDRs described herein above are transferred to the human
antibody framework (see for example, Kashmiri et al., 2005,
Methods, 36, 25-34). In one embodiment only the specificity
determining residues from one or more of the CDRs described herein
above are transferred to the human antibody framework. In another
embodiment only the specificity determining residues from each of
the CDRs described herein above are transferred to the human
antibody framework.
[0294] When the CDRs or specificity determining residues are
grafted, any appropriate acceptor variable region framework
sequence may be used having regard to the class/type of the donor
antibody from which the CDRs are derived, including mouse, primate
and human framework regions. Suitably, the humanised antibody
according to the present invention has a variable domain comprising
human acceptor framework regions as well as one or more of the CDRs
provided herein.
[0295] Thus, provided in one embodiment is a humanised antibody
which binds toxin A or toxin B wherein the variable domain
comprises human acceptor framework regions and non-human donor
CDRs.
[0296] Examples of human frameworks which can be used in the
present invention are KOL, NEWM, REI, EU, TUR, TEI, LAY and POM
(Kabat et al., supra). For example, KOL and NEWM can be used for
the heavy chain, REI can be used for the light chain and EU, LAY
and POM can be used for both the heavy chain and the light chain.
Alternatively, human germline sequences may be used; these are
available at: http://vbase.mrc-cpe.cam.ac.uk/
[0297] In a humanised antibody of the present invention, the
acceptor heavy and light chains do not necessarily need to be
derived from the same antibody and may, if desired, comprise
composite chains having framework regions derived from different
chains.
[0298] Also, in a humanised antibody of the present invention, the
framework regions need not have exactly the same sequence as those
of the acceptor antibody. For instance, unusual residues may be
changed to more frequently-occurring residues for that acceptor
chain class or type. Alternatively, selected residues in the
acceptor framework regions may be changed so that they correspond
to the residue found at the same position in the donor antibody
(see Reichmann et al., 1998, Nature, 332, 323-324). Such changes
should be kept to the minimum necessary to recover the affinity of
the donor antibody. A protocol for selecting residues in the
acceptor framework regions which may need to be changed is set
forth in WO 91/09967.
[0299] Generally the antibody sequences disclosed in the present
specification are humanised.
[0300] The invention also provides sequences which are 80%, 90%,
91%, 92%, 93% 94%, 95% 96%, 97%, 98% or 99% similar to a sequence
or antibody disclosed herein.
[0301] "Identity", as used herein, indicates that at any particular
position in the aligned sequences, the amino acid residue is
identical between the sequences. "Similarity", as used herein,
indicates that, at any particular position in the aligned
sequences, the amino acid residue is of a similar type between the
sequences. For example, leucine may be substituted for isoleucine
or valine. Other amino acids which can often be substituted for one
another include but are not limited to: [0302] phenylalanine,
tyrosine and tryptophan (amino acids having aromatic side chains);
[0303] lysine, arginine and histidine (amino acids having basic
side chains); [0304] aspartate and glutamate (amino acids having
acidic side chains); [0305] asparagine and glutamine (amino acids
having amide side chains); and [0306] cysteine and methionine
(amino acids having sulphur-containing side chains).
[0307] Degrees of identity and similarity can be readily calculated
(Computational Molecular Biology, Lesk, A. M., ed., Oxford
University Press, New York, 1988; Biocomputing. Informatics and
Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;
Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and
Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence
Analysis in Molecular Biology, von Heinje, G., Academic Press,
1987, Sequence Analysis Primer, Gribskov, M. and Devereux, J.,
eds., M Stockton Press, New York, 1991, the BLAST.TM. software
available from NCBI (Altschul, S. F. et al., 1990, J. Mol. Biol.
215:403-410; Gish, W. & States, D. J. 1993, Nature Genet.
3:266-272. Madden, T. L. et al., 1996, Meth. Enzymol. 266:131-141;
Altschul, S. F. et al., 1997, Nucleic Acids Res. 25:3389-3402;
Zhang, J. & Madden, T. L. 1997, Genome Res. 7:649-656,).
[0308] The antibody molecules of the present invention include a
complete antibody molecule having full length heavy and light
chains or a fragment thereof and may be, but are not limited to
Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv,
Fab-dsFv, single domain antibodies (e.g. VH or VL or VHH), scFv,
bi, tri or tetra-valent antibodies, Bis-scFv, diabodies,
triabodies, tetrabodies and epitope-binding fragments of any of the
above (see for example Holliger and Hudson, 2005, Nature Biotech.
23(9):1126-1136; Adair and Lawson, 2005, Drug Design
Reviews--Online 2(3), 209-217). The methods for creating and
manufacturing these antibody fragments are well known in the art
(see for example Verma et al., 1998, Journal of Immunological
Methods, 216, 165-181). Other antibody fragments for use in the
present invention include the Fab and Fab' fragments described in
International patent applications WO2005/003169, WO2005/003170 and
WO2005/003171. Multi-valent antibodies may comprise multiple
specificities e.g bispecific or may be monospecific (see for
example WO 92/22853 and WO05/113605). Bispecific and multispecific
antibody variants are especially considered in this example since
the aim is to neutralise two independent target proteins: TcdA and
TcdB. Variable regions from antibodies disclosed herein may be
configured in such a way as to produce a single antibody variant
which is capable of binding to and neutralising TcdA and TcdB.
[0309] In one embodiment the antibody according to the present
disclosure is provided as TcdA or TcdB binding antibody fusion
protein which comprises an immunoglobulin moiety, for example a Fab
or Fab' fragment, and one or two single domain antibodies (dAb)
linked directly or indirectly thereto, for example as described in
WO2009/040562.
[0310] In one embodiment the fusion protein comprises two domain
antibodies, for example as a variable heavy (VH) and variable light
(VL) pairing, optionally linked by a disulphide bond, for example
as described in WO2010/035012.
[0311] In one embodiment the Fab or Fab' element of the fusion
protein has the same or similar specificity to the single domain
antibody or antibodies. In one embodiment the Fab or Fab' has a
different specificity to the single domain antibody or antibodies,
that is to say the fusion protein is multivalent. In one embodiment
a multivalent fusion protein according to the present invention has
an albumin binding site, for example a VH/VL pair therein provides
an albumin binding site.
[0312] In one embodiment the multivalent fusion protein according
to the invention binds TcdA and TcdB.
[0313] In one embodiment the multivalent fusion protein according
to the invention binds TcdB in multiple positions, for example has
distinct binding regions specific for two different epitopes.
[0314] The constant region domains of the antibody molecule of the
present invention, if present, may be selected having regard to the
proposed function of the antibody molecule, and in particular the
effector functions which may be required. For example, the constant
region domains may be human IgA, IgD, IgE, IgG or IgM domains. In
particular, human IgG constant region domains may be used,
especially of the IgG1 and IgG3 isotypes when the antibody molecule
is intended for therapeutic uses and antibody effector functions
are required. Alternatively, IgG2 and IgG4 isotypes may be used
when the antibody molecule is intended for therapeutic purposes and
antibody effector functions are not required, e.g. for simply
neutralising or agonising an antigen. It will be appreciated that
sequence variants of these constant region domains may also be
used. For example IgG4 molecules in which the serine at position
241 has been changed to proline as described in Angal et al.,
Molecular Immunology, 1993, 30 (1), 105-108 may be used. It will
also be understood by one skilled in the art that antibodies may
undergo a variety of posttranslational modifications. The type and
extent of these modifications often depends on the host cell line
used to express the antibody as well as the culture conditions.
Such modifications may include variations in glycosylation,
methionine oxidation, diketopiperazine formation, aspartate
isomerization and asparagine deamidation. A frequent modification
is the loss of a carboxy-terminal basic residue (such as lysine or
arginine) due to the action of carboxypeptidases (as described in
Harris, R J. Journal of Chromatography 705:129-134, 1995).
[0315] In one embodiment the antibody heavy chain comprises a CH1
domain and the antibody light chain comprises a CL domain, either
kappa or lambda.
[0316] Biological molecules, such as antibodies or fragments,
contain acidic and/or basic functional groups, thereby giving the
molecule a net positive or negative charge. The amount of overall
"observed" charge will depend on the absolute amino acid sequence
of the entity, the local environment of the charged groups in the
3D structure and the environmental conditions of the molecule. The
isoelectric point (pI) is the pH at which a particular molecule or
solvent accessible surface thereof carries no net electrical
charge. In one example, the antibody and fragments of the invention
may be engineered to have an appropriate isoelectric point. This
may lead to antibodies and/or fragments with more robust
properties, in particular suitable solubility and/or stability
profiles and/or improved purification characteristics.
[0317] Thus in one aspect the invention provides a humanised
antibody engineered to have an isoelectric point different to that
of the originally identified antibody from which it is derived. The
antibody may, for example be engineered by replacing an amino acid
residue such as replacing an acidic amino acid residue with one or
more basic amino acid residues. Alternatively, basic amino acid
residues may be introduced or acidic amino acid residues can be
removed. Alternatively, if the molecule has an unacceptably high pI
value acidic residues may be introduced to lower the pI, as
required. It is important that when manipulating the pI care must
be taken to retain the desirable activity of the antibody or
fragment. Thus in one embodiment the engineered antibody or
fragment has the same or substantially the same activity as the
"unmodified" antibody or fragment.
[0318] Programs such as ** ExPASY
http://www.expasy.ch/tools/pi_tool.html, and
http://www.iut-arles.up.univ-mrs.fr/w3bb/d_abim/compo-p.html, may
be used to predict the isoelectric point of the antibody or
fragment.
[0319] It will be appreciated that the affinity of antibodies
provided by the present invention may be altered using any suitable
method known in the art. The affinity of the antibodies or variants
thereof may be measured using any suitable method known in the art,
including BIAcore, using an appropriate isolated natural or
recombinant protein or a suitable fusion protein/polypeptide.
[0320] The present invention therefore also relates to variants of
the antibody molecules of the present invention, which have an
improved affinity for TcdA or TcdB, as appropriate. Such variants
can be obtained by a number of affinity maturation protocols
including mutating the CDRs (Yang et al., J. Mol. Biol., 254,
392-403, 1995), chain shuffling (Marks et al., Bio/Technology, 10,
779-783, 1992), use of mutator strains of E. coli (Low et al., J.
Mol. Biol., 250, 359-368, 1996), DNA shuffling (Patten et al.,
Curr. Opin. Biotechnol., 8, 724-733, 1997), phage display (Thompson
et al., J. Mol. Biol., 256, 77-88, 1996) and sexual PCR (Crameri et
al., Nature, 391, 288-291, 1998). Vaughan et al. (supra) discusses
these methods of affinity maturation.
[0321] Improved affinity as employed herein in this context refers
to an improvement refers to an improvement over the starting
molecule.
[0322] If desired an antibody for use in the present invention may
be conjugated to one or more effector molecule(s). It will be
appreciated that the effector molecule may comprise a single
effector molecule or two or more such molecules so linked as to
form a single moiety that can be attached to the antibodies of the
present invention. Where it is desired to obtain an antibody
fragment linked to an effector molecule, this may be prepared by
standard chemical or recombinant DNA procedures in which the
antibody fragment is linked either directly or via a coupling agent
to the effector molecule. Techniques for conjugating such effector
molecules to antibodies are well known in the art (see, Hellstrom
et al., Controlled Drug Delivery, 2nd Ed., Robinson et al., eds.,
1987, pp. 623-53; Thorpe et al., 1982, Immunol. Rev., 62:119-58 and
Dubowchik et al., 1999, Pharmacology and Therapeutics, 83, 67-123).
Particular chemical procedures include, for example, those
described in WO 93/06231, WO 92/22583, WO 89/00195, WO 89/01476 and
WO03031581. Alternatively, where the effector molecule is a protein
or polypeptide the linkage may be achieved using recombinant DNA
procedures, for example as described in WO 86/01533 and
EP0392745.
[0323] The term effector molecule as used herein includes, for
example, biologically active proteins, for example enzymes, other
antibody or antibody fragments, synthetic or naturally occurring
polymers, nucleic acids and fragments thereof e.g. DNA, RNA and
fragments thereof, radionuclides, particularly radioiodide,
radioisotopes, chelated metals, nanoparticles and reporter groups
such as fluorescent compounds or compounds which may be detected by
NMR or ESR spectroscopy.
[0324] Other effector molecules may include chelated radionuclides
such as 111In and 90Y, Lu177, Bismuth213, Californium252,
Iridium192 and Tungsten188/Rhenium188; or drugs such as but not
limited to, alkylphosphocholines, topoisomerase I inhibitors,
taxoids and suramin.
[0325] Other effector molecules include proteins, peptides and
enzymes. Enzymes of interest include, but are not limited to,
proteolytic enzymes, hydrolases, lyases, isomerases, transferases.
Proteins, polypeptides and peptides of interest include, but are
not limited to, immunoglobulins, toxins such as abrin, ricin A,
Pseudomonas exotoxin, or diphtheria toxin, a protein such as
insulin, tumour necrosis factor, .alpha.-interferon,
.beta.-interferon, nerve growth factor, platelet derived growth
factor or tissue plasminogen activator, a thrombotic agent or an
anti-angiogenic agent, e.g. angiostatin or endostatin, or, a
biological response modifier such as a lymphokine, interleukin-1
(IL-1), interleukin-2 (IL-2), granulocyte macrophage colony
stimulating factor (GM-CSF), granulocyte colony stimulating factor
(G-CSF), nerve growth factor (NGF) or other growth factor and
immunoglobulins.
[0326] Other effector molecules may include detectable substances
useful for example in diagnosis. Examples of detectable substances
include various enzymes, prosthetic groups, fluorescent materials,
luminescent materials, bioluminescent materials, radioactive
nuclides, positron emitting metals (for use in positron emission
tomography), and nonradioactive paramagnetic metal ions. See
generally U.S. Pat. No. 4,741,900 for metal ions which can be
conjugated to antibodies for use as diagnostics. Suitable enzymes
include horseradish peroxidase, alkaline phosphatase,
beta-galactosidase, or acetylcholinesterase; suitable prosthetic
groups include streptavidin, avidin and biotin; suitable
fluorescent materials include umbelliferone, fluorescein,
fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine
fluorescein, dansyl chloride and phycoerythrin; suitable
luminescent materials include luminol; suitable bioluminescent
materials include luciferase, luciferin, and aequorin; and suitable
radioactive nuclides include 125I, 131I, 111In and 99Tc.
[0327] In another example the effector molecule may increase the
half-life of the antibody in vivo, and/or reduce immunogenicity of
the antibody and/or enhance the delivery of an antibody across an
epithelial barrier to the immune system. Examples of suitable
effector molecules of this type include polymers, albumin, albumin
binding proteins or albumin binding compounds such as those
described in WO05/117984.
[0328] Where the effector molecule is a polymer it may, in general,
be a synthetic or a naturally occurring polymer, for example an
optionally substituted straight or branched chain polyalkylene,
polyalkenylene or polyoxyalkylene polymer or a branched or
unbranched polysaccharide, e.g. a homo- or
hetero-polysaccharide.
[0329] Specific optional substituents which may be present on the
above-mentioned synthetic polymers include one or more hydroxy,
methyl or methoxy groups.
[0330] Specific examples of synthetic polymers include optionally
substituted straight or branched chain poly(ethyleneglycol),
poly(propyleneglycol) poly(vinylalcohol) or derivatives thereof,
especially optionally substituted poly(ethyleneglycol) such as
methoxypoly(ethyleneglycol) or derivatives thereof.
[0331] Specific naturally occurring polymers include lactose,
amylose, dextran, glycogen or derivatives thereof.
[0332] "Derivatives" as used herein is intended to include reactive
derivatives, for example thiol-selective reactive groups such as
maleimides and the like. The reactive group may be linked directly
or through a linker segment to the polymer. It will be appreciated
that the residue of such a group will in some instances form part
of the product as the linking group between the antibody fragment
and the polymer.
[0333] The size of the polymer may be varied as desired, but will
generally be in an average molecular weight range from 500 Da to
50000 Da, for example from 5000 to 40000 Da such as from 20000 to
40000 Da. The polymer size may in particular be selected on the
basis of the intended use of the product for example ability to
localize to certain tissues such as tumors or extend circulating
half-life (for review see Chapman, 2002, Advanced Drug Delivery
Reviews, 54, 531-545). Thus, for example, where the product is
intended to leave the circulation and penetrate tissue, for example
for use in the treatment of a tumour, it may be advantageous to use
a small molecular weight polymer, for example with a molecular
weight of around 5000 Da. For applications where the product
remains in the circulation, it may be advantageous to use a higher
molecular weight polymer, for example having a molecular weight in
the range from 20000 Da to 40000 Da.
[0334] Suitable polymers include a polyalkylene polymer, such as a
poly(ethyleneglycol) or, especially, a methoxypoly(ethyleneglycol)
or a derivative thereof, and especially with a molecular weight in
the range from about 15000 Da to about 40000 Da.
[0335] In one example antibodies for use in the present invention
are attached to poly(ethyleneglycol) (PEG) moieties. In one
particular example the antibody is an antibody fragment and the PEG
molecules may be attached through any available amino acid
side-chain or terminal amino acid functional group located in the
antibody fragment, for example any free amino, imino, thiol,
hydroxyl or carboxyl group. Such amino acids may occur naturally in
the antibody fragment or may be engineered into the fragment using
recombinant DNA methods (see for example U.S. Pat. Nos. 5,219,996;
5,667,425; WO98/25971, WO2008/038024). In one example the antibody
molecule of the present invention is a modified Fab fragment
wherein the modification is the addition to the C-terminal end of
its heavy chain one or more amino acids to allow the attachment of
an effector molecule. Suitably, the additional amino acids form a
modified hinge region containing one or more cysteine residues to
which the effector molecule may be attached. Multiple sites can be
used to attach two or more PEG molecules.
[0336] Suitably PEG molecules are covalently linked through a thiol
group of at least one cysteine residue located in the antibody
fragment. Each polymer molecule attached to the modified antibody
fragment may be covalently linked to the sulphur atom of a cysteine
residue located in the fragment. The covalent linkage will
generally be a disulphide bond or, in particular, a sulphur-carbon
bond. Where a thiol group is used as the point of attachment
appropriately activated effector molecules, for example thiol
selective derivatives such as maleimides and cysteine derivatives
may be used. An activated polymer may be used as the starting
material in the preparation of polymer-modified antibody fragments
as described above. The activated polymer may be any polymer
containing a thiol reactive group such as an .alpha.-halocarboxylic
acid or ester, e.g. iodoacetamide, an imide, e.g. maleimide, a
vinyl sulphone or a disulphide. Such starting materials may be
obtained commercially (for example from Nektar, formerly Shearwater
Polymers Inc., Huntsville, Ala., USA) or may be prepared from
commercially available starting materials using conventional
chemical procedures. Particular PEG molecules include 20K
methoxy-PEG-amine (obtainable from Nektar, formerly Shearwater;
Rapp Polymere; and SunBio) and M-PEG-SPA (obtainable from Nektar,
formerly Shearwater).
[0337] In one embodiment, the antibody is a modified Fab fragment
or diFab which is PEGylated, i.e. has PEG (poly(ethyleneglycol))
covalently attached thereto, e.g. according to the method disclosed
in EP 0948544 or EP1090037 [see also "Poly(ethyleneglycol)
Chemistry, Biotechnical and Biomedical Applications", 1992, J.
Milton Harris (ed), Plenum Press, New York, "Poly(ethyleneglycol)
Chemistry and Biological Applications", 1997, J. Milton Harris and
S. Zalipsky (eds), American Chemical Society, Washington D.C. and
"Bioconjugation Protein Coupling Techniques for the Biomedical
Sciences", 1998, M. Aslam and A. Dent, Grove Publishers, New York;
Chapman, A. 2002, Advanced Drug Delivery Reviews 2002, 54:531-545].
In one example PEG is attached to a cysteine in the hinge region.
In one example, a PEG modified Fab fragment has a maleimide group
covalently linked to a single thiol group in a modified hinge
region. A lysine residue may be covalently linked to the maleimide
group and to each of the amine groups on the lysine residue may be
attached a methoxypoly(ethyleneglycol) polymer having a molecular
weight of approximately 20,000 Da. The total molecular weight of
the PEG attached to the Fab fragment may therefore be approximately
40,000 Da.
[0338] Particular PEG molecules include
2-[3-(N-maleimido)propionamido]ethyl amide of
N,N'-bis(methoxypoly(ethylene glycol) MW 20,000) modified lysine,
also known as PEG2MAL40K (obtainable from Nektar, formerly
Shearwater).
[0339] Alternative sources of PEG linkers include NOF who supply
GL2-400MA2 (wherein m in the structure below is 5) and GL2-400MA
(where m is 2) and n is approximately 450:
##STR00001##
[0340] That is to say each PEG is about 20,000 Da.
[0341] Further alternative PEG effector molecules of the following
type:
##STR00002##
are available from Dr Reddy, NOF and Jenkem.
[0342] In one embodiment there is provided an antibody which is
PEGylated (for example with a PEG described herein), attached
through a cysteine amino acid residue at or about amino acid 226 in
the chain, for example amino acid 226 of the heavy chain (by
sequential numbering).
[0343] In one embodiment one certain antibodies according to the
present disclosure have the following properties:
TABLE-US-00052 Affinity (pM) Valency of binding Antibody
TcdA.sub.123 TcdA.sub.456 TcdA, est. EC.sub.50 (ng/ml) CA922 4.06
2.59 16 1.21 CA923 64.7 312 12 160.42 CA995 nil 119 1 37.64 CA997
132 66.8 12 6.25 CA1000 73.3 84.1 2 19.73
[0344] The present invention also provides compositions such as a
pharmaceutical composition of antibody or combination of antibodies
defined herein.
[0345] The present invention also provides a composition that
comprises at least two antibodies according to the invention, for
example wherein at least one antibody therein is specific to TcdA
and at least one antibody therein is specific to TcdB or
alternatively at least two antibodies specific to TcdA or at least
two antibodies specific to TcdB.
[0346] In one embodiment there is provided a composition that
comprises multiple antibodies specific to TcdA and optionally one
or more antibodies specific to TcdB.
[0347] In one embodiment there is provided a composition that
comprises multiple antibodies specific to TcdB and optionally one
or more antibodies specific to TcdA.
[0348] Thus in one embodiment there is provided a composition
comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15
antibodies according to the invention i.e. distinct antibodies.
[0349] The invention describes one particular mixture comprising 3
Mabs, one Mab of which is specific for TcdA and two Mabs of which
are specific for TcdB. This mixture demonstrated very high levels
of protection from death and gut inflammation from a lethal
infective oral dose of Clostridium difficile in hamsters.
[0350] In particular there is provided a composition comprising a
combination of one anti-TcdA antibody comprising a heavy variable
region with a sequence as shown in SEQ ID NO:49 and a light
variable region with a sequence shown in SEQ ID NO: 47 and two
anti-TcdB the first with a heavy variable region shown in SEQ ID
NO: 129 and a light variable region shown in SEQ ID NO: 127, and
the second with a heavy variable region shown in SEQ ID NO: 159 and
light variable region shown in SEQ ID NO: 157.
[0351] In one embodiment wherein the composition comprises 3
antibodies, such as one anti-TcdA and two anti-TcdB antibodies the
antibodies are in the ratio of 50%, 25% and 25% respectively of the
total antibody content thereof.
[0352] In one embodiment there is provided a composition comprising
2, 3, 4 or 5 antibodies specific to TcdA and optionally 1, 2, 3, 4
or 5 antibodies specific to TcdB.
[0353] In one embodiment the compositions provided according to the
invention are well defined, for example are mixtures of monoclonal
antibodies rather than simply polyclonal compositions derived from
an immunised or immune competent host.
[0354] In one embodiment the composition of antibodies has an
EC.sub.50 of 200 ng/ml or less, for example 150 ng/ml or less, such
as 100 ng/ml or less, such as 0.1 to 10 ng/ml.
[0355] Advantageously the antibodies described herein have very
high levels of biophysical stability and so are suitable for
inclusion in mixtures of antibodies.
[0356] In one aspect a pharmaceutical formulation or composition
according to the invention further comprises a pharmaceutically
acceptable excipient.
[0357] Pharmaceutically acceptable carriers in therapeutic
compositions may additionally contain liquids such as water,
saline, glycerol and ethanol. Additionally, auxiliary substances,
such as wetting or emulsifying agents or pH buffering substances,
may be present in such compositions. Such carriers enable the
pharmaceutical compositions to be formulated as tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries and suspensions,
for ingestion by the patient.
[0358] Suitable forms for administration include forms suitable for
parenteral administration, e.g. by injection or infusion, for
example by bolus injection or continuous infusion. Where the
product is for injection or infusion, it may take the form of a
suspension, solution or emulsion in an oily or aqueous vehicle and
it may contain formulatory agents, such as suspending,
preservative, stabilising and/or dispersing agents. Alternatively,
the antibody molecule may be in dry form, for reconstitution before
use with an appropriate sterile liquid.
[0359] Once formulated, the compositions of the invention can be
administered directly to the subject. The subjects to be treated
can be animals. However, in one or more embodiments the
compositions are adapted for administration to human subjects.
[0360] Suitably in formulations according to the present
disclosure, the pH of the final formulation is not similar to the
value of the isoelectric point of the antibody or fragment, for
example if the pH of the formulation is 7 then a pI of from 8-9 or
above may be appropriate. Whilst not wishing to be bound by theory
it is thought that this may ultimately provide a final formulation
with improved stability, for example the antibody or fragment
remains in solution.
[0361] In one embodiment the composition or formulation of the
present disclosure comprises 1-200 mg/mL of antibodies, that this
to say the combined antibody content, for example 150 mg/mL or
less, such as 100 mg/mL or less, in particular 90, 80, 70, 60, 50,
40, 30, 20, 10 mg/mL or less.
[0362] In one embodiment a composition or formulation according to
the present disclosure comprises 20 mg/mL of each antibody
therein.
[0363] In one embodiment there is provided a formulation
comprising: [0364] 33 mg/mL or less of one anti-TcdA antibody
comprising a heavy variable region with a sequence as shown in SEQ
ID NO: 49 and a light variable region with a sequence shown in SEQ
ID NO: 47, and [0365] 28 mg/mL or less of a first anti-TcdB with a
heavy variable region shown in SEQ ID NO: 129 and a light variable
region shown in SEQ ID NO: 127, and 25 mg/mL of a second anti-TcdB
with a heavy variable region shown in SEQ ID NO: 159 and light
variable region shown in SEQ ID NO: 157.
[0366] In one embodiment the pharmaceutical formulation at a pH in
the range of 4.0 to 7.0 comprises: 1 to 200 mg/mL of an antibody
according to the present disclosure, 1 to 100 mM of a buffer, 0.001
to 1% of a surfactant,
[0367] a) 10 to 500 mM of a stabiliser,
[0368] b) 5 to 500 mM of a tonicity agent, or
[0369] c) 10 to 500 mM of a stabiliser and 5 to 500 mM of a
tonicity agent.
[0370] In one embodiment the composition or formulation according
to the present disclosure comprises the buffer phosphate buffered
saline.
[0371] For example the formulation at approximately pH6 may
comprise 1 to 50 mg/mL of antibody, 20 mM L-histidine HCl, 240 mM
trehalose and 0.02% polysorbate 20. Alternatively a formulation at
approximately pH 5.5 may comprise 1 to 50 mg/mL of antibody, 20 mM
citrate buffer, 240 mM sucrose, 20 mM arginine, and 0.02%
polysorbate 20.
[0372] The pharmaceutical compositions of this invention may be
administered by any number of routes including, but not limited to,
oral, intravenous, intramuscular, intra-arterial, intramedullary,
intrathecal, intraventricular, transdermal, transcutaneous (for
example, see WO98/20734), subcutaneous, intraperitoneal,
intranasal, enteral, topical, sublingual, intravaginal or rectal
routes. Hyposprays may also be used to administer the
pharmaceutical compositions of the invention. Typically, the
therapeutic compositions may be prepared as injectables, either as
liquid solutions or suspensions. Solid forms suitable for solution
in, or suspension in, liquid vehicles prior to injection may also
be prepared.
[0373] Direct delivery of the compositions will generally be
accomplished by injection, subcutaneously, intraperitoneally,
intravenously or intramuscularly, or delivered to the interstitial
space of a tissue.
[0374] The compositions can also be administered into a lesion or
directly into the gastrointestinal tract by for examples,
encapsulated oral dosage for swallowing, through a nasogastric tube
to the stomach or ileum, through a rectal tube or enema solutions
or by rectal capsule. Dosage treatment may be a single dose
schedule or a multiple dose schedule.
[0375] It will be appreciated that the active ingredient in the
composition will be an antibody molecule. As such, it will be
susceptible to degradation in the gastrointestinal tract. Thus, if
the composition is to be administered by a route using the
gastrointestinal tract, the composition will need to contain agents
which protect the antibody from degradation but which release the
antibody once it has been absorbed from the gastrointestinal
tract.
[0376] A thorough discussion of pharmaceutically acceptable
carriers is available in Remington's Pharmaceutical Sciences (Mack
Publishing Company, N.J. 1991).
[0377] The present invention also provides an antibody or antibody
combination or a composition comprising the same, as described
herein, for treatment, for example for the treatment or prophylaxis
of C. difficile infection or complications associated with the same
such as diarrhoea, colitis in particular pseudomembranous colitis,
bloating, abdominal pain and toxic megacolon.
[0378] Prophylaxis can also be achieved by the administration of
pre-formed complexes of inactivated toxin antigen (toxoid) and
antibody in order to create a vaccine.
[0379] In one embodiment the antibodies, combinations thereof and
compositions comprising the same according to the invention are
suitable for treating infection with so-called super strains of C.
difficile, i.e. hypervirulent strains such as ribotype 027.
[0380] The antibodies and compositions according to the present
invention are suitable for use in the treatment or prophylaxis of
acute and/or chronic effects of the relevant C. difficile toxins
during primary infection.
[0381] The antibodies and compositions according to the present
invention are suitable for use in the treatment or prophylaxis of
effects of the relevant C. difficile toxins during secondary
infection or re-infection. International guidelines enshrine time
intervals after a primary infection which hence defines a secondary
(or recurrent) infection as being distinct from a continuation of
existing symptoms sometimes described as a relapse (29). Research
has shown that secondary infections can be the result of the same
strain or ribotype as the primary infection. In such cases
recurrence rather than relapse relies on agreed temporal
constraints. However, research also clearly shows that secondary
infection can also be the result of infection of a strain or
ribotype distinct from that of the primary infection. In one study,
48% of disease recurrences were the result of a second strain
distinct from that having caused the first infection (30). In
another study, more than 56% of disease recurrences were the result
of a second strain distinct from that having caused the first
infection (31).
[0382] In one embodiment the antibodies, combinations thereof and
compositions comprising the same according to the invention are
suitable for use in the prevention of damage, for example long term
structural damage to the epithelium of the colon.
[0383] In one embodiment the antibodies, combinations and
composition are suitable for preventing C. difficile infection
including recurrence of infection, in particular nosocomial
infection.
[0384] In one embodiment the antibodies, combinations thereof and
compositions comprising the same according to the invention are
suitable for reducing the risk of recurrence of C. difficile
infection.
[0385] Advantageously, the antibodies of the present disclosure can
be administered prophylactically to prevent infection or
re-infection because in the absence of toxin to which the antibody
is specific the antibody is simply to be cleared from the body
without causing adverse interactions with the subjects body
tissues.
[0386] Advantageously the antibodies of the present disclosure seem
to elicit a rapid response after administration, for example within
one or two days of administration rapid clearance of the target
toxin is invoked, this may prevent vital organs such as the lungs,
heart and kidneys being damaged. This is the first time that agents
have been made available with can be employed to prevent damage or
injury to a patient by toxins A and/or B in the acute C. difficile
infection stage.
[0387] Thus in one embodiment the antibodies, combinations thereof
and compositions comprising the same according to the invention are
suitable for preventing damage to vital organs.
[0388] In one embodiment the antibody, combinations or formulations
described herein are suitable for preventing death of an infected
patient, if administered within an appropriate time frame before
irreparable damage has been done by the toxins.
[0389] The antibodies of the present disclosure have fast on-rates,
which facilitates the rapid effect in vivo.
[0390] In one embodiment the patient population is over 60, such as
over 65 years of age.
[0391] In one embodiment the patient population is 5 years old or
less.
[0392] The antibodies according the invention may be employed in
combination with antibiotic treatment for example metronidazole,
vancomycin or fidaxomicin.
[0393] A range of in vitro data exemplify the properties of the
Mabs and Mab mixtures. We show that one mixture of 3 Mabs (50%
molar quantities of anti-TcdA and 50% molar quantities of anti-TcdB
components) was able to protect hamsters from a lethal CDI.
[0394] In one embodiment there is provided a method of treating a
patient in need thereof by administering a therapeutically
effective amount of an antibody as described herein or antibody
combination or a composition comprising the same, for example in
the treatment or prophylaxis of C. difficile infection or
complications associated with the same such as diarrhoea, colitis
in particular pseudomembranous colitis, bloating, abdominal pain
and toxic megacolon.
[0395] In one embodiment the antibody, combination or formulation
is administered by a parenteral route, for example subcutaneously,
intraperitoneally, intravenously or intramuscularly. The data in
the Examples generated in hamsters indicates that the doses
administered by this route reach the gut and thus are able to
generate a therapeutic effect.
[0396] In one embodiment the antibody, combination or formulation
is administered orally, for example an enterically coated
formulation.
[0397] In one embodiment there is provided use of an antibody,
combination or formulation as described herein for the manufacture
of a medicament for the treatment or prophylaxis of C. difficile
infection.
[0398] In one embodiment the dose administered is in the range 1 to
1000 mg/Kg, for example 10 to 75 mg/Kg, such 20 to 50 mg/Kg.
[0399] In one embodiment the half-life of the antibody or
antibodies in mice and hamsters in vivo is in the range 6 to 8 days
in healthy (uninfected) animals and hence are expected to have
half-lives in humans in the range of 14-28 days.
[0400] In one embodiment the antibody or antibodies are given as
one dose only.
[0401] In one embodiment the antibody or antibodies are given as a
weekly or biweekly dose.
[0402] In one embodiment the antibody or antibodies are given as
once daily doses.
[0403] In one embodiment there is provided complex comprising TcdA
or an immunogenic fragment thereof, complexed with one or more
anti-TcdA antibodies defined herein. The complex may be employed as
the antigen in a vaccine formulation, for example suitable for
generating protective antibodies to toxin A in vivo after
administration to a human.
[0404] In one embodiment there is provided complex comprising TcdB
or an immunogenic fragment thereof, complexed with one or more
anti-TcdB antibodies defined herein. The complex may be employed as
the antigen in a vaccine formulation, for example suitable for
generating protective antibodies to toxin B in vivo after
administration to a human.
[0405] Th1-type immunostimulants which may be formulated to produce
adjuvants suitable for use in the present invention include and are
not restricted to the following.
[0406] In one embodiment there is provided a complex comprising
TcdA or an immunogenic fragment thereof and TcdB or an immunogenic
fragment thereof, wherein each toxin or fragment is complexed with
one or more antibodies specific thereto, wherein the complex is
suitable for administration as a vaccine formulation.
[0407] Antibody:antigen complexes are known to be taken up by the
immune system in an Fc receptor mediated process (27, 28) and
pre-formed complexes of antibody:antigen complexes have been
successfully use as vaccines in human clinical trials (22).
[0408] In one or more embodiments the vaccine formulation further
comprises an adjuvant as an immunostimulant.
[0409] Monophosphoryl lipid A, in particular 3-de-O-acylated
monophosphoryl lipid A (3D-MPL), is a preferred Th1-type
immunostimulant for use in the invention. 3D-MPL is a well known
adjuvant manufactured by Ribi Immunochem, Montana. Chemically it is
often supplied as a mixture of 3-de-O-acylated monophosphoryl lipid
A with either 4, 5, or 6 acylated chains. It can be purified and
prepared by the methods taught in GB 2122204B, which reference also
discloses the preparation of diphosphoryl lipid A, and
3-O-deacylated variants thereof. Other purified and synthetic
lipopolysaccharides have been described (U.S. Pat. No. 6,005,099
and EP 0 729 473 B1; Hilgers et al., 1986, Int. Arch. Allergy.
Immunol., 79(4):392-6; Hilgers et al., 1987, Immunology,
60(1):141-6; and EP 0 549 074 B1). A preferred form of 3D-MPL is in
the form of a particulate formulation having a small particle size
less than 0.2 mm in diameter, and its method of manufacture is
disclosed in EP 0 689 454.
[0410] Saponins are also preferred Th1 immunostimulants in
accordance with the invention. Saponins are well known adjuvants
and are taught in: Lacaille-Dubois, M and Wagner H. (1996. A review
of the biological and pharmacological activities of saponins.
Phytomedicine vol 2 pp 363-386). For example, Quil A (derived from
the bark of the South American tree Quillaja Saponaria Molina), and
fractions thereof, are described in U.S. Pat. No. 5,057,540 and
"Saponins as vaccine adjuvants", Kensil, C. R., Crit Rev Ther Drug
Carrier Syst, 1996, 12 (1-2):1-55; and EP 0 362 279 B1. The
haemolytic saponins QS21 and QS17 (HPLC purified fractions of Quil
A) have been described as potent systemic adjuvants, and the method
of their production is disclosed in U.S. Pat. No. 5,057,540 and EP
0 362 279 B1. Also described in these references is the use of QS7
(a non-haemolytic fraction of Quil-A) which acts as a potent
adjuvant for systemic vaccines. Use of QS21 is further described in
Kensil et al. (1991. J. Immunology vol 146, 431-437). Combinations
of QS21 and polysorbate or cyclodextrin are also known (WO
99/10008). Particulate adjuvant systems comprising fractions of
QuilA, such as QS21 and QS7 are described in WO 96/33739 and WO
96/11711. One such system is known as an Iscorn and may contain one
or more saponins.
[0411] Another preferred immunostimulant is an immunostimulatory
oligonucleotide containing unmethylated CpG dinucleotides ("CpG").
CpG is an abbreviation for cytosine-guanosine dinucleotide motifs
present in DNA. CpG is known in the art as being an adjuvant when
administered by both systemic and mucosal routes (WO 96/02555, EP
468520, Davis et al., J. Immunol, 1998, 160(2):870-876; McCluskie
and Davis, J. Immunol., 1998, 161(9):4463-6). Historically, it was
observed that the DNA fraction of BCG could exert an anti-tumour
effect. In further studies, synthetic oligonucleotides derived from
BCG gene sequences were shown to be capable of inducing
immunostimulatory effects (both in vitro and in vivo). The authors
of these studies concluded that certain palindromic sequences,
including a central CG motif, carried this activity. The central
role of the CG motif in immunostimulation was later elucidated in a
publication by Krieg, Nature 374, p 546 1995. Detailed analysis has
shown that the CG motif has to be in a certain sequence context,
and that such sequences are common in bacterial DNA but are rare in
vertebrate DNA. The immunostimulatory sequence is often: Purine,
Purine, C, G, pyrimidine, pyrimidine; wherein the CG motif is not
methylated, but other unmethylated CpG sequences are known to be
immunostimulatory and may be used in the present invention.
[0412] In certain combinations of the six nucleotides a palindromic
sequence is present. Several of these motifs, either as repeats of
one motif or a combination of different motifs, can be present in
the same oligonucleotide. The presence of one or more of these
immunostimulatory sequences containing oligonucleotides can
activate various immune subsets, including natural killer cells
(which produce interferon g and have cytolytic activity) and
macrophages (Wooldrige et al Vol 89 (no. 8), 1977). Other
unmethylated CpG containing sequences not having this consensus
sequence have also now been shown to be immunomodulatory.
[0413] CpG when formulated into vaccines, is generally administered
in free solution together with free antigen (WO 96/02555; McCluskie
and Davis, supra) or covalently conjugated to an antigen (WO
98/16247), or formulated with a carrier such as aluminium hydroxide
((Hepatitis surface antigen) Davis et al. supra; Brazolot-Millan et
al., Proc. Natl. Acad. Sci., USA, 1998, 95(26), 15553-8).
[0414] Such immunostimulants as described above may be formulated
together with carriers, such as for example liposomes, oil in water
emulsions, and or metallic salts, including aluminium salts (such
as aluminium hydroxide). For example, 3D-MPL may be formulated with
aluminium hydroxide (EP 0 689 454) or oil in water emulsions (WO
95/17210); QS21 may be advantageously formulated with cholesterol
containing liposomes (WO 96/33739), oil in water emulsions (WO
95/17210) or alum (WO 98/15287); CpG may be formulated with alum
(Davis et al. supra; Brazolot-Millan supra) or with other cationic
carriers.
[0415] Combinations of immunostimulants are also preferred, in
particular a combination of a monophosphoryl lipid A and a saponin
derivative (WO 94/00153; WO 95/17210; WO 96/33739; WO 98/56414; WO
99/12565; WO 99/11241), more particularly the combination of QS21
and 3D-MPL as disclosed in WO 94/00153. Alternatively, a
combination of CpG plus a saponin such as QS21 also forms a potent
adjuvant for use in the present invention.
[0416] Alternatively the saponin may be formulated in a liposome or
in an Iscorn and combined with an immunostimulatory
oligonucleotide.
[0417] Thus, suitable adjuvant systems include, for example, a
combination of monophosphoryl lipid A, preferably 3D-MPL, together
with an aluminium salt.
[0418] Thus is one embodiment the adjuvant is a combination of QS21
and 3D-MPL in an oil in water or liposomal formulation.
[0419] An enhanced system involves the combination of a
monophosphoryl lipid A and a saponin derivative particularly the
combination of QS21 and 3D-MPL as disclosed in WO 94/00153, or a
less reactogenic composition where the QS21 is quenched in
cholesterol containing liposomes (DQ) as disclosed in WO 96/33739.
This combination may additionally comprise an immunostimulatory
oligonucleotide.
[0420] A particularly potent adjuvant formulation involving QS21,
3D-MPL & tocopherol in an oil in water emulsion is described in
WO 95/17210 and is another preferred formulation for use in the
invention.
[0421] Another preferred formulation comprises a CpG
oligonucleotide alone or together with an aluminium salt.
[0422] In a further aspect of the present invention there is
provided a method of manufacture of a vaccine formulation as herein
described, wherein the method comprises admixing a polypeptide
according to the invention with a suitable adjuvant.
[0423] Particularly suitable adjuvant combinations for use in the
formulations according to the invention are as follows:
i) 3D-MPL+QS21 in a liposome
ii) Alum+3D-MPL
[0424] iii) Alum+QS21 in a liposome+3D-MPL
iv) Alum+CpG
[0425] v) 3D-MPL+QS21+oil in water emulsion
vi) CpG
[0426] As used herein, the term "comprising" in context of the
present specification should be interpreted as "including".
[0427] Embodiments and preferences may be combined as technically
appropriate.
[0428] The disclosure herein describes embodiments comprising
certain integers. The disclosure also extends to the same
embodiments consisting or consisting essentially of said
integers.
FIGURES
[0429] FIG. 1-10 shows various antibody and fragment sequences
[0430] FIG. 11 shows sera titres for TcdA and TcdB
[0431] FIG. 12 shows anti TcdA (Ribotype 003) in-vitro
neutralization data for single Mabs
[0432] FIG. 13 shows anti TcdA (Ribotype 003) in-vitro
neutralization data for single and paired Mabs
[0433] FIG. 14-15 shows anti TcdA (Ribotype 003) in-vitro
neutralization data for paired Mabs
[0434] FIG. 16-18 shows anti TcdA (Ribotype 003) in-vitro
neutralization data for three Mab mixtures
[0435] FIG. 19-20 shows anti TcdA (Ribotype 003) in-vitro
neutralization data for four and five Mab mixtures
[0436] FIG. 21-22 shows anti TcdA (Ribotype 003) in-vitro
neutralization data for single and paired Mabs at different TcdA
concentrations
[0437] FIG. 23-24 shows anti TcdA (Ribotype 003) in-vitro
neutralization data for single and to five Mab mixtures at
different TcdA concentrations
[0438] FIG. 25-26 shows anti TcdB (Ribotype 003) in-vitro
neutralization data for single Mabs
[0439] FIG. 27-30 shows anti TcdB (Ribotype 003) in-vitro
neutralization data for paired Mabs
[0440] FIG. 31-33 shows anti TcdB (Ribotype 003) in-vitro
neutralization data for three Mab mixtures
[0441] FIG. 34-40 shows anti TcdB (Ribotype 003) in-vitro
neutralization data for two Mab mixtures at different toxin
concentrations
[0442] FIG. 41-45 shows anti TcdB (Ribotype 003) in-vitro
neutralization data for two Mab mixtures at different relative Mab
ratios and different toxin concentrations
[0443] FIG. 46-59 shows TcdB neutralisation data for single
antibodies and pairs of antibodies
[0444] FIG. 60 shows the amino acid sequence for TcdA
[0445] FIG. 61 shows the amino acid sequence for TcdB
[0446] FIG. 62 shows TEER assay data for TcdA in a histogram
format
[0447] FIG. 62A shows TEER assay data for TcdA in line graph
format
[0448] FIG. 63 shows a meier-kaplan curve for the combination of
antibodies 997, 1125 and 1151, high concentration is 50 mg/Kg and
low concentration is 5 mg/Kg 50 mg/kg' dose gave 100% protection to
day 11, .about.82% protection to day 28. 5 mg/kg' dose resulted in
non-durable and incomplete protection.
[0449] FIG. 64 shows bodyweight changes for vancomycin and vehicle
treated hamsters
[0450] FIG. 65 shows the bodyweight for low dose antibodies 5 mg/Kg
and high dose antibodies 50 mg/Kg
[0451] FIG. 66 shows photographs of a colon where the animal
received treatment with antibodies according to the present
disclosure vs a control
[0452] FIG. 67-68 show effects of vortexing on antibody
stability
[0453] FIG. 69 shows a comparison of aggregation stability for
various antibodies
[0454] FIG. 70-73 show neutralisation of TcdA for various
ribotypes
EXAMPLES
Antibody Generation
[0455] A range of different immunogens and screening reagents were
either purchased or produced by conventional E. coli expression
techniques in order to provide a diverse and broad immune response
and to facilitate identification and characterisation of monoclonal
antibodies (listed in Table 1). In cases where recombinant proteins
or peptides were generated, sequences were based on ribotype 027.
The sequence for TcdA from ribotype 027 is given in SEQ ID NO: 171
(Uniprot accession number C9YJ37) and the sequence for TcdB from
ribotype 027 is given is SEQ ID NO: 172 (Uniprot accession number
C9YJ35).
[0456] Sprague Dawley rats and half-lop rabbits were immunised with
either synthetic peptides mapping to regions common to both TcdA
and TdcdB full-length toxin, formaldehyde-inactivated toxoid A,
binding domain fragments of Toxin A (CROPs1,2,3 or CROPs4,5,6) or
binding domain fragment of Toxin B (CROPs1,2,3,4), or in some
cases, a combination of the above. Following 2 to 6 immunisations,
animals were sacrificed and PBMC, spleen and bone marrow harvested.
Sera were monitored for binding to Toxin A domains, toxin B
domains, toxin or toxoid by ELISA. Sera titres of 2 such
immunisations are shown in FIG. 11. UCB SLAM was used as a means to
generate monoclonal antibodies. B cells were cultured directly from
immunised animals (Zubler et al., 1985). This step enabled sampling
of a large percentage of the B cell repertoire. By incorporating
the selected lymphocyte antibody method (SLAM) (Babcook et al.,
1996) it was possible to deconvolute positive culture wells and
identify antigen-specific antibody-secreting cells. Here we used a
modified version of SLAM (UCB SLAM (Tickle et al. 2009)) that
utilises a fluorescence-based method to identify antigen-specific B
cells from culture wells. B cell cultures were set up and
supernatants were first screened for their ability to bind a
relevant purified toxin domain (binding, translocation or
catalytic) in a bead-based assay using an Applied Biosystem 8200
cellular detection system. This was a homogeneous assay using B
cell culture supernatant containing IgG, biotinylated toxin domains
coated onto streptavidin beads and a goat anti-rat/rabbit Fc-Cy5
conjugate. Cell cultures positive for binding to TcdA or TcdB
components from this assay were selected for use in cell-based
functional assays to identify neutralisers of toxin-induced
cytotoxicity. Approximately 12,000 toxin-specific positives were
identified in the primary binding screen from a total of
40.times.50-plate experiments. This equated to the screening of
approximately 0.5 billion B cells. Heavy and light variable region
gene pairs were isolated from single cells harvested by
micromanipulation from approximately 100 toxin-neutralising wells
following reverse transcription (RT)-PCR. These V-region genes were
then cloned as mouse IgG1/kappa full-length antibodies for rat
variable regions and rabbit IgG/kappa full-length antibodies for
rabbit variable regions. Antibodies were re-expressed in a HEK-293
transient expression system. These recombinant antibodies were then
retested for their ability to neutralise toxin in cell based
assays. Recombinant antibodies were also screened by BIAcore to
determine affinity for a given toxin domain and to also determine
the specificity and approximate the number of binding events of
antibody to toxin. Based on in vitro activity in cell based assays
and affinity measurements, lead candidates were selected for
humanisation. Unless otherwise stated, all the data herein was
generated using the humanised antibodies.
[0457] A panel of recombinant, E. coli-produced toxin fragments
(TcdA), C. difficile-derived toxin or toxoid (A) and synthetic
peptides (B) were generated or purchased from commercial
sources.
TABLE-US-00053 TABLE 1 Toxin A (TcdA) sequence related reagents for
screening and immunizations. Fragment Residue number Source TcdA
catalytic M1-E659 UCB E. coli expression TcdA translocation
K577-D1350 UCB E. coli expression TcdA CROPS.sub.123 (TcdA123)
S1827-D2249 UCB E. coli expression TcdA CROPS.sub.456 (TcdA456)
G2205-R2608 UCB E. coli expression TcdA CROP.sub.1 S1827-N1978 UCB
E. coli expression TcdA CROP.sub.2 G1966-N2133 UCB E. coli
expression TcdA CROP.sub.3 G2100-D2249 UCB E. coli expression TcdA
CROP.sub.4 G2213-N2381 UCB E. coli expression TcdA CROP.sub.5
G2328-N2494 UCB E. coli expression TcdA CROP.sub.6 G2462-N2609 UCB
E. coli expression TcdA CROP.sub.7 R2554-D2701 UCB E. coli
expression TcdB catalytic M1-A593 UCB E. coli expression TcdB
translocation R576-D1349 UCB E. coli expression TcdB binding
(TcdB1234) S1833-E2366 UCB E. coli expression TcdB CROP.sub.1
S1833-S1981 UCB E. coli expression TcdB CROP.sub.2 G1968-D2113 UCB
E. coli expression TcdB CROP.sub.3 G2100-E2247 UCB E. coli
expression TcdB CROP.sub.4 G2234-E2366 UCB E. coli expression Toxin
A Full length purchased Toxin B Full length purchased Toxoid A Full
length purchased
TABLE-US-00054 TABLE 2 Toxin B (TcdB) sequence related reagents for
screening and immunizations. Toxin Domain Amino acid Sequence
Catalytic SPVEKNLHFVWIGGEVSD SEQ ID NO: 173 Catalytic NLAAASDIVRL
SEQ ID NO: 174 Catalytic CGGVYLDVDMLPGIH SEQ ID NO: 175 Catalytic
CGGVYLDVDMLPGIHSDLFK SEQ ID NO: 176 Catalytic CWEMIKLEAIMKYK SEQ ID
NO: 177 Catalytic CTNLVIEQVKNR SEQ ID NO: 178 Catalytic
PEARSTISLSGP SEQ ID NO: 179 Catalytic CSNLIVKQIENR SEQ ID NO: 180
Catalytic TEQEINSLWSFDQA SEQ ID NO: 181 Catalytic
TEQEINSLWSFDPEARSTISLSGPC SEQ ID NO: 182 Translocation
NVEETYPGKLLLC SEQ ID NO: 183 Translocation Acetyl-CANQYEVRINSEGR
SEQ ID NO: 184 Translocation VNTLNAAFFIQSLIC SEQ ID NO: 185
Translocation YAQLFSTGLNTIC SEQ ID NO: 186 Translocation
CAGISAGIPSLVNNEL SEQ ID NO: 187 Translocation DDLVISEIDFNNNSIC SEQ
ID NO: 188 Translocation MEGGSGHTVT SEQ ID NO: 189 Translocation
AVNDTINVLPTITEGIPIVSTILDGINLGAAIK EL SEQ ID NO: 190 Binding
CGFEYFAPANTDANNIEGQA SEQ ID NO: 191 Binding CGYKYFAPANTVNDNIYGQA
SEQ ID NO: 192 Binding CKYYFNTNTAEA SEQ ID NO: 193 Binding
CKYYFDEDTAEA SEQ ID NO: 194
Expression and Purification of C. difficile Anti-Toxin Mabs
[0458] Separate light chain and heavy chain mammalian expression
plasmids were combined in equimolar ratios and used to transfect
HEK-293 or CHO-S cells. For small scale expression studies
lipofectamine and HEK-293 cells were used whereas for production of
larger batches of IgG electroporation into CHO-S was preferred.
[0459] Culture supernatants were loaded onto a Mab Select SuRe
column (in PBS pH 7.4). Antibody was eluted with 100% 0.1M Sodium
Citrate pH 3.4 buffer. Samples were neutralized to pH7.4 with
Tris.Cl pH8.0. Aggregate was removed by Superdex 200 Gel Filtration
column in PBS pH 7.4.
TABLE-US-00055 TABLE 3 Cell Volume of Expression Amount Antibody
type SN (L) type purified (mg) CA164_00997.g1_P3 CHO 10 Transient
755.93 CA164_00922.g1_P3 CHO 0.5 Transient 129.36 CA164_01125.g2_P3
CHO 10 Transient 498.96 CA164_01151.g4_P3 CHO 5 Transient
262.43
Example 1 In-Vitro Neutralization of TcdA Activity by Purified
Mabs
[0460] All neutralisation screening assays were run in 96 well
polystyrene plates. The assay uses CACO-2 cells grown, and screened
in MEM+20% FCS, 2 mM Q, and NEAA. Any antibody combinations are at
equal molar ratios unless stated otherwise. Day 1: Cells were
plated @ 3000 per well in 50 .mu.l media, and incubated for 24 hrs;
Day 2: Purified samples of humanised Mab were added to 96 well
round bottomed polypropylene sterile plates; Spike PP plates with
toxin A at a concentration sufficient to generated the appropriate
lethal dose i.e. LD.sub.80 or above and incubate for 1 hr, at
37.degree. C.; Add 50 .mu.l of this mixture to cell plates and
incubate for 96 hrs; Day 5: Add Methylene blue (0.5% Methylene Blue
50% ethanol); Incubate for 1 hr at room temperature; Lyse the cells
with 1% N-Lauryl Sarcosine, and Read on the BIOTEK Synergy2 plate
reader at 405 nm. The results are shown in FIGS. 12 to 24.
EC.sub.50 and % maximum neutralization of TcdA activity shown
confirm that the selected antibodies have very high potencies as
single agents. Combinations of 2 to 5 of these did not improve upon
the best EC.sub.50 or % maximum neutralization. Lack of any synergy
when combining Mabs CA922, 923, 995, 997 and 1000 is an important
observation and may be due to the fact the each antibody alone has
exceptionally high levels of affinity and potency. Supporting data
in Example 5 also show that some of the Mabs (e.g. CA997) are
capable of binding to TcdA subdomains many times. Hence it seems
probable that these 5 Mabs represent that the maximum affinity,
potency and valency that is achievable when targeting the
C-terminal cell binding domain of TcdA. The antibodies were also
effective at neutralising very high toxin concentrations ranging
from LD80 to greater than LD.sub.95 (LD.sub.max) but some modest
increases in EC.sub.50 (i.e. decreases in potency) were observed
with very high levels of [TcdA]. These data are also surprising
since others have shown substantial reductions in potency when
testing elevated TcdA concentrations (20).
[0461] The high potency and affinity of the Mabs described here,
e.g. for CA997; is not due solely to their high valency of binding.
Others (20 and WO06/071877) describe anti-TcdA Mabs capable of
binding up to 14 times. These Mabs only had affinities in the range
0.3 to 100 nM and showed incomplete protection against TcdA
mediated cell killing, alone (26-63% protection) or in pairs
(31-73% protection). Hence it has been demon-strated that high
valency of binding to TcdA does not necessarily invoke either high
affinity of binding to or neutralisation of TcdA. Neither the
affinities nor valency of binding to TcdA were described for Mab
CDA-1 (18 and U.S. Pat. No. 7,625,559). Thus Mabs described herein
to have surprising affinity, potency and valency.
TABLE-US-00056 TABLE 4 Anti TcdA 1, 2 & 3 Mab combinations at a
single TcdA conc. (LD.sub.80) Final (highest) EC.sub.50 Antibody
Mab conc.ng/ml (ng/ml) 922 500 1.21 923 500 160.42 995 500 37.64
997 500 6.25 1000 500 19.73 922 + 923 500 3.58 922 + 925 500 3.326
922 + 997 500 2.88 922 + 1000 500 2.64 923 + 995 500 60.23 923 +
997 500 7.54 923 + 1000 500 9.24 995 + 997 500 7.29 995 + 1000 500
19.63 997 + 1000 500 4.46 922 + 923 + 995 500 4.72 922 + 923 + 997
500 3.23 922 + 923 + 1000 500 3.21 922 + 995 + 997 500 2.22 922 +
995 + 1000 500 2.85 922 + 997 + 1000 500 2.22 923 + 995 + 997 500
5.04 923 + 995 + 1000 500 9.49 995 + 997 + 1000 500 5.84 922 + 923
+ 995 + 997 500 2.75 922 + 923 + 995 + 1000 500 3.75 922 + 995 +
997 + 1000 500 3.46 923 + 995 + 997 + 1000 500 4.81 922 + 923 + 997
+ 1000 500 3.06 922 + 923 + 995 + 997 + 1000 500 4.72
TABLE-US-00057 TABLE 5 Anti TcdA single, paired, and triplet Mab
combinations at various TcdA concentrations, where TcdA is at its
LD.sub.80, LD.sub.90, LD.sub.95 and LD.sub.max. Final Mab EC.sub.50
Toxin TcdA Sample conc.ng/ml (ng/ml) @ 3000 pg/ml 922 500 4.89
(LD.sub.MAX) 997 500 10.99 1000 500 50.17 922 + 997 500 7.18 922 +
1000 500 6.99 997 + 1000 500 9.437 922 + 997 + 1000 500 10.80 922 +
997 + 1000 + 995 500 15.03 922 + 997 + 1000 + 995 + 923 500 7.16 @
1000 pg/ml 922 500 1.24 (LD.sub.95) 997 500 3.42 1000 500 9.60 922
+ 997 500 1.85 922 + 1000 500 2.51 997 + 1000 500 3.61 922 + 997 +
1000 500 2.40 922 + 997 + 1000 + 995 500 2.74 922 + 997 + 1000 +
995 + 923 500 2.38 @ 700 pg/ml 922 500 0.84 (LD.sub.90) 997 500
2.40 1000 500 6.23 922 + 997 500 1.19 922 + 1000 500 1.33 997 +
1000 500 2.68 922 + 997 + 1000 500 1.84 922 + 997 + 1000 + 995 500
2.17 922 + 997 + 1000 + 995 + 923 500 2.06 @ 350 pg/ml 922 500 0.39
(LD.sub.80) 997 500 1.18 1000 500 2.76 922 + 997 500 0.67 922 +
1000 500 0.85 997 + 1000 500 2.06 922 + 997 + 1000 500 0.83 922 +
997 + 1000 + 995 500 0.97 922 + 997 + 1000 + 995 + 923 500 0.98
Example 2 Anti TcdB In-Vitro Neutralization by Purified Mab
Assay Methods Description:
[0462] All neutralisation screening assays were run in 96 well
polystyrene plates.
[0463] The assay uses CACO-2 cells grown, and screened in MEM+20%
FCS, 2 mM Q, and NEAA. Unless stated all Ab combinations are in
equal ratios. [0464] Day 1: Cells are plated @ 3000 per well in 50
.mu.l media, and incubated for 24 hrs [0465] Day 2: Purified
samples of humanised Mab were added to 96 well round bottomed
polypropylene sterile plates [0466] Spike PP plates with toxin B
lot #031 and incubate for 1 hr, at 37.degree. C. [0467] Add 50
.mu.l of this mixture to cell plates [0468] Incubate for 96 hrs
[0469] Day 5: Add Methylene blue (0.5% Methylene Blue 50% ethanol)
[0470] Incubate for 1 hr at room temperature [0471] Lyse the cells
with 1% N-Lauryl Sarcosine [0472] Read on the BIOTEK Synergy2 plate
reader at 405 nm
[0473] The data in FIGS. 25 to 33 show that single Mabs alone were
relatively ineffective at neutralizing TcdB, both in terms of %
maximum neutralization and activity (EC.sub.50). However, when the
antibodies were combined in two's and three's considerable
improvements in both % maximum neutralization and activity
(EC.sub.50) were observed. 1125 and 1151 were selected as a best
pairing, although other good pairings were observed: 1125+1153,
1125+1134.
[0474] The most effective pairs of Mabs were selected empirically
and were found retrospectively to make unexpected and surprising
combinations when regarding the individual potencies of each Mab.
For example, in Table 6 only CA927 had a TcdB neutralisation
potential which could result in a defined EC.sub.50 whilst the TcdB
neutralisation potential of both CA1125 and CA1151 were
insufficient under these assay conditions to result in a defined
EC.sub.50. However, CA927 was not found to be the most effective
Mab to use within a combination. The best CA927 containing
combination had an EC.sub.50 of 13.5 ng/ml whereas other two Mab
combinations had EC.sub.50's as low as 2.59 and 4.71 ng/ml. In
another example, in Table 8 CA1099 had the lowest TcdB
neutralisation EC.sub.50 under the assay conditions used. However,
CA1099 was not found to be the most effective Mab to use within a
combination. The best CA1099 containing combination had an
EC.sub.50 of Eng/ml whereas other two Mab combinations had
EC.sub.50's as low as 2 and 1 ng/ml. We might speculate that the
most effective pairings of Mabs are defined by their cooperative
binding modalities especially as defined by having non-overlapping
epitopes.
TABLE-US-00058 TABLE 6 Anti-TcdB Mab combinations and relative Mab
ratios at constant toxin concentration. Final Mab Sample conc.ng/ml
EC.sub.50(ng/ml) 1125.g2 1000 >1000 1134.g5 1000 >1000 927.g2
1000 12.89 1153.g8 1000 >1000 1102.g4 1000 >1000 927 + 1099
1000 >1000 927 + 1102 1000 >1000 927 + 1114 1000 >111.111
927 + 1125 1000 13.55 927 + 1134 1000 51.58 1099 + 1114 1000
>1000 1102 + 1114 1000 >333.333 1102 + 1125 1000 15.51 1114 +
1134 1000 19.70 1114 + 1151 1000 25.69 1114 + 1153 1000 27.48 1125
+ 1134 1000 2.59 1125 + 1151 1000 4.71 1125 + 1153 1000 21.23 1125
+ 1134 + 1114 1000 3.77 1125 + 1134 + 927 1000 2.63 1125 + 1151 +
1114 1000 4.90 1125 + 1151 + 927 1000 5.69 1125.g2 + 1134.g5 +
927.g2 1000 5.83 1125.g2 + 1134.g5 + 1153.g8 1000 9.89 1125.g2 +
1134.g5 + 1102.g4 1000 2.72
Example 3 Neutralisation of TcdB by Combinations of Purified
Mab
[0475] All neutralisation screening assays were run in 96 well
polystyrene plates.
[0476] The assay uses CACO-2 cells grown, and screened in MEM+20%
FCS, 2 mM Q, and NEAA. [0477] Day 1: Cells are plated @ 3000 per
well in 50 .mu.l media, and incubated for 24 hrs [0478] Day 2:
Purified samples of humanised Mab were added to 96 well round
bottomed polypropylene sterile plates [0479] Spike PP plates with
toxin B (VPI 10463) and incubate for 1 hr, at 37.degree. C. [0480]
Add 50 .mu.l of this mixture to cell plates [0481] Incubate for 72
hrs [0482] Day 5: Add Methylene blue (0.5% Methylene Blue 50% ETOH)
[0483] Incubate for 1 hr at room temperature [0484] Lyse the cells
with 1% N-Lauryl Sarcosine [0485] Read on the BIOTEK Synergy2 plate
reader at 405 nm
[0486] The results are shown in FIGS. 34 to 45.
[0487] These data show that the best pair of Mabs for neutralizing
TcdB at a range of toxin concentrations was CA1125 and CA1151.
Moreover, the 1125+1151 combination was largely unaffected by
changes in the relative molar ratios which is in contrast to
1125+1153.
TABLE-US-00059 TABLE 7 Anti-TcdB Mab combinations and relative Mab
ratios at 3 different toxin cones. EC50 values (ng/ml) Antibody
combination TcdB LD60 TcdB LD77 TcdB LD85 1125.g2 + 927.g2 (50:50)
2.8 6 11.3 1125.g2 + 1102.g4 (50:50) 4 13 44 1125.g2 + 1114.g8
(50:50) 3.5 7.1 25.4 1125.g2 + 1134.g5 (50:50) 0.48 1.4 4 1125.g2 +
1151.g4 (50:50) 0.85 0.85 1.5 1125.g2 + 1153.g8 (50:50) 2.7 5.2
25.2 1125.g2 + 1134.g5 (25:75) <0.15 0.84 7.2 1125.g2 + 1151.g4
(25:75) 0.73 1 2.1 1125.g2 + 1153.g8 (25:75) 7 10 27 1125.g2 +
1134.g5 (75:25) 0.66 1.2 2.5 1125.g2 + 1151.g4 (75:25) 1.4 1.2 8.3
1125.g2 + 1153.g8 (75:25) 2.9 7.5 30
[0488] The data show that even the most active specific paired
combinations have surprisingly and non-predictably different
properties relative to each other. The EC.sub.50 of the preferred
combination of CA1125 and CA1151 in equimolar ratios is largely
unaffected by an increasing [TcdB]. The three relative molar ratios
of Mabs tested (i.e. 25:75 vs 50:50 vs 75:25) have very similar
EC.sub.50's to each other, suggesting that CA1125 and CA1151 have
especially complementary modes of action. This is in contrast to
the combination of CA1125 with CA1134 where the increase in
EC.sub.50 (i.e. reduction of potency) with higher [TcdB] is more
substantial and where the three Mab molar ratios are not equally
effective: The CA1125:CA1134 ratio of 25:75 is notably less potent
than 50:50 and 75:25. This suggests that the combined potency of
CA1125+CA1134 is more dependent upon the CA1125 component. The
EC.sub.50 of all three molar combinations of CA1125 and CA1153 is
substantially affected by increasing [TcdB] suggesting that CA1153
is a less suitable partner for combination with CA1125. In toto,
these data show that CA1125 and CA1151 are a particularly
favourable combination since the highest potency is maintained
across a range of Mab and TcdB molar ratios.
TABLE-US-00060 TABLE 8 TcdB neutralisation - 1 or 2 anti-TcdB Mabs
at constant toxin dose (LD.sub.80). Antibody IC50 (ng/ml) 1099 2
1102 N/A 1114 103 1125 N/A 1134 8 1151 182 1153 260 926 N/A 927 N/A
1099 + 1125 6 1114 + 1125 7 1151 + 1125 2 1134 + 1125 1 1102 + 1125
6 1125 + 1153 12 926 + 1125 42 927 + 1125 4
TABLE-US-00061 TABLE 9 TcdB neutralisation - 1 or 2 anti-TcdB Mabs
at various TcdB doses. EC50 values (ng/ml) Maximum neutralisation
Antibody combination TcdB LD75 TcdB LD86 TcdB LD90 TcdB LD75 TcdB
LD86 TcdB LD90 1125.g2 n/a n/a n/a 40% 25% 15% 1114.g8 n/a n/a n/a
45% 25% 15% 1134.g5 n/a n/a n/a 45% 25% 15% 1151.g4 n/a n/a n/a 45%
25% 20% 1153.g8 28.3 n/a n/a 65% 35% 28% 1125.g2 + 1114.g8 (50:50)
10.1 243.8 n/a 85% 65% 40% 1125.g2 + 1134.g5 (50:50) 1.7 22.6 n/a
87% 60% 40% 1125.g2 + 1153.g8 (50:50) 6.1 32.2 n/a 95% 75% 48%
1125.g2 + 1151.g4 (50:50) 0.8 2.8 19.1 85% 80% 55% 1125.g2 +
1151.g4 (25:75) 1.2 2.8 47.2 85% 75% 60% 1125.g2 + 1151.g4 (75:25)
2.9 3.8 2.6 75% 70% 60%
[0489] These data show that combination of Mabs, especially CA1125
and CA1151 improve both the potency as measured by EC.sub.50 but
also as measured by % maximum protection. The % maximum protection
is of particular relevance in this assay method since the Mab:TcdB
mixture is incubated with cells for a long time (72 h). Since TcdB
is toxic to Caco-2 cells in the range of pg/ml in 2-4 h this
measure may be considered to be a very difficult test of Mab
neutralisation ability and may reflect the ability of Mab mixture
with regard to their binding kinetics or modalities. In turn this
may reflect the ability of Mab mixtures to protect against the
effects of TcdB during an established infection when there may be
substantial quantities of TcdB within tissues for many hours.
Selected data from Tables 6-9 are further illustrated in FIGS.
46-59.
Example 4 Valency of Binding of Mabs to TcdB Sub-Domains
[0490] The number of moles of binding events of anti-C. difficile
TcdB antibodies to TcdB.sub.1234 was determined by Surface Plasmon
Resonance (SPR) on a Biacore 3000 (GE Healthcare). Streptavidin was
immobilized on a CM5 sensor chip (GE Healthcare) to a level of
.about.4000RU via amine coupling and biotinylated TcdB.sub.1234 was
bound at 500-600RU. Two 20 .mu.l injections of the same anti-TcdB
antibody mixtures (final concentration of each antibody was 500 nM)
were injected over this surface at 10 .mu.l/min and the saturating
binding response recorded. The surface was regenerated after every
cycle using HCl. All the data was corrected for background binding
using the response to the streptavidin only reference flowcell.
TABLE-US-00062 TABLE 10 Surface plasmon resonance analysis of the
number of IgG binding sites on TcdB.sub.1234 No. of Binding Binding
relative Antibody binding Response to CA927 combination cycle
repeats (RU) average response CA1125.g2 10 750 0.9 CA1151.g4 10
1232 1.6 CA1125_CA1151 4 1941 2.5 CA1125_CA927 3 1570 2.0
CA1151_CA927 3 1959 2.5 CA927 8 791 1.0
[0491] All responses have been expressed relative to a multiple of
CA927 average response (final column table 10) since CA927 appears
to be representative of a Mab which binds to TcdB.sub.1234 once
only.
[0492] Immobilized CA1125, when bound to TcdB.sub.1234, does not
allow CA1125 to bind further supporting the idea that CA1125 has
one binding site on TcdB.sub.1234 and that after this has been
saturated that no other binding site for CA1125 can be found.
However, when TcdB.sub.1234 has been saturated by CA1125, CA1151
can still bind. This demonstrates that CA1151 binds at alternative
sites to that occupied by CA1125. Together these data show that
CA1125 is a single binder of TcdB.sub.1234 whereas 1151 IgG binds
to TcdB.sub.1234 more than once, most likely twice. Hence a mixture
of CA1125 and CA1151 can bind to TcdB.sub.1234 approximately 3
times.
[0493] All antibodies combinations have an additive binding
response showing that there are 2 or more non-competitive sites on
TcdB.sub.1234 bound by these combinations.
Example 5 Valency of Binding of Mabs to TcdA Sub-Domains
[0494] The number of moles of binding events of anti-C. difficile
TcdA antibodies to TcdA.sub.123 and A.sub.456 were determined by
Surface Plasmon Resonance (SPR) on a Biacore 3000 (GE Healthcare).
Streptavidin was immobilized on a CM5 sensor chip (GE Healthcare)
via amine coupling to a level of .about.4000RU and biotinylated
TcdA.sub.123 was bound to one flowcell and TcdA.sub.456 was bound
to a different flowcell to a response of .about.500RU. Two 30 .mu.l
injections of the same anti-TcdA antibody at 1 .mu.M were injected
over both flowcells at 10 .mu.l/min and the saturating binding
response recorded. The surface was regenerated after every cycle
using HCl. All the data was corrected for background binding using
the response to the streptavidin only reference flowcell.
TABLE-US-00063 TABLE 11 SPR analysis of the binding responses of
IgGs to immobilised TcdA.sub.123 and TcdA.sub.456 CA997 CA1000
CA997/CA1000 ratio TcdA123 1069 166 6 TcdA456 1285 407 3
[0495] Antibodies CA997 and CA1000 bind to TcdA.sub.123 in a ratio
of six CA997's to one CA1000 whereas they bind to TcdA.sub.456 in a
ratio of three CA997's to one CA1000 (Table 2).
[0496] The maximum antibody response for CA997, corrected for
molecular weight and immobilized toxin level is similar for
TcdA.sub.123 and TcdA.sub.456. This suggests that CA997 binds
TcdA.sub.456 six times and CA1000 binds twice to TcdA.sub.456.
Hence antibody CA997 likely binds to TcdA whole toxin (TcdA)
approximately 12 times.
[0497] Overall CA997 binds six times or more to A.sub.123 and six
times or more to A.sub.456, whereas CA1000 binds at least once to
A.sub.123 and twice to A.sub.456.
[0498] Increased valency of binding to TcdA and TcdB may have two
important effects in vivo. The first is that any Mab or Mab mixture
which is capable of binding TcdB more than once will have increased
potential to form inter-toxin binding events and hence
immunoprecipitation. Immunoprecipitation can contribute to potency
by reducing the solubility of toxin and forming very large
macromolecular complexes which hence reduce the effective working
concentration of toxin. Such large protein complexes may be taken
up by macrophages and monocytes resident in the tissue and may
contribute to an augmented host immune response. Antigen:antibody
complexes bearing Fc fragments have been specifically shown to be
capable of priming a host immune response against a gut pathogen
(21). Also, soluble antigen:antibody complexes have been
successfully used as a vaccine directed against the antigen in
human clinical trials (22). In addition, immune decoration of toxin
with Fc bearing IgG may contribute to immune clearance using normal
mechanisms through the liver and spleen. In general, higher levels
of Fc decoration of antigen lead to faster and more complete levels
of clearance (23) Critically, it may be that presence of 2 or more
Mab Fc domains per toxin, especially 3 Fc domains per toxin may
represent a critical number of Fcs required for very rapid and
substantial clearance of toxin (24). The anti-TcdA Mab CA997 is
likely capable of binding to TcdA up to 12 times and the
combination of CA1125 and CA1151 is likely capable of binding to
TcdB 3 times. Hence the 3 Mab mixture is very likely to be capable
of providing for these kinds of additional potency mechanisms in
vivo.
Example 6 Mab Neutralisation of Loss of TEER Caused by TcdA
[0499] C. difficile monolayer integrity assay is performed using
the Becton-Dickinson (BD) Caco-2 BioCoat HTS plate system.
[0500] Day 1--Caco-2 cells seeded @ 2.times.10.sup.5/ml per well of
the plate insert in 500 .mu.l Basal seeding medium (provided by
BD). 35 ml of Basal seeding medium added to the feeder tray. Cells
incubated for 24 hours at 37.degree. C. Day 2--Basal seeding medium
removed from inserts and feeder tray, and replaced with Entero-STIM
differentiation medium (supplied by BD). 500 .mu.l added per well
insert and 35 ml to the feeder tray. Cells incubate for a further
72 hrs at 37.degree. C. Day 5--Antibodies prepared at 2.times.
concentration relative to that to be used in the assay well in a
polypropylene plate and toxin A. Toxin A added to antibodies at a
concentration of 125 ng/ml and plate incubated for 1 hr at
37.degree. C. 1 ml of Caco-2 growth medium (MEM+20% FCS, 2 mM Q,
NEAA) added to each well of a standard 24-well TC plate. BioCoat
insert plate transferred to the 24-well TC plate. Entero-STIM
medium removed from inserts and replaced with 400 .mu.l of toxin:Ab
mixture. Loss of tight junctions between gut cells is the key early
effect of TcdA on cell monolayers and gut tissue sections and is
the primary cause of diarrhoea. Albumin and other serum proteins
are lost into the gut lumen along with accompanying serum fluid.
The loss of trans-epithelial electrical resistance in
differentiated cultured cells which have formed a monolayer is a
useful surrogate for the protection against the acute effects of
TcdA. Three antibodies shown have good levels of protection against
TEER loss, FIG. 62. It is notable and surprising that the abilities
of these Mabs in TEER assays do not reflect those seen in toxin
neutralization as measured in a cell proliferation assay. CA922 has
the best performance in a cell proliferation assay (EC.sub.50=1.21
ng/ml) and yet this is considerably out-performed in the TEER assay
by an antibody (CA1000) which has >10.times. lower potency in a
cell proliferation assay (EC.sub.50=19.73 ng/ml). CA997 had the
best performance in the TEER assay since it had both high levels of
protection and maintained this at the lower Mab concs. CA997 had a
substantial potential to neutralize TEER loss with maximal
inhibition approaching 80% and an EC.sub.50 of approximately 80
ng/ml at 4 h. These observations are unexpected since the Mabs in
question all had high affinities for TcdA domains (CA922 .about.4
pM, CA997 .about.132 pM, CA1000 .about.73 pM). These data suggest
that CA997 and CA1000 recognise epitopes important in TEER loss or
neutralize TcdA by different mechanism to other Mabs. Furthermore,
since CA1000 is estimated to bind to holotoxin twice (once in
TcdA.sub.123 and once in TcdA.sub.456) CA1000 may define `TEER
critical` epitopes within the TcdA cell binding regions which might
have particular value for defining vaccine immunogens. Results are
shown in FIG. 62.
Example 7 Affinity of Anti-C. difficile Toxin Antibodies for
Sub-Domains of TcdA and TcdB: TcdA.sub.123, TcdA.sub.456 and
TcdB.sub.1234
[0501] Kinetic constants for the interactions of anti-C. difficile
TcdA and TcdB antibodies were determined by surface plasmon
resonance conducted on a BIAcore 3000 using CM5 sensor chips. All
experiments were performed at 25.degree. C. Affinipure F(ab').sub.2
fragment goat anti-human IgG, Fc fragment specific (Jackson
ImmunoResearch) was immobilised on a CM5 Sensor Chip (GE) via amine
coupling chemistry to a capture level of .apprxeq.7000 response
units (RUs). HBS-EP buffer (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM
EDTA, 0.005% Surfactant P20, Biacore AB) was used as the running
buffer with a flow rate of 10 .mu.L/min. A 10 .mu.L injection of
each antibody at 1 ug/ml or lower was used for capture by the
immobilised anti-human IgG, Fc. TcdA123, TcdA456 or TcdB1234 were
titrated over captured purified antibodies at doubling dilutions
from 12.5 nM at a flow rate of 30 .mu.L/min. For antibodies present
in culture supernatants, a single concentration of 12.5 nM of
TcdA123 or TcdA456 and 50 nM of TcdB1234 was passed over the
antibodies at 30 ul/min. Kinetics were calculated on n=2 The
surface was regenerated at a flowrate of 10 uL/min by two 10 .mu.L
injections of 40 mM HCl, and a 5 .mu.L injection of 5 mM NaOH.
Double referenced background subtracted binding curves were
analysed using the BIAevaluation software (version 3.2) following
standard procedures. Kinetic parameters were determined from the
fitting algorithm.
TABLE-US-00064 TABLE 12 Anti-TcdA Mab affinities and binding
kinetics Antibody ID ka (1/Ms) kd (1/s) KD (M) KD (pM)
Material/Assay TcdA123 CA164_00922.g1 1.09E+06 4.43E-06 4.06E-12
4.06 Purified Mab 5 point CA164_00923.g1 5.36E+05 3.47E-05 6.47E-11
64.7 titration CA164_00995.g1 No binding No binding CA164_00997.g1
7.84E+05 1.03E-04 1.32E-10 132 CA164_01000.g1 1.33E+05 9.78E-06
7.33E-11 73.3 CA164_00993.g1 9.00E+05 5.00E-06 5.56E-12 5.56
Supernatant 2x 1 point titration TcdA456 CA164_00922.g1 1.29E+06
3.33E-06 2.59E-12 2.59 CA164_00923.g1 6.16E+05 1.92E-04 3.12E-10
312 Purified Mab 5 point CA164_00995.g1 2.87E+05 3.42E-05 1.19E-10
119 titration CA164_00997.g1 9.21E+05 6.15E-05 6.68E-11 66.8
CA164_01000.g1 3.55E+05 2.98E-05 8.41E-11 84.1 CA164_00993.g1
1.25E+06 5.00E-06 4.00E-12 4.00 Supernatant 2x 1 point
titration
TABLE-US-00065 TABLE 13 Anti-TcdB Mab affinities and binding
kinetics Antibody ID ka (1/Ms) kd (1/s) KD (M) KD (pM)
Material/Assay TcdB1234 CA164_1125.g2 2.64E+05 3.23E-05 1.22E-10
122 Purified Mab 3 point titration CA164_1151.g4 7.49E+05 4.13E-04
5.51E-10 551 Purified Mab 3 point titration CA164_926.g1 1.38E+05
7.12E-05 5.16E-10 516 Supernatant 2x 1 point titration CA164_927.g2
3.97E+05 3.61E-05 9.11E-11 91 Purified Mab 3 point titration
CA164_1099.g2 5.24E+05 1.63E-05 3.10E-11 31 Purified Mab 3 point
titration CA164_1102.g4 1.17E+05 3.78E-04 3.25E-09 3250 Supernatant
2x 1 point titration CA164_1114.g2 2.87E+05 1.97E-03 6.87E-09 6870
Supernatant 2x 1 point titration CA164_1114.g8 2.55E+05 1.85E-03
7.25E-09 7250 Supernatant 2x 1 point titration CA164_1129.g1
1.89E+05 2.30E-04 1.22E-09 1220 Supernatant 2x 1 point titration
CA164_1134.g5 5.09E+05 2.45E-05 4.81E-11 48 Purified Mab 3 point
titration CA164_1153.g8 1.43E+05 4.48E-05 3.14E-10 314 Purified Mab
3 point titration
[0502] The anti-TcdA affinities are particularly high compared to
the published affinities of other Mabs. We demonstrate that
affinities as low as 4 pM are achievable. The preferred CA997 has
an affinity of 132 pM, CA1125 122 pM and CA115 551 pM. CA995
clearly shows that it does not bind to CROPs A.sub.123 and hence
that demonstrates that the Mab shown here have properties which are
different from each other in surprising and unexpected ways. CA922,
923, 997 and 1000 do bind at least once to CROPs A123 and A456.
Hence these 4 Mabs confirming that each must bind to holotoxin at
least twice. We have been unable to derive affinities for the
binding of these Mabs to holotoxin due to technical constraints.
However, given the high affinities and valencies demonstrated for
the anti-TcdA Mabs it is possible to speculate that the functional
affinities against holotoxin may be even stronger than those
illustrated for binding to toxin sub-domains. The anti-TcdB Mabs
also demonstrated strong affinities reaching as low as 31 pM. In
particular CA1125, 1151, 927, 1099, 1134 and 1153 show affinities
which surpass those demonstrated by others.
Example 8 Biophysical Characterisation of C. difficile Anti-Toxin
Humanised IgG1 Molecules
Molecules Analysed
Anti-TcdA IgG1:
CA164_00922.g1
CA164_0923.g1
CA164_0995.g1
CA164_0997.g1
CA164_01000.g1
Anti-TcdB IgG1
CA164_01125.g1
CA164_01125.g2
CA164_01134.g4
CA164_01134.g5
CA164_01134.g6
CA164_01102.g1
CA164_01102.g4
CA164_01151.g4
[0503] Antibody combinations need to be made up of Mabs having high
levels of stability in order to mitigate potential risks of
aggregation during long term storage. Thermal stability (Tm) is
used as one measure. Of special value to Mab mixtures is measuring
their propensity to aggregate due to physical stress such as
agitation or shaking. Aggregates are undesirable components of drug
compositions since they may reduce storage life time and may pose a
safety risk to patients at certain levels. The Tm data show that
all 5 anti-TcdA Mabs have high Tm stability, whilst three (CA922,
923 and 997) have very high Tm's in the range of 79-81.degree. C.
Of the anti-TcdB Mabs tested all but two have very high Tm's. Of
note is that CA997, CA1125 and CA1151 which were tested in the
hamster infection study (Example 9) had very high Tm's
(79.2.degree. C., 79.3.degree. C. and 80.8.degree. C. respectively)
which makes them suitable for use in a Mab mixture.
[0504] In the shaking aggregation assay, CA997 and 922 had the
lowest propensity to aggregate of the 5 anti-TcdA Mabs. Similarly,
CA115 and 1151 had the lowest aggregation propensities of the
anti-TcdB Mabs. Hence the use of CA997, 1125 and 1151 as a Mab
mixture may have special value since they are more likely to
survive co-formulation and storage at high protein
concentrations.
Estimation of Isoelectric Point (pI) by Capillary IEF
[0505] Samples were prepared by mixing the following: 30 ul Protein
sample at 2 mg/ml, 0.35% Methylcellulose, 4% pH3-10 ampholytes
(Pharmalyte), synthetic pI markers (4.65 and 9.77), 1 ul of each
stock solution, and HPLC grade water to make up the final volume to
200 ul. The mixture was then analysed using iCE280 IEF analyzer
(pre-focusing at 1500V for 1 min followed by focusing at 3000V for
6 mins). The calibrated electropherograms were then integrated
using Empower software (from Waters)
Thermal Stability (Tm) Measured Via Thermofluor Assay.
[0506] This method uses Sypro orange fluorescent dye to monitor the
unfolding process of protein domains. The dye binds to exposed
hydrophobic regions that become exposed as a consequence of
unfolding which results in a change to the emission spectrum.
[0507] The sample (5 ul at 1 mg/ml) is mixed with a 5 ul of a stock
solution of Sypro orange (30.times.) and the volume made up to 50
ul with PBS, pH 7.40.
[0508] 10 ul aliquots of this solution is applied towells in a 384
well plate (n=4).
[0509] The plate is placed in a 7900HT fast real-time PCR system
containing a heating device for accurate temperature control. The
temperature is increased from 20.degree. C. to 99.degree. C. (Ramp
rate of 1.1.degree. C./min). A CCD device simultaneously monitors
the fluorescence changes in the wells. An algorithm is used to
process intensity data and take into account multiple
transitions.
Stressing of Samples by Agitation.
[0510] During manufacture antibody samples are subjected to
mechanical stress generated by processes such as pumping and
filtration. This may cause denaturation and consequently
aggregation due to exposure of the protein to air-liquid interfaces
and shear forces resulting in the ultimate loss of bioactivity.
Stress by vortexing is a method to screen the robustness of the
antibody samples for prediction of aggregation stability.
[0511] Both anti-TcdA and anti-TcdB IgG1 molecules were subjected
to stress by agitation, by vortexing using an Eppendorf Thermomixer
Comfort at 25.degree. C., 1400 rpm. Sample size was 250 uL,
(.times.3 per sample) in a 1.5 mL conical Eppendorf-style capped
tube (plastic), in PBS pH 7.4. Each sample was brought to a
concentration of 1 mg/ml (using extinction coefficient calculated
from sequence) and aggregation was monitored by absorbance at 340
nm and/or 595 nm, by use of a Varian Cary 50-Bio spectrophotometer,
measured at intervals for up to 24 hours.
[0512] Results Table 14 provides a summary of the measured pI and
Tm data for both anti-TcdA and anti-TcdB IgG1 molecules.
TABLE-US-00066 TABLE 14 Compilation of pI and Tm Data measured
Tm(Fab) Anti-TcdA IgG1 pI in PBS Tm(CH2) CA164_00922.g1 8.8 81 69.2
CA164_0923.g1 9.2 79 69.3 CA164_0995.g1 8.5 71 no data*
CA164_0997.g1 8.3 79.2 68.4 CA164_01000.g1 7.74 70.5 no data*
Anti-TcdB IgG1 CA164_01125.g1 9.2 79.3 69.4 CA164_01125.g2 9.2 79.5
69.3 CA164_01134.g4 9.3 78.4 69.4 CA164_01134.g5 9.2 76.4 69.2
CA164_01134.g6 9.2 76.6 69.6 CA164_01102.g1 9.1 69 no data*
CA164_01102.g4 9.1 69.1 no data* CA164_01151.g4 9.2 80.8 69.8
*denotes that it was not possible to discern the Fab and CH2
domains.
Measured pI
[0513] The measured pI of the molecules were high (except for
CA164_01000.g1_P3) and away from the pH of formulation buffers such
as PBS, pH 7.4 and 50 m sodium acetate/125 mM sodium chloride, pH
5. This may mean that buffers with pH's suitable for co-formulation
of two or more Mabs can be selected.
Thermal Stability (Tm) Measured Via Thermofluor Assay
[0514] Since all of the molecules are IgG1, the Tm of the Fc domain
(Tm(CH2)) are the same. The difference in thermal stability between
the molecules can be determined by the Tm of the Fab' domain
(Tm(Fab)).
[0515] For the anti-TcdA molecules, the rank order (most stable
first) was CA922.gtoreq.997>923>995>1000 and for the
anti-TcdB molecules (most stable first) was
CA1151.g4>1125.g1,g4>1134.g4>1134.g5.gtoreq.1134.g6>1102.g1=1-
102.g4.
Stressing of Samples by Agitation It was possible to determine
different aggregation stability between the different antibodies,
FIG. 67 shows the effect of agitation via vortexing on different
anti-TcdA IgG1 molecules in PBS, pH 7.4.
[0516] It was possible to determine a ranking order (most
aggregation stable first):
CA922.gtoreq.997>923.gtoreq.995>1000
[0517] FIG. 68 shows the effect of agitation via vortexing on
different anti-TcdB molecules.
[0518] It was possible to rank the order of aggregation stability,
such that the CA1125 grafts appeared more stable than the CA1134
molecules which were more stable than the CA1102 molecules.
[0519] A further study was performed to compare directly the
aggregation stability of the anti-TcdB molecule (CA1151.g4) with
the more stable molecule CA1125.g2 (see FIG. 2) and more
aggregation stable anti-TcdA molecules (CA922.g1 and CA997.g1). The
results can be seen in FIG. 69.
[0520] Further results for these 4 Mabs are also shown in FIGS. 67
and 68.
[0521] For the anti-TcdA molecules, CA922.g1 and CA977.g1, CA922
were preferable based on the analyses above, although apart from
CA1000) all molecules could be considered suitable candidates for
use as therapeutic IgG1.
[0522] For the anti-TcdB molecules, the biophysical characteristics
could be grouped within the family of grafts based on the
aggregation stability and Tm, such that the CA1125 grafts
potentially proved more stable. The CA1102 grafts showed poorest Tm
data and also showed the greatest tendency to aggregate via stress
by agitation.
[0523] A study using CA1151.g4 showed that this molecule exhibited
slightly increased aggregation stability relative to CA11125.g2 and
seemed equivalent to the TcdA molecules (CA922.g1 and CA997.g1. All
four molecules showed equivalent Tm values. CA997, CA1125 and
CA1151 show very high levels of thermostability and very low levels
of aggregate formation after agitation.
Example 9 Anti-C. difficile Toxin Mab Hamster Infection Study
[0524] The hamster infection study was performed by Ricerca
Biosciences LLC, Cleveland, Ohio, USA. The study protocol was
approved by the Ricerca IACUC committee. Active and control
components (composition and dose) were blinded to Ricerca staff
until after completion of the planned 28 day study period.
[0525] Golden Syrian male hamsters (weight 82-103 g, 54 days old)
were individually housed in HEPA filtered disposable cages and fed
Teklad Global Diet 2016 and water ad libitum. After
acclimatisation, hamsters were pre-dosed (i.p.) with Mab mixtures
or PBS (vehicle control) once a day for each of 4 days: days -3,
-2, -1 and 0. Two doses of Mab were investigated: high dose=50
mg/kg each of anti-TcdA and anti-TcdB components and low dose 5
mg/kg each of anti-TcdA and anti-TcdB components.
[0526] The drug combination tested was composed of one anti-TcdA
antibody (CA997.g1) which constituted 50% of the injected protein
and two anti-TcdB antibodies (CA1125.g2 and CA1151.g4) which
together constituted 50% of the injected protein but which alone
constituted 25% of the injected protein. Hamsters were sensitised
(day -1) with 50 mg/kg of Clindamycin phosphate in PBS (s.c.)
before being challenged 1 day later (day 0) with 3.4.times.106
c.f.u. of vegetative cells from strain ATCC43596. Vancomycin was
dosed at 5 mg/kg twice a day for 5 days (p.o.) on days 1, 2, 3, 4,
5.
[0527] Viability checks were performed on animals twice a day,
animals found to be in extremis were euthanised and counted as
dead. Body weights were determined on each day of dosing, then
twice weekly and before euthanising survivors. Gross necropsy was
performed on all animals. Survival curves were created by the
method of Kaplan and Meier. Survival curves were analysed using the
P value from the log rank test compared to the Bonferroni corrected
threshold of P=0.005. The Vancomycin treated group were not
included in the analysis. All statistical tests were done with
Prism v5.04. All groups contained 11 animals, except the Vancomycin
control group which contained 5 animals.
[0528] Survival curves can be seen in FIG. 63. Hamsters receiving
PBS (control) all died on days +2 and +3, whilst those receiving
vancomycin treatment for 5 days all died on days +10 and +11.
Hamsters receiving the high dose of UCB Mab mixture all survived
until day +11, thereafter only two animals died until the end of
the 28 day study. Hamsters receiving the low dose of UCB Mab
mixture all survived until day +3, thereafter animals were lost
fairly steadily until day +16 when all had died. The data show
exceptional levels and duration of protection when compared to
published data for use of anti-toxin Mabs in hamsters (18). These
in vivo data support the in vitro observations of very high level
performance for neutralization and stability.
[0529] There is no apparent link between death and body weight
during the acute phase (days 1-5) of the infection, FIGS. 64-65.
Hence it may be supposed that hamsters die of overwhelming direct
and indirect effects of TcdA and TcdB. Hamsters which survive the
acute period due to partial protection (UCB low dose) of
neutralizing Mabs lose weight, presumably due to gut damage and
altered nutritional status. It was notable that many of the
hamsters which went on to survive the 28 period of the study due to
the protective effects of the UCB high dose Mabs recovered from
weight loss and indeed even gained weight. This may be taken as
evidence of the superior protective effects of the UCB Mabs
enabling the gut to function as normal.
TABLE-US-00067 TABLE 15 Gross pathology scores Anogenital Black
Dark red Red Pink Normal staining Red small Group caecum caecum
caecum caecum caecum `wet-tail` intestine PBS 1 9 1 0 0 1 1 control
UCB low 0 4 5 2 0 4 1 UCB high 0 0 1 1 9 3 0
[0530] It is clear that UCB Mabs were able to protect the large and
small intestines from the bloody effusions caused by TcdA and
TcdB.
[0531] The results are shown in FIGS. 63 to 66
[0532] The photographs in FIG. 66 show typical gross pathologies
for the swelling and bloody effusions of caeca caused by TcdA and
TcdB (left image, PBS control, animal death on day 2) and a normal
stool filled caeca after protection by UCB high dose Mabs (right
image, UCB high dose, animal surviving to day 28). These data show
that after protection with a high dose of UCB Mabs the large
intestine can return to normal morphology and function.
Example 10 Neutralisation of TcdA from Different Ribotyped Strains
by Purified Mab
[0533] Clinical infections are caused by a variety of different
strains. Strain differences are characterized using a number of
different methods of which ribotyping is a key one. Different
ribotype strains are observed to have different pathogenicity,
infection and sporulation properties. All of the TcdA
neutralization shown above used TcdA purified from strain known as
VPI10463. However, the predominant aggressively pathogenic strain
associated with out-breaks is called ribotype 027. Other key
ribotypes include 078, 001, 106. Amino acid sequence difference
have been observed between toxins produced by different ribotypes
and hence it is important that Mabs are capable of neutralizing
toxin from a diverse set of clinical isolates. CA922, 997 and 1000
were tested for their ability to neutralize TcdA from strains 027
and 078 and compared to their abilities against TcdA from VPI10463.
Mabs were tested at 4 [TcdA] and found to be capable of
neutralizing all toxins without significant difference at
LD.sub.80, LD.sub.90 and LD.sub.95
TABLE-US-00068 TABLE 16 EC50 values (ng/ml) - TcdA strain VPI 10463
Antibody LD80 LD90 LD95 LDmax CA164_922 0.27 0.9 1.2 >500
CA164_997 1 2.5 3.5 25.4 CA164_1000 3.6 13.5 19.3 >500
TABLE-US-00069 TABLE 17 EC50 values (ng/ml) - TcdA ribotype 027
Antibody LD80 LD90 LD95 LDmax CA164_922 0.19 0.25 0.41 1.46
CA164_997 0.92 1.27 1.75 7.19 CA164_1000 2.25 2.49 3.52 16.32
TABLE-US-00070 TABLE 18 EC50 values (ng/ml) - TcdA ribotype 078
Antibody LD80 LD90 LD95 LDmax CA164_922 0.11 0.12 0.25 0.68
CA164_997 0.33 0.64 1.11 2.57 CA164_1000 2.04 2.41 5.03 14.16
Example 11 PK Data
[0534] A PK study of a human IgG1 (20 mg/kg) in healthy hamsters.
The hamster PK was found a half-life similar to Mabs in mice or
rats. (t1/2 6-8 days). i.p. and s.c. dosing were essentially the
same. The pharmacokinetics and distribution to the gut of a hIgG1
Mab was studied in `normal` (non-infected) golden Syrian hamsters.
Purified Mab was administered to male hamsters (120-135 g) by CARE
Research LLC, Fort Collins, Colo., USA and samples were assayed by
UCB Pharma. The study was approved by the CARE IACUC committee.
Eight animals each received a single dose of 20 mg/kg of IgG1, four
were dosed i.p., four were dosed s.c. Blood was collected at 1, 3,
8, 24, 48, 72, 103 and 168 hours post-dose, serum was separated
before storage at -80.degree. C. Blood was also taken from two
untreated hamsters in order to provide assay controls. Following
euthanasia, a 2 cm length of colon was cut from the caeca junction
onwards from each hamster. The colon section was flushed with wash
buffer (50% (v/v) PBS containing 50% (v/v) Sigma protease inhibitor
cocktail (P2714) before being opened and separation and removal of
the mucosa from the underlying muscle. Mucosal samples were placed
in 0.5 ml of wash buffer homogenized until visually uniform and
stored at 4.degree. C. before immediate shipping on wet ice. For
the anti-human IgG1 ELISA Nunc maxisorp 96 well plates were coated
overnight in 0.1M NaHCO.sub.3 pH 8.3 with Goat F(ab')2 anti-human
IgG-Fc.gamma. fragment (Jackson 109-006-098), plates were washed
with PBS-Tween (PBS/0.1% (v/v) Tween 20) and then blocked with 1.0%
(w/v) BSA & 0.1% (v/v) Tween in PBS. Serum samples were diluted
in sample-conjugate buffer (1% (w/v) BSA, 0.2% Tween in PBS) and
after washing were revealed with goat anti-human kappa-HRP
(Cambridge Bioscience 2060-05) in sample-conjugate buffer and TMB
with a 2.5M H2504 stop solution.
Gut, Mucosa and Serum Levels:
[0535] Serum samples collected from blood taken at 168 hour time
point and colon samples were removed after this.
TABLE-US-00071 20mg/kg IP at 168 hour Sample ng/mL per cm mucosa
serum .mu.g/mL 1001 23.2 75.0 1002 13.7 90.8 1003 21.8 70.5 1004
53.8 119.4
TABLE-US-00072 20mg/kg SC at 168 hour Sample ng/mL per cm mucosa
serum .mu.g/mL 2001 41.4 108.7 2002 62.1 76.6 2003 35.6 163.7 2004
37.3 153.3
TABLE-US-00073 Serum Data Hamster i.p. Hamster s.c. SE of SE of
Mean mean Mean mean C.sub.max: .mu.g/mL 202 12 186 21 T.sub.max: hr
36 7 76 16 AUC .sub.(last): hr .mu.g/mL 22626 1378 22371 2258 AUC
.sub.(inf): hr .mu.g/mL 43287 7169 61290 17637 % Extrapolation:
43.7 9.2 54 11.7 CL/F mL/hr/kg 0.50 0.07 0.43 0.13 MRT.sub.inf h
223 53 310 88 t.sub.1/2,z: h 149.2 36.9 188.5 61.9
[0536] The data is also shown in FIGS. 70 and 71
TABLE-US-00074 Hamster ID Mean SE IP serum kinetics C.sub.max:
.mu.g/mL 202 12 T.sub.max: hr 36 7 AUC.sub.(last): hr .mu.g/mL
22626 1378 AUC.sub.(inf): hr .mu.g/mL 43287 7169 % Extrapolation:
43.7 9.2 CL/F mL/hr/kg 0.50 0.07 MRT.sub.inf h 223 53 t.sub.1/2,z:
h 149.2 36.9 SC serum kinetics Hamster ID Mean SE C.sub.max:
.mu.g/mL 186 21 T.sub.max: hr 76 16 AUC.sub.(last): hr .mu.g/mL
22371 2258 AUC.sub.(inf): hr .mu.g/mL 61290 17637 % Extrapolation:
54 11.7 CL/F mL/hr/kg 0.43 0.13 MRT.sub.inf h 310 88 t.sub.1/2: h
188.5 61.9
[0537] It was also shown that hIgG1 could be found in `scrapings`
of the gut i.e that hIgG1 gets into the vasculature of healthy
gut--and so could be protective in `prophylactic dosing`. This
effect would be even more profound in humans since they have a
cognate hFcRn.
Example 12 Serum Levels in Hamsters with C. difficile Infection
[0538] This study was to determine the serum concentration of
CA725.0, CA726.0, CA997.g1 CA1125.g2, and CA01151.g4 following i.p.
administration (various doses detailed below) in the Golden Syrian
Hamster.
[0539] Humanised Mabs were quantified using liquid chromatography
tandem mass spectrometry (LC-MS/MS) analysis following tryptic
digestion. Quantitation was achieved by comparison to authentic
standard material spiked at known concentrations into blank matrix,
with spiked horse myoglobin used as the internal standard.
[0540] A unique ("proteotypic") peptide common to all of the
humanised Mabs investigated was selected (DTLMISR, a CH2 region
peptide) and both samples and calibration samples were tryptically
digested as outlined. Tryptic digest of 5 .mu.l serum samples was
performed overnight using sequencing grade modified Trypsin
(Promega, Southampton, UK) following denaturation/reduction with
acetonitrile/Tris (2-carboxyethyl) phosphine and
carbamido-methylation with iodoacetamide (Sigma-Aldrich, Poole,
UK).
[0541] The LC-MS/MS system consisted of a CTC HTS-x Autosampler
(CTC Analytics, Zwingen, Switzerland), a Agilent 1290 LC system
(Agilent Technologies, Stockport, UK) and a Sciex 5500 QTrap MS
system (AB Sciex, Warrington, UK), equipped with a Turbo V ion
source operated in electrospray mode. Analytes were separated using
an Onyx Monolithic C18 column (100.times.4.6 mm, Phenomenex,
Macclesfield, UK) with a gradient of 2 to 95% (v/v)
water/acetonitrile (0.1% formic acid) delivered at 1.5 mL/min over
6 minutes. The injection volume was 10 .mu.L; all of the eluent was
introduced into the mass spectrometer source. The source
temperature of the mass spectrometer was maintained at 600.degree.
C. and other source parameters (e.g. collision energy, declustering
potential, curtain gas pressure etc.) were optimized to achieve
maximum sensitivity for the peptide of interest. Selective
transitions for each proteotypic peptide of interest were
monitored. Unique ("proteotypic) peptides were selected for all of
the analytes of interest; samples were analysed following tryptic
digestion.
[0542] Plasma concentrations calculated based on the peptides
monitored are outlined below.
[0543] For CA164_00997 and CA164_01151, interfering peaks were
observed in the MRM traces. For this reason, these two analytes
could not be quantified in the samples.
[0544] Total h-IgG was quantified in all samples using a peptide
common to all analytes of interest. This was done using a combined
standard curve of all five analytes. The validity of this approach
is demonstrated by the fact that the sum of the concentrations
observed for CA164_00725 and CA164_00726 are in good agreement
(within experimental error) of the concentration observed for total
h-IgG.
[0545] Using this approach, the total concentration of h-IgG in the
samples of animals dosed with CA164_00997, CA164_01125 and
CA164_01151 was determined.
[0546] Overall the data obtained indicate that the exposure of all
five analytes of interest was similar for a given dose.
TABLE-US-00075 Study groups Blinded labels Treatment components Grp
Treatment Actual Treatments Dose days Anti-toxin A Anti-toxin B 4
Treatment3 Vehicle PBS 5 mL/kg i.p. 3, -2, -1, 0 2 Vancomycin
Vancomycin 5 1, 2, 3, 4, 5 mg/kg b.i.d. p.o. 1 Treatment1 UCB LD*
3, -2, -1, 0 CA997.g1_P3 CA1125.g2_P3 CA1151.g4_P3 5 mg/kg A 5
mg/kg i.p. 5 mg/kg 2.5 mg/kg 2.5 mg/kg 5 Treatment4 UCB HD* 3, -2,
-1, 0 CA997.g1_P3 CA1125.g2_P3 CA1151.g4_P3 50 mg/kg A 50 mg/kg
i.p. 50 mg/kg 25 mg/kg 25 mg/kg 6 Treatment5 CompetitorLD* 3, -2,
-1, 0 CA726_P3 CA725_P3 5 mg/kg A 5 mg/kg i.p. 5 mg/kg 5 mg/kg 3
Treatment2 CompetitorHD* 3, -2, -1, 0 CA726_P3 CA725_P3 50 mg/kg A
50 mg/kg i.p. 50 mg/kg 50 mg/kg
TABLE-US-00076 TABLE 19 Group/ Animal Serum conc time Day No Dose
.mu.g/mLtotal h-IgG 1 1 44 5 mg/kg 997, 280 1 45 2.5 mg/kg 302 1 46
1125,2.5 182 6 45 mg/kg 1151 61 6 47 71 6 49 45 3 1 60 50 mg/kg
3040 1 61 725, 3330 1 62 50 mg/kg 2990 6 62 726 583 6 63 913 6 64
1240 28 64 199 28 65 36 4 1 71 Vehicle nd 1 72 nd 1 73 nd 5 1 82 50
mg/kg 3050 1 83 997, 2790 1 84 25 mg/kg 2370 6 82 1125,25 838 6 83
mg/kg 1151 645 6 84 855 28 82 116 28 83 65 28 84 66 28 85 44 28 86
101 28 87 89 28 88 27 28 89 31 28 90 66 6 1 93 5 mg/kg 725, 335 1
94 5 mg/kg 726 322 1 95 260 6 200 103 6 202 62 6 203 79 28 203 nd
nd - not detected (LOQ = 2.5 .mu.g/mL for all analytes na - not
analysed: interference in the sample was observed for 997 and
1151
TABLE-US-00077 TABLE 20 Antibody CA725 is prior art antibody
MDX1388. Antibody CA726 is prior art antibody CDA1 as described
(15) A summary of this data is presented in FIG. 72. Small
intestine Caecal pathology pathology Dark Dark Group Black Red Red
Pink Normal Red Red PBS control 1 9 1 0 0 0 1 MDX high 0 1 4 4 2 1
0 50 mg/Kg x4 UCB high 0 0 1 1 9 0 0 50 mg/Kg x4
REFERENCES
[0547] 1. Kuehne, S et al., "The role of toxin A and toxin B in
Clostridium difficile Infection" Nature (2010) 467: 711-713. [0548]
2. Davies A H et al., "Super toxins from a super bug: structure and
function of Clostridium difficile toxins" Biochem. J (2011) 436:
517-526. [0549] 3. Rothman, S et al., "Differential Cytotoxic
Effects of Toxins A and B Isolated from Clostridium difficile"
Infect. Imm. (1984) 46: 324-331. [0550] 4. Du, T and Alfa, M J
"Translocation of Clostridium difficile toxin B across polarized
Caco-2 cell monolayers is enhanced by toxin A" Can J Infect Dis.
(2004) 15: 83-88. [0551] 5. Kim, Iaconis and Rolfe. "Immunization
of Adult Hamsters against Clostridium difficile-Associated
Ileocecitis and Transfer of Protection to Infant Hamsters" Infect.
Imm. (1987) 55:2984-2992 [0552] 6. Rupnik J C M (2003) 41:1118-1125
[0553] 7. Chaves-Olarte J B C (1999) 274:11046-11052. [0554] 8.
Lylerly, D M et al., "Passive Immunization of Hamsters against
Disease Caused by Clostridium difficile by Use of Bovine
Immunoglobulin G Concentrate" Infection and Immunity (1991)
59:2215-2218. [0555] 9. Lylerly, D M et al., "Vaccination against
Lethal Clostridium difficile Enterocolitis with a Nontoxic
Recombinant Peptide of Toxin A" Current Microbiology (1990)
21:29-32. [0556] 10. Lylerly, D M et al., "Characterization of
Toxins A and B of Clostridium difficile with Monoclonal Antibodies"
Infect. Imm. (1986) 54:70-76. [0557] 11. Corthier et al.,
"Protection against Experimental Pseudomembranous Colitis in
Gnotobiotic Mice by Use of Monoclonal Antibodies against
Clostridium difficile Toxin A" Infect. Imm. (1991) 59: 1192-1195.
[0558] 12. Kink J A and Williams J A, "Antibodies to Recombinant
Clostridium difficile Toxins A and B Are an Effective Treatment and
Prevent Relapse of C. difficile-Associated Disease in a Hamster
Model of Infection" Infect. Imm. (1998) 66:2018-2025. [0559] 13. Ma
D, et al., Progenics inc. ASM Poster 27 May 2010 [0560] 14. Hansen,
G and Demarest, S J. WO 2006/0718877 A2 [0561] 15. Babcock G J, et
al., "Human monoclonal antibodies directed against toxins A and B
prevent Clostridium difficile-induced mortality in hamster" Infect.
Imm. (2006) 74:6339-6347. [0562] 16. Lowy I et al., "Treatment with
Monoclonal Antibodies against Clostridium difficile Toxins" NEJM
(2010) 362: 197-205. [0563] 17. Zubler, R. H., Erard, F., Lees, R.
K., Van, L. M., Mingari, C., Moretta, L. & MacDonald, H. R.
(1985). Mutant EL-4 thymoma cells polyclonally activate murine and
human B cells via direct cell interaction. J. Immunol. 134,
3662-3668 [0564] 18. Babcook, J. S., Leslie, K. B., Olsen, O. A.,
Salmon, R. A. & Schrader, J. W. (1996). A novel strategy for
generating monoclonal antibodies from single, isolated lymphocytes
producing antibodies of defined specificities. Proc. Natl. Acad.
Sci. U.S.A 93, 7843-7848 [0565] 19. Tickle, S., Adams, R., Brown,
D., Griffiths, M., Lightwood, D. & Lawson, A. (2010).
High-Throughput Screening for High Affinity Antibodies., pp.
303-307. [0566] 20. Demarest et al., mAbs (2010) 2:190-198 [0567]
21. Yoshida et al., J. Clin. Invest. (2006) 116: 2142-2151 [0568]
22. Xu et al., Vaccine (2005) 23:2658-2664. [0569] 23. Yousaf et
al., Clin. Exp. Immunol. (1986) 66:654-660 [0570] 24. Mannik et
al., J. Exp. Med. (1971) 133: 713-739 [0571] 25. Nusrat et al.,
Infection and Immunity (2001) 69:1329-1336 [0572] 26. Lima et al.,
Infect Immun (1988) 56:582-588 [0573] 27. Ravichandran et al J of
Pharmacology and Experimental Therapeutics. (2006) 318: 1343-1351
[0574] 28. Takahashi et al., (2009) Vaccine 27:2616-2619 [0575] 29.
Cohen et al., Infect. Cont. and Hosp. Epidem. (2010) 31: 431-455
[0576] 30. Barbut et al., J. Clin. Microbiol. (2000) 38: 2386-2388
[0577] 31. Wilcox et al., J. Hospital Infection (1998) 38: 93-100.
Sequence CWU 1
1
194111PRTArtificialAntibody CDR 1Gln Ala Ser Gln Ser Ile Ser Asn
Ala Leu Ala1 5 1027PRTArtificialAntibody CDR 2Ser Ala Ser Ser Leu
Ala Ser1 5312PRTArtificialAntibody CDR 3Gln Tyr Thr His Tyr Ser His
Thr Ser Lys Asn Pro1 5 10410PRTArtificialAntibody CDR 4Gly Phe Thr
Ile Ser Ser Tyr Tyr Met Ser1 5 10517PRTArtificialAntibody CDR 5Ile
Ile Ser Ser Gly Gly His Phe Thr Trp Tyr Ala Asn Trp Ala Lys1 5 10
15Gly613PRTArtificialAntibody CDR 6Ala Tyr Val Ser Gly Ser Ser Phe
Asn Gly Tyr Ala Leu1 5 107110PRTArtificialAntibody variable region
7Asp Pro Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5
10 15Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Ser Asn
Ala 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ser Ala Ser Ser Leu Ala Ser Gly Val Pro Ser Arg
Phe Lys Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Tyr
Thr His Tyr Ser His Thr 85 90 95Ser Lys Asn Pro Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys 100 105 1108330DNAArtificialPolynucleotide
encoding variable region of anti-TcdA antibody 922.g1 8gaccctgtga
tgacccagag tccgagcact ctttctgcct ccgtgggaga ccgcgtgacc 60attacatgtc
aggcttcaca aagtatctcc aatgctctgg cctggtatca gcagaaaccc
120ggcaaagccc ctaagctgct catctactct gcatcaagcc tggctagcgg
cgtgccaagc 180cgattcaagg ggagcggttc tggcactgag tttacgctga
ccatcagtag cttgcagcct 240gacgattttg caacctatta ctgccagtac
acacactact cccatacatc taaaaaccca 300ttcggagggg gtactaaggt
cgaaataaag 3309119PRTArtificialAntibody variable region for
anti-TcdA antibody 922 9Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Ile Ser Ser Tyr 20 25 30Tyr Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Ile Ile Ser Ser Gly Gly His
Phe Thr Trp Tyr Ala Asn Trp Ala 50 55 60Lys Gly Arg Phe Thr Ile Ser
Ser Asp Ser Thr Thr Val Tyr Leu Gln65 70 75 80Met Asn Ser Leu Arg
Asp Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg 85 90 95Ala Tyr Val Ser
Gly Ser Ser Phe Asn Gly Tyr Ala Leu Trp Gly Gln 100 105 110Gly Thr
Leu Val Thr Val Ser 11510357DNAArtificialPolynucleotide encoding
variable region of anti-TcdA antibody 922 (heavy chain variable
region) 10gaagtgcaat tggtggaaag tggcggagga ctggtgcaac ccgggggtag
tctgcgactg 60agctgtgctg cctccggctt taccattagc tcctactata tgagctgggt
tcgacaggcc 120cctggaaaag gactcgaatg gatcggcatc atatcttccg
gtgggcattt cacctggtac 180gcaaactggg ctaaggggag attcacgatt
agcagcgact ccacaaccgt gtacctgcaa 240atgaacagcc tgagggatga
ggacactgcc acatatttct gcgcacgcgc ttacgtgagc 300ggaagctcat
ttaatggcta tgcactgtgg gggcaaggaa cactcgtgac tgtctcg
3571111PRTArtificialAntibody CDR 11Gln Ala Ser Gln Ser Ile Ser Asn
Tyr Leu Ala1 5 10127PRTArtificialAntibody CDR 12Ser Ala Ser Thr Leu
Ala Ser1 51312PRTArtificialAntibody CDR 13Gln Tyr Ser His Tyr Gly
Thr Gly Val Phe Gly Ala1 5 101410PRTArtificialAntibody CDR 14Ala
Phe Ser Leu Ser Asn Tyr Tyr Met Ser1 5 101518PRTArtificialAntibody
CDR 15Ile Ile Ser Ser Gly Ser Asn Ala Leu Lys Trp Tyr Ala Ser Trp
Pro1 5 10 15Lys Gly1613PRTArtificialAntibody CDR 16Asn Tyr Val Gly
Ser Gly Ser Tyr Tyr Gly Met Asp Leu1 5 1017110PRTArtificialAnitbody
ariable region of anti-TcdA antibody 923 17Asp Val Val Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Gln Ala Ser Gln Ser Ile Ser Asn Tyr 20 25 30Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala
Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly 50 55 60Ser Gly
Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Val Ala Thr Tyr Tyr Cys Gln Tyr Ser His Tyr Gly Thr Gly
85 90 95Val Phe Gly Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
105 11018330DNAArtificialPolynucleotide encoding antibody variable
region for anti-TcdA antibody 923.g1 18gacgtcgtga tgactcagag
cccatctagt ctgagcgcta gcgtcggaga ccgagtcaca 60attacctgtc aagcctccca
gagcatctcc aactacctgg cctggtacca acagaaacct 120ggcaaggtgc
ccaagctgct gatctatagt gcttccacac tcgcaagcgg cgttccgtca
180cgctttaagg gatctggctc tggcactcag ttcaccttga cgatctcaag
cctgcagcca 240gaagatgtgg ccacctatta ctgccagtat tcccactacg
ggactggggt gttcggtgcc 300tttggaggtg ggaccaaagt ggagataaag
33019120PRTArtificialAntibody variable region for anti-TcdA
antibody 923 19Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Ala Phe Ser
Leu Ser Asn Tyr 20 25 30Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Ile 35 40 45Gly Ile Ile Ser Ser Gly Ser Asn Ala Leu
Lys Trp Tyr Ala Ser Trp 50 55 60Pro Lys Gly Arg Phe Thr Ile Ser Lys
Asp Ser Thr Thr Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Thr Tyr Phe Cys Ala 85 90 95Arg Asn Tyr Val Gly Ser
Gly Ser Tyr Tyr Gly Met Asp Leu Trp Gly 100 105 110Gln Gly Thr Leu
Val Thr Val Ser 115 12020360DNAArtificialPolynucleotide encoding
antibody variable region for anti-TcdA antibody 923.g1 20gaagttcaac
ttgtggaatc tggaggcggg ctcgtgcagc ctggtggaag ccttagactg 60agctgcgctg
catccgcatt ttccctgtcc aactactaca tgagctgggt gcgacaagca
120ccaggcaagg gactggaatg gattggcatc ataagctccg gttccaatgc
cctgaaatgg 180tacgcatcat ggccgaaagg ccgctttacc ataagcaagg
actccaccac cgtctatctg 240cagatgaact cattgcgtgc cgaggacact
gcaacgtact tctgtgctcg caactacgtg 300ggaagcggat cttattatgg
catggatctg tggggacaag gtacactcgt gaccgtctcg
3602111PRTArtificialAntibody CDR 21Gln Ala Ser Gln Ser Ile Ser Ser
Tyr Phe Ser1 5 10227PRTArtificialAntibody CDR 22Gly Ala Ser Thr Leu
Ala Ser1 52312PRTArtificialAntibody CDR 23Gln Cys Thr Asp Tyr Ser
Gly Ile Tyr Phe Gly Gly1 5 102410PRTArtificialAntibody CDR 24Gly
Phe Ser Leu Ser Ser Tyr Tyr Met Ser1 5 102519PRTArtificialAntibody
CDR 25Ile Ile Ser Ser Gly Ser Ser Thr Thr Phe Thr Trp Tyr Ala Ser
Trp1 5 10 15Ala Lys Gly2613PRTArtificialAntibody CDR 26Ala Tyr Val
Gly Ser Ser Ser Tyr Tyr Gly Phe Asp Pro1 5
1027110PRTArtificialAntibody variable region for anti-TcdA antibody
993 27Asp Val Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val
Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Ser
Ser Tyr 20 25 30Phe Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Gln
Leu Leu Ile 35 40 45Tyr Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser
Arg Phe Lys Gly 50 55 60Ser Gly Ser Gly Thr Glu Leu Thr Leu Thr Ile
Ser Ser Leu Gln Pro65 70 75 80Asp Asp Phe Ala Thr Tyr Tyr Cys Gln
Cys Thr Asp Tyr Ser Gly Ile 85 90 95Tyr Phe Gly Gly Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys 100 105 11028330DNAArtificialPolynucleotide
encoding antibody variable region for anti-TcdA antibody 933.g1
28gatgtcgtga tgactcagtc cccctctaca ttgagtgcct ctgtcggtga tcgagttacc
60atcacctgtc aagcaagcca gagcatcagc tcctacttct cttggtacca gcaaaagccg
120ggaaaagccc ctcaactgct gatttatggg gcctcaacac tggcttctgg
cgtgccatca 180agattcaagg gatctggctc cggcactgag cttacactga
ccattagctc cctgcaacct 240gacgattttg ctacctacta ctgccagtgc
accgactata gtgggatata tttcggcgga 300tttgggggag ggacgaaagt
ggaaatcaag 33029121PRTArtificialAntibody variable region for
anti-TcdA antibody 993 (heavy chain) 29Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys
Thr Ala Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30Tyr Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Ile Ile Ser
Ser Gly Ser Ser Thr Thr Phe Thr Trp Tyr Ala Ser 50 55 60Trp Ala Lys
Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Tyr65 70 75 80Leu
Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Thr Tyr Phe Cys 85 90
95Ala Arg Ala Tyr Val Gly Ser Ser Ser Tyr Tyr Gly Phe Asp Pro Trp
100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser 115
12030363DNAArtificialPolynucleotide encoding antibody variable
region for anti-TcdA antibody 993.g1 30gaagttcagc tggtcgagag
cggaggcgga ctggtgcaac ctggtggtag cctgaaactc 60tcttgtactg cctccgggtt
ttccctgagc tcttactata tgtcatgggt gagacaggct 120cccgggaaag
gattggaatg gatcgggatt atctcctccg gctcttccac cactttcaca
180tggtacgcct catgggcaaa ggggaggttt accataagca agacaagcac
gaccgtgtat 240cttcagatga actccctgaa gacggaggat actgccacct
acttttgcgc tcgggcctat 300gtgggctcaa gctcttacta tggcttcgac
ccatggggac agggcacact tgtgaccgtc 360tcg
3633111PRTArtificialAntibody CDR 31Gln Ala Ser Gln Ser Ile Asn Asn
Tyr Phe Ser1 5 10327PRTArtificialAntibody CDR 32Gly Ala Ala Asn Leu
Ala Ser1 53312PRTArtificialAntibody CDR 33Gln Asn Asn Tyr Gly Val
His Ile Tyr Gly Ala Ala1 5 103410PRTArtificialAntibody CDR 34Gly
Phe Ser Leu Ser Asn Tyr Asp Met Ile1 5 103516PRTArtificialAntibody
CDR 35Phe Ile Asn Thr Gly Gly Ile Thr Tyr Tyr Ala Ser Trp Ala Lys
Gly1 5 10 153612PRTArtificialAntibody CDR 36Val Asp Asp Tyr Ile Gly
Ala Trp Gly Ala Gly Leu1 5 1037110PRTArtificialAntibody variable
region for anti-TcdA antibody 995 37Asp Val Val Met Thr Gln Ser Pro
Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Gln Ala Ser Gln Ser Ile Asn Asn Tyr 20 25 30Phe Ser Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Gly Ala Ala Asn
Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly 50 55 60Ser Gly Ser Gly
Thr Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Asp Asp
Phe Ala Thr Tyr Ser Cys Gln Asn Asn Tyr Gly Val His Ile 85 90 95Tyr
Gly Ala Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
11038330DNAArtificialPoly nucleotide encoding antibody variable
region for anti-TcdA antibody 995. g1 38gacgtcgtga tgacacagag
cccttcaaca ctgtctgcaa gcgtgggcga tagggtcacc 60ataacgtgcc aggcctctca
atccatcaac aactatttta gctggtacca gcagaagcca 120ggcaaggctc
cgaaacttct gatctacgga gctgccaacc tggcaagtgg cgtgccatca
180cggttcaagg gatccgggag cggtactgag tataccctga ccatttcatc
tctccaaccc 240gacgatttcg ccacctactc ctgccagaat aattacggcg
tgcacatcta tggagctgcc 300tttggcggtg ggacaaaagt ggaaattaag
33039117PRTArtificialAntibody variable region for anti-TcdA
antibody 995 (heavy chain) 39Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Thr Ala
Ser Gly Phe Ser Leu Ser Asn Tyr 20 25 30Asp Met Ile Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Tyr Ile 35 40 45Gly Phe Ile Asn Thr Gly
Gly Ile Thr Tyr Tyr Ala Ser Trp Ala Lys 50 55 60Gly Arg Phe Thr Ile
Ser Arg Asp Ser Ser Thr Val Tyr Leu Gln Met65 70 75 80Asn Ser Leu
Arg Ala Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Val 85 90 95Asp Asp
Tyr Ile Gly Ala Trp Gly Ala Gly Leu Trp Gly Gln Gly Thr 100 105
110Leu Val Thr Val Ser 11540351DNAArtificialPolynucleotide encoding
antibody variable region for anti-TcdA antibody 40gaagttcagc
tggtcgagag tgggggaggg cttgtgcaac ctggtggctc cctccgtctg 60agctgtactg
cttctggatt ctcactgagc aattacgaca tgatctgggt gcgacaggca
120cccggcaaag gactggagta cattggcttc atcaacaccg ggggtataac
gtactatgcc 180tcatgggcta aggggcgctt tacaattagt agggattcct
ctaccgtgta cctgcagatg 240aactcactga gagccgagga cactgccaca
tatttctgcg ctcgggtgga tgactatatc 300ggggcctggg gcgccggatt
gtggggccaa ggaacactgg tcaccgtctc g 3514111PRTArtificialAntibody CDR
41Gln Ala Ser Gln Ser Ile Ser Ser Tyr Leu Ser1 5
10427PRTArtificialAntibody CDR 42Arg Ala Ser Thr Leu Ala Ser1
54313PRTArtificialAntibody CDR 43Leu Gly Val Tyr Gly Tyr Ser Asn
Asp Asp Gly Ile Ala1 5 104410PRTArtificialAntibody CDR 44Gly Ile
Asp Leu Ser Ser His His Met Cys1 5 104516PRTArtificialAntibody CDR
45Val Ile Tyr His Phe Gly Ser Thr Tyr Tyr Ala Asn Trp Ala Thr Gly1
5 10 154611PRTArtificialAntibody CDR 46Ala Ser Ile Ala Gly Tyr Ser
Ala Phe Asp Pro1 5 1047111PRTArtificialAntibody variable region for
anti-TcdA antibody 997 47Ala Leu Val Met Thr Gln Ser Pro Ser Ser
Phe Ser Ala Ser Thr Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Gln Ala
Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Ser Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Arg Ala Ser Thr Leu Ala
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu
Tyr Thr Leu Thr Ile Ser Cys Leu Gln Ser65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Leu Gly Val Tyr Gly Tyr Ser Asn 85 90 95Asp Asp Gly
Ile Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
11048333DNAArtificialPolynucleotide sequence encoding antibody
variable region for anti-TcdA antibody 997.g1 48gcactcgtga
tgacacagag cccgagtagc tttagtgctt caaccggtga tagggtcact 60attacttgcc
aagcctctca gagtatatct agctatctga gctggtacca gcaaaagccc
120gggaaggctc ctaaactgct gatctaccgg gcttccacat tggcctccgg
cgttccctca 180cgctttagcg gctccggatc cggaaccgag tacaccctga
ctatctcttg cctgcaatct 240gaggacttcg caacctacta ttgtctgggc
gtctacggat atagcaacga tgacgggatc 300gccttcggcg gcggtaccaa
agtggaaatt aag 33349116PRTArtificialAntibody variable region for
anti-TcdA antibody 997 (heavy chain) 49Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys
Thr Val Ser Gly Ile Asp Leu Ser Ser His 20 25 30His Met Cys Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile 35 40 45Gly Val Ile Tyr
His Phe Gly Ser Thr Tyr Tyr Ala Asn Trp Ala Thr 50 55 60Gly Arg Phe
Thr Ile Ser Lys Asp Ser Thr Thr Val Tyr Leu Gln Met65 70 75 80Asn
Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Ala 85 90
95Ser Ile Ala Gly Tyr Ser Ala Phe Asp Pro Trp Gly Gln Gly Thr Leu
100 105 110Val Thr Val Ser 11550348DNAArtificialPolynucleotide
sequence encoding antibody variable region for anti-TcdA anitbody
997.g1 (heavy chain) 50gaggtgcaac ttgtggaaag cgggggagga ctggtgcagc
ctgggggctc attgagactg 60agctgcaccg tttctggtat tgacctgagc tcccatcata
tgtgctgggt gcgccaggca 120cccggaaaag
gactggaata catcggcgtc atataccact ttggctctac atactatgcc
180aactgggcaa ctgggcgatt cacaattagc aaggactcaa ctaccgttta
cctgcaaatg 240aatagcctga gggctgagga tactgccacc tatttctgtg
cccgggcttc aatcgccggc 300tattctgcct ttgatccatg ggggcaagga
acactcgtga ccgtctcg 3485111PRTArtificialAntibody CDR 51Gln Ala Ser
Gln Ser Ile Tyr Ser Tyr Leu Ala1 5 10527PRTArtificialAntibody CDR
52Asp Ala Ser Thr Leu Ala Ser1 55313PRTArtificialAntibody CDR 53Gln
Gly Asn Ala Tyr Thr Ser Asn Ser His Asp Asn Ala1 5
105410PRTArtificialAntibody CDR 54Gly Ile Asp Leu Ser Ser Asp Ala
Val Gly1 5 105516PRTArtificialAntibody CDR 55Ile Ile Ala Thr Phe
Asp Ser Thr Tyr Tyr Ala Ser Trp Ala Lys Gly1 5 10
155619PRTArtificialAntibody CDR 56Thr Gly Ser Trp Tyr Tyr Ile Ser
Gly Trp Gly Ser Tyr Tyr Tyr Gly1 5 10 15Met Asp
Leu57111PRTArtificialAntibody variable region for anti-TcdA
antibody 1000 57Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser
Ile Tyr Ser Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile 35 40 45Tyr Asp Ala Ser Thr Leu Ala Ser Gly Val
Pro Ser Arg Phe Lys Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80Asp Asp Phe Ala Thr Tyr Tyr
Cys Gln Gly Asn Ala Tyr Thr Ser Asn 85 90 95Ser His Asp Asn Ala Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
11058333DNAArtificialPolynucleotide encoding antibody variable
region for anti-TcdA antibody 1000.g1 58gaaatcgtga tgacgcagtc
accaagcaca ctgagcgctt ctgtgggaga tcgggtcaca 60ataacctgtc aggcctccca
gagcatctac tcttatctgg catggtacca gcagaagcca 120gggaaagctc
ccaagctgct gatttatgac gccagcactt tggcttccgg tgttcctagt
180aggttcaaag gctccggaag cggtaccgag tttaccctga ccatctcatc
tctgcaaccc 240gatgactttg ccacatacta ttgccagggg aatgcctaca
cttccaactc acacgacaac 300gcattcgggg gaggcaccaa agtcgaaatt aag
33359125PRTArtificialAntibody variable region for anti-TcdA
antibody 1000 (heavy chain) 59Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Ile Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Thr Val
Ser Gly Ile Asp Leu Ser Ser Asp 20 25 30Ala Val Gly Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Tyr Ile 35 40 45Gly Ile Ile Ala Thr Phe
Asp Ser Thr Tyr Tyr Ala Ser Trp Ala Lys 50 55 60Gly Arg Phe Thr Ile
Ser Lys Ala Ser Ser Thr Thr Val Tyr Leu Gln65 70 75 80Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg 85 90 95Thr Gly
Ser Trp Tyr Tyr Ile Ser Gly Trp Gly Ser Tyr Tyr Tyr Gly 100 105
110Met Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120
12560375DNAArtificialPolynucleotide encoding antibody variable
region for anti-TcdA antibody 1000.g1 (heavy chain) 60gaagttcagc
tggtcgagag cggagggggt ttgattcagc ccggtggctc acttagattg 60agctgcaccg
tgtccggaat cgatctgtca tctgatgccg tgggctgggt gcgacaggca
120cctgggaaag gactggagta tatagggatc atcgccacct tcgactccac
atactacgct 180agctgggcaa aagggcgctt tacgattagc aaggcctcct
ctactaccgt gtacctccaa 240atgaactcac tgagggccga ggacactgcc
acttatttct gtgctcggac cggtagctgg 300tactacatct ctggctgggg
ctcctactat tatggcatgg acctgtgggg acaggggaca 360ctcgtgaccg tctcg
3756111PRTArtificialAntibody CDR 61Arg Ala Ser Lys Ser Val Ser Thr
Leu Met His1 5 10627PRTArtificialAntibody CDR 62Leu Ala Ser Asn Leu
Glu Ser1 5639PRTArtificialAntibody CDR 63Gln Gln Thr Trp Asn Asp
Pro Trp Thr1 56410PRTArtificialAntibody CDR 64Gly Phe Thr Phe Ser
Asn Tyr Gly Met Ala1 5 106517PRTArtificialAntibody CDR 65Ser Ile
Ser Ser Ser Gly Gly Ser Thr Tyr Tyr Arg Asp Ser Val Lys1 5 10
15Gly669PRTArtificialAntibody CDR 66Val Ile Arg Gly Tyr Val Met Asp
Ala1 567107PRTArtificialAntibody variable region for anti-TcdB
antibody 926 67Asp Thr Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu
Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Ser
Val Ser Thr Leu 20 25 30Met His Trp Phe Gln Gln Lys Pro Gly Gln Ala
Pro Lys Leu Leu Ile 35 40 45Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val
Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr
Cys Gln Gln Thr Trp Asn Asp Pro Trp 85 90 95Thr Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys 100 10568321DNAArtificialPolynucleotide
encoding antibody variable region for anti-TcdB antibody 926.g1
68gataccgtgc tgacccagag ccctgctaca ttgtcactga gccccgggga gagggccaca
60ttgagctgcc gggcttcaaa atccgtgtcc accctcatgc actggtttca gcaaaagccc
120gggcaggccc caaaactgct gatctacctc gcatctaacc ttgaatctgg
cgtgccggcc 180cgctttagtg gctccggaag cggaaccgac ttcacactga
cgattagctc cctggagcct 240gaggatttcg ccgtgtacta ttgccagcaa
acttggaatg acccttggac tttcgggggc 300ggtactaagg tcgaaataaa g
32169117PRTArtificialAntibody variable region for anti-TcdB
antibody 926 (heavy chain) 69Glu Val Glu Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Glu Ala
Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30Gly Met Ala Trp Val Arg Gln
Ala Pro Thr Lys Gly Leu Glu Trp Val 35 40 45Thr Ser Ile Ser Ser Ser
Gly Gly Ser Thr Tyr Tyr Arg Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Ser Ser Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95Thr Thr
Val Ile Arg Gly Tyr Val Met Asp Ala Trp Gly Gln Gly Thr 100 105
110Leu Val Thr Val Ser 11570351DNAArtificialPolynucleotide encoding
antibody variable region for anti-TcdBantibody 926.g1 (heavy chain)
70gaggtggaac tgctcgaatc tggtggtggg ctggtgcagc ccggtggatc tctgagattg
60tcatgcgagg catccggctt taccttttcc aactacggaa tggcctgggt gagacaggcc
120ccaacgaagg ggctcgaatg ggttacaagc atcagctctt ctgggggatc
tacttactat 180cgcgatagcg tcaaaggccg gtttaccatt agccgagata
atgccaaatc aagcctgtat 240ctgcaaatga acagcctgag ggctgaggac
accgccacat actattgtac aaccgtgata 300aggggctacg tgatggacgc
atggggacag gggacattgg ttaccgtctc g 3517111PRTArtificialAntibody CDR
71Arg Ala Ser Gly Ser Val Ser Thr Leu Met His1 5
10727PRTArtificialAntibody CDR 72Lys Ala Ser Asn Leu Ala Ser1
5738PRTArtificialAntibody CDR 73His Gln Ser Trp Asn Ser Asp Thr1
57410PRTArtificialAntibody CDR 74Gly Phe Thr Phe Ser Asn Tyr Gly
Met Ala1 5 107517PRTArtificialAntibody CDR 75Thr Ile Asn Tyr Asp
Gly Arg Thr Thr His Tyr Arg Asp Ser Val Lys1 5 10
15Gly769PRTArtificialAntibody CDR 76Ile Ser Arg Ser His Tyr Phe Asp
Cys1 577106PRTArtificialAntibody variable region for anti-TcdB
antibody 927 77Asp Thr Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gly Ser
Val Ser Thr Leu 20 25 30Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile 35 40 45Tyr Lys Ala Ser Asn Leu Ala Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80Asp Asp Phe Ala Thr Tyr Tyr
Cys His Gln Ser Trp Asn Ser Asp Thr 85 90 95Phe Gly Gln Gly Thr Arg
Leu Glu Ile Lys 100 10578318DNAArtificialPolynucleotide sequence
encoding antbody variable region for anti-TcdB antibody 927.g2
78gacacacaga tgacccagag cccatccact ttgtctgcat ccgtgggcga ccgagtgaca
60atcacctgta gagcaagcgg ttccgtgagc acactgatgc attggtacca gcagaagcct
120gggaaggctc ccaagctgct gatctacaaa gccagcaacc ttgcctccgg
cgttccaagc 180cggtttagcg gttccggatc tggaaccgag ttcaccctga
ccatatcaag cctgcaaccc 240gacgacttcg ccacctacta ttgccaccag
agctggaata gcgacacgtt cgggcaaggc 300acaaggctgg aaatcaaa
31879117PRTArtificialAntibody variable region for anti-TcdB
antibody 927 (heavy chain) 79Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30Gly Met Ala Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Thr Ile Asn Tyr Asp
Gly Arg Thr Thr His Tyr Arg Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Ser Thr Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Ser
Ile Ser Arg Ser His Tyr Phe Asp Cys Trp Gly Gln Gly Thr 100 105
110Leu Val Thr Val Ser 11580351DNAArtificialPolynucleotide encoding
antibody variable region for anti-TcdB antibody 927.g2 (heavy
chain) 80gaggtgcaac ttgtggaaag cggagggggc gtggtccaac ccggaagaag
tctccgtctt 60tcttgcgccg caagtggctt caccttttcc aactacggaa tggcctgggt
tcgacaagct 120cctgggaaag gattggagtg ggtggccact atcaactatg
acggacgcac gacacactac 180cgagactctg ttaaggggcg ctttacgatt
tcccgcgaca atagcaagag caccctctac 240ctgcaaatga atagcctccg
ggccgaggat actgctgtgt actattgtac ctccatctca 300cggagccact
acttcgattg ctggggacaa ggcacactcg tgactgtctc g
3518111PRTArtificialAntibody CDR 81Lys Ala Ser Lys Ser Ile Ser Asn
His Leu Ala1 5 10827PRTArtificialAntibody CDR 82Ser Gly Ser Thr Leu
Gln Ser1 5839PRTArtificialAntibody CDR 83Gln Gln Tyr Asp Glu Tyr
Pro Tyr Thr1 58410PRTArtificialAntibody CDR 84Gly Phe Ser Leu Gln
Ser Tyr Thr Ile Ser1 5 108516PRTArtificialAntibody CDR 85Ala Ile
Ser Gly Gly Gly Ser Thr Tyr Tyr Asn Leu Pro Leu Lys Ser1 5 10
158611PRTArtificialAntibody CDR 86Pro Arg Trp Tyr Pro Arg Ser Tyr
Phe Asp Tyr1 5 1087109PRTArtificialAntibody variable region for
anti-TcdB antibody 1099 87Asp Val Gln Leu Thr Gln Ser Pro Ser Phe
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala
Ser Lys Ser Ile Ser Asn His 20 25 30Leu Ala Trp Tyr Gln Glu Lys Pro
Gly Lys Ala Asn Lys Leu Leu Ile 35 40 45His Ser Gly Ser Thr Leu Gln
Ser Gly Thr Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Tyr Asp Glu Tyr Pro Tyr 85 90 95Thr Phe Gly
Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr 100
10588327DNAArtificialPolynucleotide encoding antibody variable
region for anti-TcdB antibody 1099.g2 88gacgtccagc tcactcaatc
tccctccttt ctgtctgctt ctgtgggcga tcgcgtgaca 60ataacctgca aggcctccaa
atcaattagc aaccatctgg catggtatca ggagaagcct 120ggcaaagcca
ataagctgct gatccactcc ggctcaactc tgcaatccgg taccccaagc
180cgatttagcg gatctgggag cggaaccgag ttcacactta ccattagctc
cctgcaaccg 240gaggacttcg ccacctatta ctgccagcaa tacgacgaat
acccctatac gttcggccaa 300gggacaagat tggaaatcaa gcgtacg
32789118PRTArtificialAntibody variable region for anti-TcdB
antibody 1099 (heavy chain) 89Glu Val Gln Leu Gln Glu Ser Gly Pro
Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val
Ser Gly Phe Ser Leu Gln Ser Tyr 20 25 30Thr Ile Ser Trp Val Arg Gln
Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Ala Ala Ile Ser Gly Gly
Gly Ser Thr Tyr Tyr Asn Leu Pro Leu Lys 50 55 60Ser Arg Val Thr Ile
Ser Arg Asp Thr Ser Lys Ser Gln Val Ser Leu65 70 75 80Lys Leu Ser
Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Thr 85 90 95Arg Pro
Arg Trp Tyr Pro Arg Ser Tyr Phe Asp Tyr Trp Gly Arg Gly 100 105
110Thr Leu Val Thr Val Ser 11590354DNAArtificialPolynucleotide
encoding antibody variable region for anti-TcdB antibody 1099.g2
(heavh chain) 90gaagttcagc tgcaggaatc tggacctggc ttggtgaaac
caagcgagac acttagtctc 60acttgcaccg tttccggctt ctcccttcaa tcctacacga
tctcttgggt gcggcaacca 120cccgggaaag gactggaatg gatcgcagcc
attagcgggg gagggagcac ctattacaac 180ttgcctctca agagccgcgt
gaccatatcc cgtgacacaa gcaagagcca ggtttccctg 240aagctgagct
ccgtgactgc tgccgatacg gctgtttact attgcacccg acctcgctgg
300tatccccgtt cctatttcga ctactgggga agaggcacac tggttaccgt ctcg
3549111PRTArtificialAntibody CDR 91Arg Ala Ser Gln Arg Ile Ser Thr
Ser Ile His1 5 10927PRTArtificialAntibody CDR 92Tyr Ala Ser Gln Ser
Ile Ser1 5939PRTArtificialAntibody CDR 93Gln Gln Ser Tyr Ser Ser
Leu Tyr Thr1 59410PRTArtificialAntibody CDRs 94Gly Phe Thr Phe Ser
Asp Ser Tyr Met Ala1 5 109517PRTArtificialAntibody CDR 95Ser Ile
Ser Tyr Gly Gly Thr Ile Ile Gln Tyr Gly Asp Ser Val Lys1 5 10
15Gly9611PRTArtificialAntibody CDR 96Arg Gln Gly Thr Tyr Ala Arg
Tyr Leu Asp Phe1 5 1097107PRTArtificialAntibody variable region for
anti-TcdB antibody 1102 97Asn Ile Val Leu Thr Gln Ser Pro Ala Thr
Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Arg Ile Ser Thr Ser 20 25 30Ile His Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Lys Tyr Ala Ser Gln Ser Ile
Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75 80Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Ser Tyr Ser Ser Leu Tyr 85 90 95Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys 100
10598321DNAArtificialPolynucleotide encoding antibody variable
region for anti-TcdB antibody 1102.g4 98aacatcgtgc tgacacagtc
tcctgcaacc ctttcactgt ctccaggtga acgagcaacc 60ctgagttgta gagccagtca
gaggatctcc acgagcattc actggtatca gcaaaagcct 120gggcaagctc
ccagactctt gatcaagtac gcctctcaga gcataagtgg cattccagct
180aggtttagcg gctcaggctc aggaacagac ttcactctga ccatcagctc
cctggaaccg 240gaggactttg ccgtctatta ctgccagcaa tcctactcca
gtctgtacac cttcgggcag 300ggtactaaac tggagataaa g
32199119PRTArtificialAntibody variabl region for anti-TcdB antibody
1102 (heavy chain) 99Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Val Ser Gly
Phe Thr Phe Ser Asp Ser 20 25 30Tyr Met Ala Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Ile 35 40 45Ala Ser Ile Ser Tyr Gly Gly Thr
Ile Ile Gln Tyr Gly Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Ser Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Gln
Gly Thr Tyr Ala Arg Tyr Leu Asp Phe Trp Gly Gln 100 105 110Gly Thr
Leu Val Thr Val Ser 115100357DNAArtificialPolynucleotide encoding
antibody variable region for anti-TcdB antibody 1002.g4 (heavy
chain) 100gaagtgcagc
tggtcgaatc cgggggaggt ttggtgcaac caggtggctc actgagactg 60agctgtgccg
tttccggctt tacgttctca gacagttata tggcctgggt gcgtcaagca
120cctggaaaag ggctggagtg gattgccagt atcagctatg gtgggaccat
aatccagtac 180ggcgatagcg tcaagggcag gtttactatc tccagggaca
acgccaagtc aagcctttac 240ctgcagatga attctctccg cgcagaggat
accgctgtgt attactgcgc tagacggcag 300ggaacctacg ctcgatacct
ggacttctgg ggtcagggaa cactcgttac agtctcg
35710111PRTArtificialAntibody CDR 101Arg Ala Ser Glu Ser Val Ser
Thr Leu Leu His1 5 101027PRTArtificialAntibody CDR 102Lys Ala Ser
Asn Leu Ala Ser1 51039PRTArtificialAntibody CDR 103His Gln Ser Trp
Asn Ser Pro Pro Thr1 510410PRTArtificialAnitbody CDR 104Gly Phe Thr
Phe Ser Asn Tyr Gly Met Ala1 5 1010517PRTArtificialAntibody CDR
105Ile Ile Asn Tyr Asp Ala Ser Thr Thr His Tyr Arg Asp Ser Val Lys1
5 10 15Gly1069PRTArtificialAntibody CDR 106Tyr Gly Arg Ser His Tyr
Phe Asp Tyr1 5107107PRTArtificialAntibody variable region for
anti-TcdB anitbody 1114 107Ala Thr Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Glu Ser Val Ser Thr Leu 20 25 30Leu His Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Lys Ala Ser Asn Leu Ala
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys His Gln Ser Trp Asn Ser Pro Pro 85 90 95Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys 100
105108321DNAArtificialPolynucleotide sequence encoding antibody
variable region for anti-TcdB antibody 1114.g2 108gcgacgcaaa
tgactcagtc gccctcatcg cttagcgcgt ccgtcggaga tagagtgacg 60atcacctgcc
gcgcatcaga gtcggtgtcc acactcctcc actggtatca gcagaaaccg
120gggaaggcac caaaactctt gatctacaaa gccagcaacc ttgcgtccgg
tgtcccgtca 180aggttctccg ggagcggttc ggggacagac tttactttga
ccatttcgtc gcttcagccg 240gaggacttcg ccacctatta ctgtcatcag
tcatggaact cacctcccac atttggccag 300ggaacgaaac tcgaaatcaa g
321109117PRTArtificialAntibody variable region for anti-TcdB
antibody 1114 (heavy chain) 109Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30Gly Met Ala Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ile Ile Asn Tyr Asp
Ala Ser Thr Thr His Tyr Arg Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Ser Ser Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Arg
Tyr Gly Arg Ser His Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105
110Leu Val Thr Val Ser 115110351DNAArtificialPolynucleotide
encoding anitbody variable region for anti-TcdB antibody 1114.g2
(heavy chain) 110gaagtacaac tcgtagagtc agggggtggg ctggtccaac
ctggcggctc ccttcggctt 60tcgtgtgccg cctcgggatt cacgtttagc aattacggta
tggcctgggt gaggcaggca 120ccagggaagg gtcttgagtg ggtagcgatc
atcaactatg atgcaagcac cacccactac 180agggatagcg tcaagggacg
ctttactatc agccgggata atgcgaaatc ctcgctctat 240ctgcagatga
actccctcag agccgaggac accgcagtgt actattgcac acgatacgga
300cgctcgcact atttcgacta ttggggacag gggacgctcg taactgtctc g
35111111PRTArtificialAntibody CDR 111Arg Ala Ser Glu Ser Val Ser
Thr Leu Leu His1 5 101127PRTArtificialAnitbody CDR 112Lys Ala Ser
Asn Leu Ala Ser1 51139PRTArtificialAntibody CDR 113His Gln Ser Trp
Asn Ser Pro Pro Thr1 511410PRTArtificialAntibody CDR 114Gly Phe Thr
Phe Ser Asn Tyr Gly Met Ala1 5 1011516PRTArtificialAntibody CDR
115Ile Ile Asn Tyr Asp Ala Ser Thr Thr His Tyr Arg Asp Ser Val Lys1
5 10 151169PRTArtificialAntibody CDR 116Tyr Gly Arg Ser His Tyr Phe
Asp Tyr1 5117107PRTArtificialAntibody variable region for anti-TcdB
antibody 1114 graft 8 117Asp Thr Val Leu Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Glu Ser Val Ser Thr Leu 20 25 30Leu His Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Lys Ala Ser Asn Leu Ala
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys His Gln Ser Trp Asn Ser Pro Pro 85 90 95Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys 100
105118321DNAArtificialPolynucleotide encoding antibody variable
region for anti-TcdB antibody 1114.g8 118gacacggtcc tgactcagtc
gccctcatcg cttagcgcgt ccgtcggaga tagagtgacg 60atcacctgcc gcgcatcaga
gtcggtgtcc acactcctcc actggtatca gcagaaaccg 120gggaaggcac
caaaactctt gatctacaaa gccagcaacc ttgcgtccgg tgtcccgtca
180aggttctccg ggagcggttc ggggacagac tttactttga ccatttcgtc
gcttcagccg 240gaggacttcg ccacctatta ctgtcatcag tcatggaact
cacctcccac atttggccag 300ggaacgaaac tcgaaatcaa g
321119117PRTArtificialAntibody variable region for anti-TcdB
antibody 1114 graft 8 (heavy chain) 119Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30Gly Met Ala Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ile Ile Asn
Tyr Asp Ala Ser Thr Thr His Tyr Arg Asp Ser Val 50 55 60Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Ser Leu Tyr65 70 75 80Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Thr Arg Tyr Gly Arg Ser His Tyr Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110Leu Val Thr Val Ser 115120351DNAArtificialPolynucleotide
encoding antibody variable region for anti-TcdB antibody 1114.g8
120gaagtacaac tcgtagagtc agggggtggg ctggtccaac ctggcggctc
ccttcggctt 60tcgtgtgccg cctcgggatt cacgtttagc aattacggta tggcctgggt
gaggcaggca 120ccagggaagg gtcttgagtg ggtagcgatc atcaactatg
atgcaagcac cacccactac 180agggatagcg tcaagggacg ctttactatc
agccgggata atgcgaaatc ctcgctctat 240ctgcagatga actccctcag
agccgaggac accgcagtgt actattgcac acgatacgga 300cgctcgcact
atttcgacta ttggggacag gggacgctcg taactgtctc g
35112111PRTArtificialAntibody CDR 121Lys Ala Ser Gln Asn Ile Tyr
Met Tyr Leu Asn1 5 101227PRTArtificialAntibody CDR 122Asn Thr Asn
Lys Leu His Thr1 51239PRTArtificialAntibody CDR 123Leu Gln His Lys
Ser Phe Pro Tyr Thr1 512410PRTArtificialAntibody CDR 124Gly Phe Thr
Phe Arg Asp Ser Phe Met Ala1 5 1012517PRTArtificialAntibody CDR
125Ser Ile Ser Tyr Glu Gly Asp Lys Thr Tyr Tyr Gly Asp Ser Val Lys1
5 10 15Gly1269PRTArtificialAntibody CDR 126Leu Thr Ile Thr Thr Ser
Gly Asp Ser1 5127107PRTArtificialAntibody variable region for
anti-TcdB antibody 1125 127Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala
Ser Gln Asn Ile Tyr Met Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45Tyr Asn Thr Asn Lys Leu His
Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu
Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Leu Gln His Lys Ser Phe Pro Tyr 85 90 95Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys 100
105128321DNAArtificialPolynucleotide sequence encoding antibody
variable region for anti-TcdB antibody 1125.g2 128gatatacaaa
tgactcagag ccctagctca ctgagcgctt ctgtgggcga tcgtgtgaca 60atcacttgca
aagcaagcca gaacatctat atgtacctga attggtacca gcaaaaaccg
120ggaaaagctc ccaagcgcct gatttacaac accaataagc tgcataccgg
cgtgccaagc 180cgttttagcg gatctggctc tggaaccgaa tatacactga
ccataagctc cctgcaaccg 240gaagactttg caacttacta ttgcctccag
cacaaatcct tcccctatac gttcggacaa 300gggaccaaac tggaaatcaa a
321129118PRTArtificialAntibody variable region for anti-TcdB
antibody 1125 (heavy chain) 129Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Arg Asp Ser 20 25 30Phe Met Ala Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Ile Ser Tyr Glu
Gly Asp Lys Thr Tyr Tyr Gly Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Leu Thr Ile Thr Thr Ser Gly Asp Ser Trp Gly Gln Gly Thr 100 105
110Met Val Thr Val Ser Ser 115130354DNAArtificialPolynucleotide
encoding antibody variable region for anti-TcdB antibody 1125.g2
(heavy chain) 130gaagtgcagc tggtcgaaag cggcggagga ttggtgcaac
ctggtggctc tcttcgcctg 60tcttgcgctg caagcggctt tacgttccgc gatagcttta
tggcttgggt gcgacaagct 120cctgggaaag ggctggaatg ggtcgctagc
ataagctacg aaggcgacaa gacttactat 180ggggactctg tgaaaggccg
attcaccatt agccgagaca acgcaaagaa ctccctgtac 240ctgcagatga
actccctgcg tgccgaagat accgccgtgt actattgcgc taggctgacg
300atcactacaa gcggagatag ctggggacaa gggacaatgg tgaccgtctc gagc
35413111PRTArtificialAntibody CDR 131Lys Ala Ser Gln His Val Gly
Thr Asn Val Asp1 5 101327PRTArtificialAntibody CDR 132Gly Ala Ser
Ile Arg Tyr Thr1 51339PRTArtificialAntibody CDR 133Leu Gln Tyr Asn
Tyr Asn Pro Tyr Thr1 513410PRTArtificialAntibody CDR 134Gly Phe Ile
Phe Ser Asn Phe Gly Met Ser1 5 1013517PRTArtificialAntibody CDR
135Ser Ile Ser Pro Ser Gly Gly Asn Ala Tyr Tyr Arg Asp Ser Val Lys1
5 10 15Gly1369PRTArtificialAntibody CDR 136Arg Ala Tyr Ser Ser Pro
Phe Ala Phe1 5137107PRTArtificialAntibody variable region for
anti-TcdB antibody 1129 137Asp Thr Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala
Ser Gln His Val Gly Thr Asn 20 25 30Val Asp Trp Tyr Gln Gln Lys Pro
Gly Lys Val Pro Lys Leu Leu Ile 35 40 45Tyr Gly Ala Ser Ile Arg Tyr
Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Val Ala
Thr Tyr Tyr Cys Leu Gln Tyr Asn Tyr Asn Pro Tyr 85 90 95Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys 100
105138321DNAArtificialPolynucleotide sequence encoding antibody
variable region for anti-TcdB antibody 1129.g1 138gacacccaga
tgactcagtc tccgtcaagc ctttctgcct ctgttggaga tcgagtcaca 60attacgtgca
aggcaagcca acacgtgggt accaacgtgg actggtatca acagaagcca
120gggaaggtcc ccaaactgct gatctacggt gccagtattc gctataccgg
cgtgcctgat 180cgcttcaccg gaagcgggtc agggaccgat ttcacactga
caatcagctc cctgcaacct 240gaagacgtgg ctacttacta ctgcctgcag
tacaactata atccctacac ctttggccag 300ggcaccaaac tggagataaa g
321139118PRTArtificialAntibody variable region for anti-TcdB
antibody 1129 (heavy chain) 139Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Thr
Ser Gly Phe Ile Phe Ser Asn Phe 20 25 30Gly Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Ile Ser Pro Ser
Gly Gly Asn Ala Tyr Tyr Arg Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Thr Thr Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Arg
Arg Ala Tyr Ser Ser Pro Phe Ala Phe Trp Gly Gln Gly Thr 100 105
110Leu Val Thr Val Ser Ser 115140354DNAArtificialPolynucleotide
sequence encoding antibody variable region for anti-TcdB antibody
1129.g1(heavy chain) 140gaggtgcaac ttgtggaatc aggaggtggc gtggttcagc
ccggtagatc acttcgtctg 60agttgtgcaa caagcggctt tatcttctcc aacttcggga
tgtcttgggt tagacaggct 120cctggtaagg gcctcgaatg ggtggctagt
attagcccaa gcgggggaaa cgcctactat 180agggacagcg tgaaaggacg
cttcactatc agccgagata actccaagac cacgctgtat 240ctgcagatga
atagtctgag ggccgaggat accgcagtgt actactgcac tcgacgggcc
300tattcttccc cttttgcctt ttggggacag gggactctgg tgacagtctc gagc
35414111PRTArtificialAntibody CDR 141Lys Ala Ser Lys Ser Ile Ser
Asn His Leu Ala1 5 101427PRTArtificialAntibody CDR 142Ser Gly Ser
Thr Leu Gln Pro1 51439PRTArtificialAntibody CDR 143Gln Gln Tyr Asp
Glu Tyr Pro Tyr Thr1 514410PRTArtificialAntibody CDR 144Gly Phe Ser
Leu Asn Ser Tyr Thr Ile Thr1 5 1014516PRTArtificialAntibody CDR
145Ala Ile Ser Gly Gly Gly Ser Thr Tyr Phe Asn Ser Ala Leu Lys Ser1
5 10 1514611PRTArtificialAntibody CDR 146Pro Arg Trp Tyr Pro Arg
Ser Tyr Phe Asp Tyr1 5 10147107PRTArtificialAntibody variable
region for anti-TcdB antibody 1134 147Asp Val Gln Leu Thr Gln Ser
Pro Ser Phe Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Lys Ala Ser Lys Ser Ile Ser Asn His 20 25 30Leu Ala Trp Tyr Gln
Glu Lys Pro Gly Lys Ala Asn Lys Leu Leu Ile 35 40 45His Ser Gly Ser
Thr Leu Gln Pro Gly Thr Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Glu Tyr Pro Tyr 85 90
95Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100
105148321DNAArtificialPolynucleotide encoding antibody variable
region for anti-TcdB antibody 1134.g5 148gacgtccagc tcactcaatc
tccctccttt ctgtctgctt ctgtgggcga tcgcgtgaca 60ataacctgca aggcctccaa
atcaattagc aaccatctgg catggtatca ggagaagcct 120ggcaaagcca
ataagctgct gatccactcc ggctcaactc tgcaacccgg taccccaagc
180cgatttagcg gatctgggag cggaaccgag ttcacactta ccattagctc
cctgcaaccg 240gaggacttcg ccacctatta ctgccagcaa tacgacgaat
acccctatac gttcggccaa 300gggacaagat tggaaatcaa g
321149118PRTArtificialAntibody variable region for anti-TcdB
antibody 1134 (heavy chain) 149Glu Val Gln Leu Gln Glu Ser Gly Pro
Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val
Ser Gly Phe Ser Leu Asn Ser Tyr 20 25 30Thr Ile Thr Trp Val Arg Gln
Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Ala Ala Ile Ser Gly Gly
Gly Ser Thr Tyr Phe Asn Ser Ala Leu Lys 50 55 60Ser Arg Val Thr Ile
Ser Arg Asp Thr Ser Lys Ser Gln Val Ser Leu65 70 75 80Lys Leu Ser
Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Thr 85 90 95Arg Pro
Arg Trp Tyr Pro Arg Ser Tyr Phe Asp Tyr Trp Gly Arg Gly 100 105
110Thr Leu Val Thr Val Ser 115150354DNAArtificialPolynucleotide
encoding antibody variable region for anti-TcdB antibody 1134.g5
(heavy chain) 150gaagttcagc tgcaggaatc tggacctggc ttggtgaaac
caagcgagac
acttagtctc 60acttgcaccg tttccggctt ctcccttaat tcctacacga tcacttgggt
gcggcaacca 120cccgggaaag gactggaatg gatcgcagcc attagcgggg
gagggagcac ctatttcaac 180tcggctctca agagccgcgt gaccatatcc
cgtgacacaa gcaagagcca ggtttccctg 240aagctgagct ccgtgactgc
tgccgatacg gctgtttact attgcacccg acctcgctgg 300tatccccgtt
cctatttcga ctactgggga agaggcacac tggttaccgt ctcg
35415111PRTArtificialAntibody CDR 151Lys Ala Ser Gln Asn Val Gly
Asn Asn Val Ala1 5 101527PRTArtificialAntibody CDR 152Tyr Ala Ser
Asn Arg Phe Thr1 51539PRTArtificialAntibody CDR 153Gln Arg Val Tyr
Gln Ser Thr Trp Thr1 515410PRTArtificialAntibody CDR 154Gly Phe Ser
Leu Thr Ser Tyr Tyr Val His1 5 1015516PRTArtificialAntibody CDR
155Cys Ile Arg Thr Gly Gly Asn Thr Glu Tyr Gln Ser Glu Phe Lys Ser1
5 10 151567PRTArtificialAntibody CDR 156Gly Asn Tyr Gly Phe Ala
Tyr1 5157107PRTArtificialAntibody variable region for anti-TcdB
antibody 1151 157Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln
Asn Val Gly Asn Asn 20 25 30Val Ala Trp Tyr Gln His Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45Tyr Tyr Ala Ser Asn Arg Phe Thr Gly
Val Pro Ser Arg Phe Thr Gly 50 55 60Gly Gly Tyr Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Arg Val Tyr Gln Ser Thr Trp 85 90 95Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 105158321DNAArtificialPolynucleotide
encoding antibody variable region for anti-TcdB antibody 1151.g1
158gcgattcaaa tgactcagtc gccctcatcg cttagcgcgt ccgtcggaga
tagagtgacg 60atcacgtgca aagcatcaca aaatgtcggg aacaatgtgg catggtatca
gcataaaccg 120gggaaggcac caaaactctt gatctactac gccagcaaca
ggtttactgg tgtcccgtca 180aggttcacgg gagggggtta cgggacagac
tttactttga ccatttcgtc gcttcagccg 240gaggacttcg ccacctatta
ctgtcagagg gtctaccagt caacgtggac atttggccag 300ggaacgaaag
tggaaatcaa g 321159114PRTArtificialAntibody variable region for
anti-TcdB antibody 1151 (heavy chain) 159Glu Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr
Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30Tyr Val His Trp
Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Cys Ile
Arg Thr Gly Gly Asn Thr Glu Tyr Gln Ser Glu Phe Lys 50 55 60Ser Arg
Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu65 70 75
80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Arg Gly Asn Tyr Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val
Thr 100 105 110Val Ser160342DNAArtificialPolynucleotide sequence
encoding antibody variable region for anti-TcdB antibody 1151.g4
(heavy chain) 160gaagtacaac tccaagagtc ggggcctggt ctggtcaagc
cgtccgaaac actttcgctg 60acgtgtacgg tatcaggatt ctcacttaca tcatactacg
tccactgggt gaggcagcca 120cccgggaagg gtcttgagtg gatgggctgc
attagaaccg gagggaatac cgagtaccag 180agcgaattta agagccgcgt
cactatcagc cgggatacgt ccaaaaacca ggtgtcgctc 240aaattgtcct
ccgtgacggc cgctgacacc gcagtgtact attgcgcgcg aggaaactat
300ggctttgcgt attggggaca ggggacgctc gtaactgtct cg
34216111PRTArtificialAntibody CDR 161Lys Ala Ser Gln Asn Ile Asn
Lys Tyr Leu Asp1 5 101627PRTArtificialAntibody CDR 162Asn Ile Gln
Ser Leu His Thr1 51637PRTArtificialAntibody CDR 163Phe Gln His Asn
Ser Gly Trp1 516410PRTArtificialAntibody CDR 164Gly Phe Thr Phe Thr
Gln Ala Ala Met Phe1 5 1016519PRTArtificialAntibody CDR 165Arg Ile
Ser Thr Lys Ser Asn Asn Phe Ala Thr Tyr Tyr Pro Asp Ser1 5 10 15Val
Lys Gly16613PRTArtificialAntibody CDR 166Pro Ala Tyr Tyr Tyr Asp
Gly Thr Val Pro Phe Ala Tyr1 5 10167106PRTArtificialAntibody
variable region for anti-TcdB antibody 1153.g8 167Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Lys Ala Ser Gln Asn Ile Asn Lys Tyr 20 25 30Leu Asp
Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45Tyr
Asn Ile Gln Ser Leu His Thr Gly Ile Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Val Ala Thr Tyr Tyr Cys Phe Gln His Asn Ser Gly Trp
Thr 85 90 95Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100
105168318DNAArtificialPolynucleotide sequence encoding antibody
variable region for anti-TcdB antibody 1153.g8 168gatatacaga
tgactcagtc cccttctagc ctttcagctt ccgtgggcga tagagtgact 60atcacgtgta
aggctagtca gaacattaac aagtatctgg actggtacca gcagaaaccc
120gggaaggttc ccaagctgct gatctacaac atccagtccc tgcatacagg
cattcctagc 180cggtttagcg gatctggttc agggaccgac ttcaccctga
caatcagctc tctgcaacca 240gaagacgtgg ccacctatta ctgcttccag
cacaatagtg gctggacttt tggacaaggt 300accaggctgg agatcaaa
318169123PRTArtificialAntibody variable region for anti-TcdB
antibody 1153 (graft 8 heavy chain) 169Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Thr Gln Ala 20 25 30Ala Met Phe Trp Val
Arg Gln Ala Ser Gly Lys Gly Leu Glu Gly Ile 35 40 45Ala Arg Ile Ser
Thr Lys Ser Asn Asn Phe Ala Thr Tyr Tyr Pro Asp 50 55 60Ser Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr65 70 75 80Val
Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90
95Tyr Cys Thr Ala Pro Ala Tyr Tyr Tyr Asp Gly Thr Val Pro Phe Ala
100 105 110Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115
120170369DNAArtificialPolynucleotide sequence encoding antibody
variable region for anti-TcdB antibody 1153.g8 (heavy chain)
170gaggttcagc tggtggaatc aggagggggt ctggtgcaac caggaggctc
cctgaaactg 60tcttgcgccg caagcggctt tacgtttacc caggccgcta tgttctgggt
taggcaggcc 120agtgggaagg gtcttgaagg catcgcaaga atcagcacca
agagcaacaa tttcgctacg 180tactatccgg actccgtgaa aggccggttt
accatttctc gcgatgacag caagaacacc 240gtgtacctgc agatgaacag
tctcaagacc gaggacacag ccgtgtacta ttgtactgct 300cccgcctatt
attacgatgg cacagtgcct ttcgcatact ggggacaggg tactttggtg 360actgtctcg
3691712710PRTClostridia 171Met Ser Leu Ile Ser Lys Glu Glu Leu Ile
Lys Leu Ala Tyr Ser Ile1 5 10 15Arg Pro Arg Glu Asn Glu Tyr Lys Thr
Ile Leu Thr Asn Leu Asp Glu 20 25 30Tyr Asn Lys Leu Thr Thr Asn Asn
Asn Glu Asn Lys Tyr Leu Gln Leu 35 40 45Lys Lys Leu Asn Glu Ser Ile
Asp Val Phe Met Asn Lys Tyr Lys Thr 50 55 60Ser Ser Arg Asn Arg Ala
Leu Ser Asn Leu Lys Lys Asp Ile Leu Lys65 70 75 80Glu Val Ile Leu
Ile Lys Asn Ser Asn Thr Ser Pro Val Glu Lys Asn 85 90 95Leu His Phe
Val Trp Ile Gly Gly Glu Val Ser Asp Ile Ala Leu Glu 100 105 110Tyr
Ile Lys Gln Trp Ala Asp Ile Asn Ala Glu Tyr Asn Ile Lys Leu 115 120
125Trp Tyr Asp Ser Glu Ala Phe Leu Val Asn Thr Leu Lys Lys Ala Ile
130 135 140Val Glu Ser Ser Thr Thr Glu Ala Leu Gln Leu Leu Glu Glu
Glu Ile145 150 155 160Gln Asn Pro Gln Phe Asp Asn Met Lys Phe Tyr
Lys Lys Arg Met Glu 165 170 175Phe Ile Tyr Asp Arg Gln Lys Arg Phe
Ile Asn Tyr Tyr Lys Ser Gln 180 185 190Ile Asn Lys Pro Thr Val Pro
Thr Ile Asp Asp Ile Ile Lys Ser His 195 200 205Leu Val Ser Glu Tyr
Asn Arg Asp Glu Thr Val Leu Glu Ser Tyr Arg 210 215 220Thr Asn Ser
Leu Arg Lys Ile Asn Ser Asn His Gly Ile Asp Ile Arg225 230 235
240Ala Asn Ser Leu Phe Thr Glu Gln Glu Leu Leu Asn Ile Tyr Ser Gln
245 250 255Glu Leu Leu Asn Arg Gly Asn Leu Ala Ala Ala Ser Asp Ile
Val Arg 260 265 270Leu Leu Ala Leu Lys Asn Phe Gly Gly Val Tyr Leu
Asp Val Asp Met 275 280 285Leu Pro Gly Ile His Ser Asp Leu Phe Lys
Thr Ile Ser Arg Pro Ser 290 295 300Ser Ile Gly Leu Asp Arg Trp Glu
Met Ile Lys Leu Glu Ala Ile Met305 310 315 320Lys Tyr Lys Lys Tyr
Ile Asn Asn Tyr Thr Ser Glu Asn Phe Asp Lys 325 330 335Leu Asp Gln
Gln Leu Lys Asp Asn Phe Lys Leu Ile Ile Glu Ser Lys 340 345 350Ser
Glu Lys Ser Glu Ile Phe Ser Lys Leu Glu Asn Leu Asn Val Ser 355 360
365Asp Leu Glu Ile Lys Ile Ala Phe Ala Leu Gly Ser Val Ile Asn Gln
370 375 380Ala Leu Ile Ser Lys Gln Gly Ser Tyr Leu Thr Asn Leu Val
Ile Glu385 390 395 400Gln Val Lys Asn Arg Tyr Gln Phe Leu Asn Gln
His Leu Asn Pro Ala 405 410 415Ile Glu Ser Asp Asn Asn Phe Thr Asp
Thr Thr Lys Ile Phe His Asp 420 425 430Ser Leu Phe Asn Ser Ala Thr
Ala Glu Asn Ser Met Phe Leu Thr Lys 435 440 445Ile Ala Pro Tyr Leu
Gln Val Gly Phe Met Pro Glu Ala Arg Ser Thr 450 455 460Ile Ser Leu
Ser Gly Pro Gly Ala Tyr Ala Ser Ala Tyr Tyr Asp Phe465 470 475
480Ile Asn Leu Gln Glu Asn Thr Ile Glu Lys Thr Leu Lys Ala Ser Asp
485 490 495Leu Ile Glu Phe Lys Phe Pro Glu Asn Asn Leu Ser Gln Leu
Thr Glu 500 505 510Gln Glu Ile Asn Ser Leu Trp Ser Phe Asp Gln Ala
Ser Ala Lys Tyr 515 520 525Gln Phe Glu Lys Tyr Val Arg Asp Tyr Thr
Gly Gly Ser Leu Ser Glu 530 535 540Asp Asn Gly Val Asp Phe Asn Lys
Asn Thr Ala Leu Asp Lys Asn Tyr545 550 555 560Leu Leu Asn Asn Lys
Ile Pro Ser Asn Asn Val Glu Glu Ala Gly Ser 565 570 575Lys Asn Tyr
Val His Tyr Ile Ile Gln Leu Gln Gly Asp Asp Ile Ser 580 585 590Tyr
Glu Ala Thr Cys Asn Leu Phe Ser Lys Asn Pro Lys Asn Ser Ile 595 600
605Ile Ile Gln Arg Asn Met Asn Glu Ser Ala Lys Ser Tyr Phe Leu Ser
610 615 620Asp Asp Gly Glu Ser Ile Leu Glu Leu Asn Lys Tyr Arg Ile
Pro Glu625 630 635 640Arg Leu Lys Asn Lys Glu Lys Val Lys Val Thr
Phe Ile Gly His Gly 645 650 655Lys Asp Glu Phe Asn Thr Ser Glu Phe
Ala Arg Leu Ser Val Asp Ser 660 665 670Leu Ser Asn Glu Ile Ser Ser
Phe Leu Asp Thr Ile Lys Leu Asp Ile 675 680 685Ser Pro Lys Asn Val
Glu Val Asn Leu Leu Gly Cys Asn Met Phe Ser 690 695 700Tyr Asp Phe
Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu Leu Leu Ser705 710 715
720Ile Met Asp Lys Ile Thr Ser Thr Leu Pro Asp Val Asn Lys Asn Ser
725 730 735Ile Thr Ile Gly Ala Asn Gln Tyr Glu Val Arg Ile Asn Ser
Glu Gly 740 745 750Arg Lys Glu Leu Leu Ala His Ser Gly Lys Trp Ile
Asn Lys Glu Glu 755 760 765Ala Ile Met Ser Asp Leu Ser Ser Lys Glu
Tyr Ile Phe Phe Asp Ser 770 775 780Ile Asp Asn Lys Leu Lys Ala Lys
Ser Lys Asn Ile Pro Gly Leu Ala785 790 795 800Ser Ile Ser Glu Asp
Ile Lys Thr Leu Leu Leu Asp Ala Ser Val Ser 805 810 815Pro Asp Thr
Lys Phe Ile Leu Asn Asn Leu Lys Leu Asn Ile Glu Ser 820 825 830Ser
Ile Gly Asp Tyr Ile Tyr Tyr Glu Lys Leu Glu Pro Val Lys Asn 835 840
845Ile Ile His Asn Ser Ile Asp Asp Leu Ile Asp Glu Phe Asn Leu Leu
850 855 860Glu Asn Val Ser Asp Glu Leu Tyr Glu Leu Lys Lys Leu Asn
Asn Leu865 870 875 880Asp Glu Lys Tyr Leu Ile Ser Phe Glu Asp Ile
Ser Lys Asn Asn Ser 885 890 895Thr Tyr Ser Val Arg Phe Ile Asn Lys
Ser Asn Gly Glu Ser Val Tyr 900 905 910Val Glu Thr Glu Lys Glu Ile
Phe Ser Lys Tyr Ser Glu His Ile Thr 915 920 925Lys Glu Ile Ser Thr
Ile Lys Asn Ser Ile Ile Thr Asp Val Asn Gly 930 935 940Asn Leu Leu
Asp Asn Ile Gln Leu Asp His Thr Ser Gln Val Asn Thr945 950 955
960Leu Asn Ala Ala Phe Phe Ile Gln Ser Leu Ile Asp Tyr Ser Ser Asn
965 970 975Lys Asp Val Leu Asn Asp Leu Ser Thr Ser Val Lys Val Gln
Leu Tyr 980 985 990Ala Gln Leu Phe Ser Thr Gly Leu Asn Thr Ile Tyr
Asp Ser Ile Gln 995 1000 1005Leu Val Asn Leu Ile Ser Asn Ala Val
Asn Asp Thr Ile Asn Val 1010 1015 1020Leu Pro Thr Ile Thr Glu Gly
Ile Pro Ile Val Ser Thr Ile Leu 1025 1030 1035Asp Gly Ile Asn Leu
Gly Ala Ala Ile Lys Glu Leu Leu Asp Glu 1040 1045 1050His Asp Pro
Leu Leu Lys Lys Glu Leu Glu Ala Lys Val Gly Val 1055 1060 1065Leu
Ala Ile Asn Met Ser Leu Ser Ile Ala Ala Thr Val Ala Ser 1070 1075
1080Ile Val Gly Ile Gly Ala Glu Val Thr Ile Phe Leu Leu Pro Ile
1085 1090 1095Ala Gly Ile Ser Ala Gly Ile Pro Ser Leu Val Asn Asn
Glu Leu 1100 1105 1110Ile Leu His Asp Lys Ala Thr Ser Val Val Asn
Tyr Phe Asn His 1115 1120 1125Leu Ser Glu Ser Lys Lys Tyr Gly Pro
Leu Lys Thr Glu Asp Asp 1130 1135 1140Lys Ile Leu Val Pro Ile Asp
Asp Leu Val Ile Ser Glu Ile Asp 1145 1150 1155Phe Asn Asn Asn Ser
Ile Lys Leu Gly Thr Cys Asn Ile Leu Ala 1160 1165 1170Met Glu Gly
Gly Ser Gly His Thr Val Thr Gly Asn Ile Asp His 1175 1180 1185Phe
Phe Ser Ser Pro Ser Ile Ser Ser His Ile Pro Ser Leu Ser 1190 1195
1200Ile Tyr Ser Ala Ile Gly Ile Glu Thr Glu Asn Leu Asp Phe Ser
1205 1210 1215Lys Lys Ile Met Met Leu Pro Asn Ala Pro Ser Arg Val
Phe Trp 1220 1225 1230Trp Glu Thr Gly Ala Val Pro Gly Leu Arg Ser
Leu Glu Asn Asp 1235 1240 1245Gly Thr Arg Leu Leu Asp Ser Ile Arg
Asp Leu Tyr Pro Gly Lys 1250 1255 1260Phe Tyr Trp Arg Phe Tyr Ala
Phe Phe Asp Tyr Ala Ile Thr Thr 1265 1270 1275Leu Lys Pro Val Tyr
Glu Asp Thr Asn Ile Lys Ile Lys Leu Asp 1280 1285 1290Lys Asp Thr
Arg Asn Phe Ile Met Pro Thr Ile Thr Thr Asn Glu 1295 1300 1305Ile
Arg Asn Lys Leu Ser Tyr Ser Phe Asp Gly Ala Gly Gly Thr 1310 1315
1320Tyr Ser Leu Leu Leu Ser Ser Tyr Pro Ile Ser Thr Asn Ile Asn
1325 1330 1335Leu Ser Lys Asp Asp Leu Trp Ile Phe Asn Ile Asp Asn
Glu Val 1340 1345 1350Arg Glu Ile Ser Ile Glu Asn Gly Thr Ile Lys
Lys Gly Lys Leu 1355 1360 1365Ile Lys Asp Val Leu Ser Lys Ile Asp
Ile Asn Lys Asn Lys Leu 1370 1375 1380Ile Ile Gly Asn Gln Thr Ile
Asp Phe Ser Gly Asp Ile Asp Asn 1385 1390 1395Lys Asp Arg Tyr Ile
Phe Leu Thr Cys Glu Leu Asp Asp Lys Ile 1400
1405 1410Ser Leu Ile Ile Glu Ile Asn Leu Val Ala Lys Ser Tyr Ser
Leu 1415 1420 1425Leu Leu Ser Gly Asp Lys Asn Tyr Leu Ile Ser Asn
Leu Ser Asn 1430 1435 1440Thr Ile Glu Lys Ile Asn Thr Leu Gly Leu
Asp Ser Lys Asn Ile 1445 1450 1455Ala Tyr Asn Tyr Thr Asp Glu Ser
Asn Asn Lys Tyr Phe Gly Ala 1460 1465 1470Ile Ser Lys Thr Ser Gln
Lys Ser Ile Ile His Tyr Lys Lys Asp 1475 1480 1485Ser Lys Asn Ile
Leu Glu Phe Tyr Asn Asp Ser Thr Leu Glu Phe 1490 1495 1500Asn Ser
Lys Asp Phe Ile Ala Glu Asp Ile Asn Val Phe Met Lys 1505 1510
1515Asp Asp Ile Asn Thr Ile Thr Gly Lys Tyr Tyr Val Asp Asn Asn
1520 1525 1530Thr Asp Lys Ser Ile Asp Phe Ser Ile Ser Leu Val Ser
Lys Asn 1535 1540 1545Gln Val Lys Val Asn Gly Leu Tyr Leu Asn Glu
Ser Val Tyr Ser 1550 1555 1560Ser Tyr Leu Asp Phe Val Lys Asn Ser
Asp Gly His His Asn Thr 1565 1570 1575Ser Asn Phe Met Asn Leu Phe
Leu Asp Asn Ile Ser Phe Trp Lys 1580 1585 1590Leu Phe Gly Phe Glu
Asn Ile Asn Phe Val Ile Asp Lys Tyr Phe 1595 1600 1605Thr Leu Val
Gly Lys Thr Asn Leu Gly Tyr Val Glu Phe Ile Cys 1610 1615 1620Asp
Asn Asn Lys Asn Ile Asp Ile Tyr Phe Gly Glu Trp Lys Thr 1625 1630
1635Ser Ser Ser Lys Ser Thr Ile Phe Ser Gly Asn Gly Arg Asn Val
1640 1645 1650Val Val Glu Pro Ile Tyr Asn Pro Asp Thr Gly Glu Asp
Ile Ser 1655 1660 1665Thr Ser Leu Asp Phe Ser Tyr Glu Pro Leu Tyr
Gly Ile Asp Arg 1670 1675 1680Tyr Ile Asn Lys Val Leu Ile Ala Pro
Asp Leu Tyr Thr Ser Leu 1685 1690 1695Ile Asn Ile Asn Thr Asn Tyr
Tyr Ser Asn Glu Tyr Tyr Pro Glu 1700 1705 1710Ile Ile Val Leu Asn
Pro Asn Thr Phe His Lys Lys Val Asn Ile 1715 1720 1725Asn Leu Asp
Ser Ser Ser Phe Glu Tyr Lys Trp Ser Thr Glu Gly 1730 1735 1740Ser
Asp Phe Ile Leu Val Arg Tyr Leu Glu Glu Ser Asn Lys Lys 1745 1750
1755Ile Leu Gln Lys Ile Arg Ile Lys Gly Ile Leu Ser Asn Thr Gln
1760 1765 1770Ser Phe Asn Lys Met Ser Ile Asp Phe Lys Asp Ile Lys
Lys Leu 1775 1780 1785Ser Leu Gly Tyr Ile Met Ser Asn Phe Lys Ser
Phe Asn Ser Glu 1790 1795 1800Asn Glu Leu Asp Arg Asp His Leu Gly
Phe Lys Ile Ile Asp Asn 1805 1810 1815Lys Thr Tyr Tyr Tyr Asp Glu
Asp Ser Lys Leu Val Lys Gly Leu 1820 1825 1830Ile Asn Ile Asn Asn
Ser Leu Phe Tyr Phe Asp Pro Ile Glu Phe 1835 1840 1845Asn Leu Val
Thr Gly Trp Gln Thr Ile Asn Gly Lys Lys Tyr Tyr 1850 1855 1860Phe
Asp Ile Asn Thr Gly Ala Ala Leu Thr Ser Tyr Lys Ile Ile 1865 1870
1875Asn Gly Lys His Phe Tyr Phe Asn Asn Asp Gly Val Met Gln Leu
1880 1885 1890Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr Phe Ala
Pro Ala 1895 1900 1905Asn Thr Gln Asn Asn Asn Ile Glu Gly Gln Ala
Ile Val Tyr Gln 1910 1915 1920Ser Lys Phe Leu Thr Leu Asn Gly Lys
Lys Tyr Tyr Phe Asp Asn 1925 1930 1935Asn Ser Lys Ala Val Thr Gly
Trp Arg Ile Ile Asn Asn Glu Lys 1940 1945 1950Tyr Tyr Phe Asn Pro
Asn Asn Ala Ile Ala Ala Val Gly Leu Gln 1955 1960 1965Val Ile Asp
Asn Asn Lys Tyr Tyr Phe Asn Pro Asp Thr Ala Ile 1970 1975 1980Ile
Ser Lys Gly Trp Gln Thr Val Asn Gly Ser Arg Tyr Tyr Phe 1985 1990
1995Asp Thr Asp Thr Ala Ile Ala Phe Asn Gly Tyr Lys Thr Ile Asp
2000 2005 2010Gly Lys His Phe Tyr Phe Asp Ser Asp Cys Val Val Lys
Ile Gly 2015 2020 2025Val Phe Ser Thr Ser Asn Gly Phe Glu Tyr Phe
Ala Pro Ala Asn 2030 2035 2040Thr Tyr Asn Asn Asn Ile Glu Gly Gln
Ala Ile Val Tyr Gln Ser 2045 2050 2055Lys Phe Leu Thr Leu Asn Gly
Lys Lys Tyr Tyr Phe Asp Asn Asn 2060 2065 2070Ser Lys Ala Val Thr
Gly Leu Gln Thr Ile Asp Ser Lys Lys Tyr 2075 2080 2085Tyr Phe Asn
Thr Asn Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr 2090 2095 2100Ile
Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ala Glu Ala 2105 2110
2115Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn
2120 2125 2130Thr Asn Thr Ala Ile Ala Ser Thr Gly Tyr Thr Ile Ile
Asn Gly 2135 2140 2145Lys His Phe Tyr Phe Asn Thr Asp Gly Ile Met
Gln Ile Gly Val 2150 2155 2160Phe Lys Gly Pro Asn Gly Phe Glu Tyr
Phe Ala Pro Ala Asn Thr 2165 2170 2175Asp Ala Asn Asn Ile Glu Gly
Gln Ala Ile Leu Tyr Gln Asn Glu 2180 2185 2190Phe Leu Thr Leu Asn
Gly Lys Lys Tyr Tyr Phe Gly Ser Asp Ser 2195 2200 2205Lys Ala Val
Thr Gly Trp Arg Ile Ile Asn Asn Lys Lys Tyr Tyr 2210 2215 2220Phe
Asn Pro Asn Asn Ala Ile Ala Ala Ile His Leu Cys Thr Ile 2225 2230
2235Asn Asn Asp Lys Tyr Tyr Phe Ser Tyr Asp Gly Ile Leu Gln Asn
2240 2245 2250Gly Tyr Ile Thr Ile Glu Arg Asn Asn Phe Tyr Phe Asp
Ala Asn 2255 2260 2265Asn Glu Ser Lys Met Val Thr Gly Val Phe Lys
Gly Pro Asn Gly 2270 2275 2280Phe Glu Tyr Phe Ala Pro Ala Asn Thr
His Asn Asn Asn Ile Glu 2285 2290 2295Gly Gln Ala Ile Val Tyr Gln
Asn Lys Phe Leu Thr Leu Asn Gly 2300 2305 2310Lys Lys Tyr Tyr Phe
Asp Asn Asp Ser Lys Ala Val Thr Gly Trp 2315 2320 2325Gln Thr Ile
Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn Thr Ala 2330 2335 2340Glu
Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr 2345 2350
2355Phe Asn Leu Asn Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile
2360 2365 2370Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe Ile
Ala Ser 2375 2380 2385Thr Gly Tyr Thr Ser Ile Asn Gly Lys His Phe
Tyr Phe Asn Thr 2390 2395 2400Asp Gly Ile Met Gln Ile Gly Val Phe
Lys Gly Pro Asn Gly Phe 2405 2410 2415Glu Tyr Phe Ala Pro Ala Asn
Thr Asp Ala Asn Asn Ile Glu Gly 2420 2425 2430Gln Ala Ile Leu Tyr
Gln Asn Lys Phe Leu Thr Leu Asn Gly Lys 2435 2440 2445Lys Tyr Tyr
Phe Gly Ser Asp Ser Lys Ala Val Thr Gly Leu Arg 2450 2455 2460Thr
Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ala Val 2465 2470
2475Ala Val Thr Gly Trp Gln Thr Ile Asn Gly Lys Lys Tyr Tyr Phe
2480 2485 2490Asn Thr Asn Thr Ser Ile Ala Ser Thr Gly Tyr Thr Ile
Ile Ser 2495 2500 2505Gly Lys His Phe Tyr Phe Asn Thr Asp Gly Ile
Met Gln Ile Gly 2510 2515 2520Val Phe Lys Gly Pro Asp Gly Phe Glu
Tyr Phe Ala Pro Ala Asn 2525 2530 2535Thr Asp Ala Asn Asn Ile Glu
Gly Gln Ala Ile Arg Tyr Gln Asn 2540 2545 2550Arg Phe Leu Tyr Leu
His Asp Asn Ile Tyr Tyr Phe Gly Asn Asn 2555 2560 2565Ser Lys Ala
Ala Thr Gly Trp Val Thr Ile Asp Gly Asn Arg Tyr 2570 2575 2580Tyr
Phe Glu Pro Asn Thr Ala Met Gly Ala Asn Gly Tyr Lys Thr 2585 2590
2595Ile Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gln Ile
2600 2605 2610Gly Val Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Ala
Pro Ala 2615 2620 2625Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala
Ile Arg Tyr Gln 2630 2635 2640Asn Arg Phe Leu His Leu Leu Gly Lys
Ile Tyr Tyr Phe Gly Asn 2645 2650 2655Asn Ser Lys Ala Val Thr Gly
Trp Gln Thr Ile Asn Gly Lys Val 2660 2665 2670Tyr Tyr Phe Met Pro
Asp Thr Ala Met Ala Ala Ala Gly Gly Leu 2675 2680 2685Phe Glu Ile
Asp Gly Val Ile Tyr Phe Phe Gly Val Asp Gly Val 2690 2695 2700Lys
Ala Pro Gly Ile Tyr Gly 2705 27101722366PRTClostridia 172Met Ser
Leu Val Asn Arg Lys Gln Leu Glu Lys Met Ala Asn Val Arg1 5 10 15Phe
Arg Thr Gln Glu Asp Glu Tyr Val Ala Ile Leu Asp Ala Leu Glu 20 25
30Glu Tyr His Asn Met Ser Glu Asn Thr Val Val Glu Lys Tyr Leu Lys
35 40 45Leu Lys Asp Ile Asn Ser Leu Thr Asp Ile Tyr Ile Asp Thr Tyr
Lys 50 55 60Lys Ser Gly Arg Asn Lys Ala Leu Lys Lys Phe Lys Glu Tyr
Leu Val65 70 75 80Thr Glu Val Leu Glu Leu Lys Asn Asn Asn Leu Thr
Pro Val Glu Lys 85 90 95Asn Leu His Phe Val Trp Ile Gly Gly Gln Ile
Asn Asp Thr Ala Ile 100 105 110Asn Tyr Ile Asn Gln Trp Lys Asp Val
Asn Ser Asp Tyr Asn Val Asn 115 120 125Val Phe Tyr Asp Ser Asn Ala
Phe Leu Ile Asn Thr Leu Lys Lys Thr 130 135 140Val Val Glu Ser Ala
Ile Asn Asp Thr Leu Glu Ser Phe Arg Glu Asn145 150 155 160Leu Asn
Asp Pro Arg Phe Asp Tyr Asn Lys Phe Phe Arg Lys Arg Met 165 170
175Glu Ile Ile Tyr Asp Lys Gln Lys Asn Phe Ile Asn Tyr Tyr Lys Ala
180 185 190Gln Arg Glu Glu Asn Pro Glu Leu Ile Ile Asp Asp Ile Val
Lys Thr 195 200 205Tyr Leu Ser Asn Glu Tyr Ser Lys Glu Ile Asp Glu
Leu Asn Thr Tyr 210 215 220Ile Glu Glu Ser Leu Asn Lys Ile Thr Gln
Asn Ser Gly Asn Asp Val225 230 235 240Arg Asn Phe Glu Glu Phe Lys
Asn Gly Glu Ser Phe Asn Leu Tyr Glu 245 250 255Gln Glu Leu Val Glu
Arg Trp Asn Leu Ala Ala Ala Ser Asp Ile Leu 260 265 270Arg Ile Ser
Ala Leu Lys Glu Ile Gly Gly Met Tyr Leu Asp Val Asp 275 280 285Met
Leu Pro Gly Ile Gln Pro Asp Leu Phe Glu Ser Ile Glu Lys Pro 290 295
300Ser Ser Val Thr Val Asp Phe Trp Glu Met Thr Lys Leu Glu Ala
Ile305 310 315 320Met Lys Tyr Lys Glu Tyr Ile Pro Glu Tyr Thr Ser
Glu His Phe Asp 325 330 335Met Leu Asp Glu Glu Val Gln Ser Ser Phe
Glu Ser Val Leu Ala Ser 340 345 350Lys Ser Asp Lys Ser Glu Ile Phe
Ser Ser Leu Gly Asp Met Glu Ala 355 360 365Ser Pro Leu Glu Val Lys
Ile Ala Phe Asn Ser Lys Gly Ile Ile Asn 370 375 380Gln Gly Leu Ile
Ser Val Lys Asp Ser Tyr Cys Ser Asn Leu Ile Val385 390 395 400Lys
Gln Ile Glu Asn Arg Tyr Lys Ile Leu Asn Asn Ser Leu Asn Pro 405 410
415Ala Ile Ser Glu Asp Asn Asp Phe Asn Thr Thr Thr Asn Thr Phe Ile
420 425 430Asp Ser Ile Met Ala Glu Ala Asn Ala Asp Asn Gly Arg Phe
Met Met 435 440 445Glu Leu Gly Lys Tyr Leu Arg Val Gly Phe Phe Pro
Asp Val Lys Thr 450 455 460Thr Ile Asn Leu Ser Gly Pro Glu Ala Tyr
Ala Ala Ala Tyr Gln Asp465 470 475 480Leu Leu Met Phe Lys Glu Gly
Ser Met Asn Ile His Leu Ile Glu Ala 485 490 495Asp Leu Arg Asn Phe
Glu Ile Ser Lys Thr Asn Ile Ser Gln Ser Thr 500 505 510Glu Gln Glu
Met Ala Ser Leu Trp Ser Phe Asp Asp Ala Arg Ala Lys 515 520 525Ala
Gln Phe Glu Glu Tyr Lys Arg Asn Tyr Phe Glu Gly Ser Leu Gly 530 535
540Glu Asp Asp Asn Leu Asp Phe Ser Gln Asn Ile Val Val Asp Lys
Glu545 550 555 560Tyr Leu Leu Glu Lys Ile Ser Ser Leu Ala Arg Ser
Ser Glu Arg Gly 565 570 575Tyr Ile His Tyr Ile Val Gln Leu Gln Gly
Asp Lys Ile Ser Tyr Glu 580 585 590Ala Ala Cys Asn Leu Phe Ala Lys
Thr Pro Tyr Asp Ser Val Leu Phe 595 600 605Gln Lys Asn Ile Glu Asp
Ser Glu Ile Ala Tyr Tyr Tyr Asn Pro Gly 610 615 620Asp Gly Glu Ile
Gln Glu Ile Asp Lys Tyr Lys Ile Pro Ser Ile Ile625 630 635 640Ser
Asp Arg Pro Lys Ile Lys Leu Thr Phe Ile Gly His Gly Lys Asp 645 650
655Glu Phe Asn Thr Asp Ile Phe Ala Gly Phe Asp Val Asp Ser Leu Ser
660 665 670Thr Glu Ile Glu Ala Ala Ile Asp Leu Ala Lys Glu Asp Ile
Ser Pro 675 680 685Lys Ser Ile Glu Ile Asn Leu Leu Gly Cys Asn Met
Phe Ser Tyr Ser 690 695 700Ile Asn Val Glu Glu Thr Tyr Pro Gly Lys
Leu Leu Leu Lys Val Lys705 710 715 720Asp Lys Ile Ser Glu Leu Met
Pro Ser Ile Ser Gln Asp Ser Ile Ile 725 730 735Val Ser Ala Asn Gln
Tyr Glu Val Arg Ile Asn Ser Glu Gly Arg Arg 740 745 750Glu Leu Leu
Asp His Ser Gly Glu Trp Ile Asn Lys Glu Glu Ser Ile 755 760 765Ile
Lys Asp Ile Ser Ser Lys Glu Tyr Ile Ser Phe Asn Pro Lys Glu 770 775
780Asn Lys Ile Thr Val Lys Ser Lys Asn Leu Pro Glu Leu Ser Thr
Leu785 790 795 800Leu Gln Glu Ile Arg Asn Asn Ser Asn Ser Ser Asp
Ile Glu Leu Glu 805 810 815Glu Lys Val Met Leu Thr Glu Cys Glu Ile
Asn Val Ile Ser Asn Ile 820 825 830Asp Thr Gln Ile Val Glu Glu Arg
Ile Glu Glu Ala Lys Asn Leu Thr 835 840 845Ser Asp Ser Ile Asn Tyr
Ile Lys Asp Glu Phe Lys Leu Ile Glu Ser 850 855 860Ile Ser Asp Ala
Leu Cys Asp Leu Lys Gln Gln Asn Glu Leu Glu Asp865 870 875 880Ser
His Phe Ile Ser Phe Glu Asp Ile Ser Glu Thr Asp Glu Gly Phe 885 890
895Ser Ile Arg Phe Ile Asn Lys Glu Thr Gly Glu Ser Ile Phe Val Glu
900 905 910Thr Glu Lys Thr Ile Phe Ser Glu Tyr Ala Asn His Ile Thr
Glu Glu 915 920 925Ile Ser Lys Ile Lys Gly Thr Ile Phe Asp Thr Val
Asn Gly Lys Leu 930 935 940Val Lys Lys Val Asn Leu Asp Thr Thr His
Glu Val Asn Thr Leu Asn945 950 955 960Ala Ala Phe Phe Ile Gln Ser
Leu Ile Glu Tyr Asn Ser Ser Lys Glu 965 970 975Ser Leu Ser Asn Leu
Ser Val Ala Met Lys Val Gln Val Tyr Ala Gln 980 985 990Leu Phe Ser
Thr Gly Leu Asn Thr Ile Thr Asp Ala Ala Lys Val Val 995 1000
1005Glu Leu Val Ser Thr Ala Leu Asp Glu Thr Ile Asp Leu Leu Pro
1010 1015 1020Thr Leu Ser Glu Gly Leu Pro Ile Ile Ala Thr Ile Ile
Asp Gly 1025 1030 1035Val Ser Leu Gly Ala Ala Ile Lys Glu Leu Ser
Glu Thr Ser Asp 1040 1045 1050Pro Leu Leu Arg Gln Glu Ile Glu Ala
Lys Ile Gly Ile Met Ala 1055 1060 1065Val Asn Leu Thr Thr Ala Thr
Thr Ala Ile Ile Thr Ser Ser Leu 1070 1075 1080Gly Ile Ala Ser Gly
Phe Ser Ile Leu Leu Val Pro Leu Ala Gly 1085 1090 1095Ile Ser Ala
Gly Ile Pro Ser Leu Val Asn Asn Glu Leu Val Leu 1100 1105 1110Arg
Asp Lys Ala Thr Lys Val Val Asp Tyr Phe Lys His Val Ser 1115 1120
1125Leu Val Glu Thr Glu Gly Val Phe Thr Leu Leu Asp Asp Lys Ile
1130 1135 1140Met Met Pro Gln Asp Asp Leu Val Ile
Ser Glu Ile Asp Phe Asn 1145 1150 1155Asn Asn Ser Ile Val Leu Gly
Lys Cys Glu Ile Trp Arg Met Glu 1160 1165 1170Gly Gly Ser Gly His
Thr Val Thr Asp Asp Ile Asp His Phe Phe 1175 1180 1185Ser Ala Pro
Ser Ile Thr Tyr Arg Glu Pro His Leu Ser Ile Tyr 1190 1195 1200Asp
Val Leu Glu Val Gln Lys Glu Glu Leu Asp Leu Ser Lys Asp 1205 1210
1215Leu Met Val Leu Pro Asn Ala Pro Asn Arg Val Phe Ala Trp Glu
1220 1225 1230Thr Gly Trp Thr Pro Gly Leu Arg Ser Leu Glu Asn Asp
Gly Thr 1235 1240 1245Lys Leu Leu Asp Arg Ile Arg Asp Asn Tyr Glu
Gly Glu Phe Tyr 1250 1255 1260Trp Arg Tyr Phe Ala Phe Ile Ala Asp
Ala Leu Ile Thr Thr Leu 1265 1270 1275Lys Pro Arg Tyr Glu Asp Thr
Asn Ile Arg Ile Asn Leu Asp Ser 1280 1285 1290Asn Thr Arg Ser Phe
Ile Val Pro Ile Ile Thr Thr Glu Tyr Ile 1295 1300 1305Arg Glu Lys
Leu Ser Tyr Ser Phe Tyr Gly Ser Gly Gly Thr Tyr 1310 1315 1320Ala
Leu Ser Leu Ser Gln Tyr Asn Met Gly Ile Asn Ile Glu Leu 1325 1330
1335Ser Glu Ser Asp Val Trp Ile Ile Asp Val Asp Asn Val Val Arg
1340 1345 1350Asp Val Thr Ile Glu Ser Asp Lys Ile Lys Lys Gly Asp
Leu Ile 1355 1360 1365Glu Gly Ile Leu Ser Thr Leu Ser Ile Glu Glu
Asn Lys Ile Ile 1370 1375 1380Leu Asn Ser His Glu Ile Asn Phe Ser
Gly Glu Val Asn Gly Ser 1385 1390 1395Asn Gly Phe Val Ser Leu Thr
Phe Ser Ile Leu Glu Gly Ile Asn 1400 1405 1410Ala Ile Ile Glu Val
Asp Leu Leu Ser Lys Ser Tyr Lys Leu Leu 1415 1420 1425Ile Ser Gly
Glu Leu Lys Ile Leu Met Leu Asn Ser Asn His Ile 1430 1435 1440Gln
Gln Lys Ile Asp Tyr Ile Gly Phe Asn Ser Glu Leu Gln Lys 1445 1450
1455Asn Ile Pro Tyr Ser Phe Val Asp Ser Glu Gly Lys Glu Asn Gly
1460 1465 1470Phe Ile Asn Gly Ser Thr Lys Glu Gly Leu Phe Val Ser
Glu Leu 1475 1480 1485Pro Asp Val Val Leu Ile Ser Lys Val Tyr Met
Asp Asp Ser Lys 1490 1495 1500Pro Ser Phe Gly Tyr Tyr Ser Asn Asn
Leu Lys Asp Val Lys Val 1505 1510 1515Ile Thr Lys Asp Asn Val Asn
Ile Leu Thr Gly Tyr Tyr Leu Lys 1520 1525 1530Asp Asp Ile Lys Ile
Ser Leu Ser Leu Thr Leu Gln Asp Glu Lys 1535 1540 1545Thr Ile Lys
Leu Asn Ser Val His Leu Asp Glu Ser Gly Val Ala 1550 1555 1560Glu
Ile Leu Lys Phe Met Asn Arg Lys Gly Asn Thr Asn Thr Ser 1565 1570
1575Asp Ser Leu Met Ser Phe Leu Glu Ser Met Asn Ile Lys Ser Ile
1580 1585 1590Phe Val Asn Phe Leu Gln Ser Asn Ile Lys Phe Ile Leu
Asp Ala 1595 1600 1605Asn Phe Ile Ile Ser Gly Thr Thr Ser Ile Gly
Gln Phe Glu Phe 1610 1615 1620Ile Cys Asp Glu Asn Asp Asn Ile Gln
Pro Tyr Phe Ile Lys Phe 1625 1630 1635Asn Thr Leu Glu Thr Asn Tyr
Thr Leu Tyr Val Gly Asn Arg Gln 1640 1645 1650Asn Met Ile Val Glu
Pro Asn Tyr Asp Leu Asp Asp Ser Gly Asp 1655 1660 1665Ile Ser Ser
Thr Val Ile Asn Phe Ser Gln Lys Tyr Leu Tyr Gly 1670 1675 1680Ile
Asp Ser Cys Val Asn Lys Val Val Ile Ser Pro Asn Ile Tyr 1685 1690
1695Thr Asp Glu Ile Asn Ile Thr Pro Val Tyr Glu Thr Asn Asn Thr
1700 1705 1710Tyr Pro Glu Val Ile Val Leu Asp Ala Asn Tyr Ile Asn
Glu Lys 1715 1720 1725Ile Asn Val Asn Ile Asn Asp Leu Ser Ile Arg
Tyr Val Trp Ser 1730 1735 1740Asn Asp Gly Asn Asp Phe Ile Leu Met
Ser Thr Ser Glu Glu Asn 1745 1750 1755Lys Val Ser Gln Val Lys Ile
Arg Phe Val Asn Val Phe Lys Asp 1760 1765 1770Lys Thr Leu Ala Asn
Lys Leu Ser Phe Asn Phe Ser Asp Lys Gln 1775 1780 1785Asp Val Pro
Val Ser Glu Ile Ile Leu Ser Phe Thr Pro Ser Tyr 1790 1795 1800Tyr
Glu Asp Gly Leu Ile Gly Tyr Asp Leu Gly Leu Val Ser Leu 1805 1810
1815Tyr Asn Glu Lys Phe Tyr Ile Asn Asn Phe Gly Met Met Val Ser
1820 1825 1830Gly Leu Ile Tyr Ile Asn Asp Ser Leu Tyr Tyr Phe Lys
Pro Pro 1835 1840 1845Val Asn Asn Leu Ile Thr Gly Phe Val Thr Val
Gly Asp Asp Lys 1850 1855 1860Tyr Tyr Phe Asn Pro Ile Asn Gly Gly
Ala Ala Ser Ile Gly Glu 1865 1870 1875Thr Ile Ile Asp Asp Lys Asn
Tyr Tyr Phe Asn Gln Ser Gly Val 1880 1885 1890Leu Gln Thr Gly Val
Phe Ser Thr Glu Asp Gly Phe Lys Tyr Phe 1895 1900 1905Ala Pro Ala
Asn Thr Leu Asp Glu Asn Leu Glu Gly Glu Ala Ile 1910 1915 1920Asp
Phe Thr Gly Lys Leu Ile Ile Asp Glu Asn Ile Tyr Tyr Phe 1925 1930
1935Asp Asp Asn Tyr Arg Gly Ala Val Glu Trp Lys Glu Leu Asp Gly
1940 1945 1950Glu Met His Tyr Phe Ser Pro Glu Thr Gly Lys Ala Phe
Lys Gly 1955 1960 1965Leu Asn Gln Ile Gly Asp Tyr Lys Tyr Tyr Phe
Asn Ser Asp Gly 1970 1975 1980Val Met Gln Lys Gly Phe Val Ser Ile
Asn Asp Asn Lys His Tyr 1985 1990 1995Phe Asp Asp Ser Gly Val Met
Lys Val Gly Tyr Thr Glu Ile Asp 2000 2005 2010Gly Lys His Phe Tyr
Phe Ala Glu Asn Gly Glu Met Gln Ile Gly 2015 2020 2025Val Phe Asn
Thr Glu Asp Gly Phe Lys Tyr Phe Ala His His Asn 2030 2035 2040Glu
Asp Leu Gly Asn Glu Glu Gly Glu Glu Ile Ser Tyr Ser Gly 2045 2050
2055Ile Leu Asn Phe Asn Asn Lys Ile Tyr Tyr Phe Asp Asp Ser Phe
2060 2065 2070Thr Ala Val Val Gly Trp Lys Asp Leu Glu Asp Gly Ser
Lys Tyr 2075 2080 2085Tyr Phe Asp Glu Asp Thr Ala Glu Ala Tyr Ile
Gly Leu Ser Leu 2090 2095 2100Ile Asn Asp Gly Gln Tyr Tyr Phe Asn
Asp Asp Gly Ile Met Gln 2105 2110 2115Val Gly Phe Val Thr Ile Asn
Asp Lys Val Phe Tyr Phe Ser Asp 2120 2125 2130Ser Gly Ile Ile Glu
Ser Gly Val Gln Asn Ile Asp Asp Asn Tyr 2135 2140 2145Phe Tyr Ile
Asp Asp Asn Gly Ile Val Gln Ile Gly Val Phe Asp 2150 2155 2160Thr
Ser Asp Gly Tyr Lys Tyr Phe Ala Pro Ala Asn Thr Val Asn 2165 2170
2175Asp Asn Ile Tyr Gly Gln Ala Val Glu Tyr Ser Gly Leu Val Arg
2180 2185 2190Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Thr
Ile Glu 2195 2200 2205Thr Gly Trp Ile Tyr Asp Met Glu Asn Glu Ser
Asp Lys Tyr Tyr 2210 2215 2220Phe Asn Pro Glu Thr Lys Lys Ala Cys
Lys Gly Ile Asn Leu Ile 2225 2230 2235Asp Asp Ile Lys Tyr Tyr Phe
Asp Glu Lys Gly Ile Met Arg Thr 2240 2245 2250Gly Leu Ile Ser Phe
Glu Asn Asn Asn Tyr Tyr Phe Asn Glu Asn 2255 2260 2265Gly Glu Met
Gln Phe Gly Tyr Ile Asn Ile Glu Asp Lys Met Phe 2270 2275 2280Tyr
Phe Gly Glu Asp Gly Val Met Gln Ile Gly Val Phe Asn Thr 2285 2290
2295Pro Asp Gly Phe Lys Tyr Phe Ala His Gln Asn Thr Leu Asp Glu
2300 2305 2310Asn Phe Glu Gly Glu Ser Ile Asn Tyr Thr Gly Trp Leu
Asp Leu 2315 2320 2325Asp Glu Lys Arg Tyr Tyr Phe Thr Asp Glu Tyr
Ile Ala Ala Thr 2330 2335 2340Gly Ser Val Ile Ile Asp Gly Glu Glu
Tyr Tyr Phe Asp Pro Asp 2345 2350 2355Thr Ala Gln Leu Val Ile Ser
Glu 2360 236517318PRTArtificialFragment from C. difficile toxin
TcdB 173Ser Pro Val Glu Lys Asn Leu His Phe Val Trp Ile Gly Gly Glu
Val1 5 10 15Ser Asp17411PRTArtificialFragment from C. difficile
toxin TcdB 174Asn Leu Ala Ala Ala Ser Asp Ile Val Arg Leu1 5
1017515PRTArtificialFragment of C. difficile toxin TcdB 175Cys Gly
Gly Val Tyr Leu Asp Val Asp Met Leu Pro Gly Ile His1 5 10
1517620PRTArtificialFragment from C. difficile toxin TcdB 176Cys
Gly Gly Val Tyr Leu Asp Val Asp Met Leu Pro Gly Ile His Ser1 5 10
15Asp Leu Phe Lys 2017714PRTArtificialFragment of C. difficile
toxin TcdB 177Cys Trp Glu Met Ile Lys Leu Glu Ala Ile Met Lys Tyr
Lys1 5 1017812PRTArtificialFragment of C. difficile toxin TcdB
178Cys Thr Asn Leu Val Ile Glu Gln Val Lys Asn Arg1 5
1017912PRTArtificialFragment of C. difficile toxin TcdB 179Pro Glu
Ala Arg Ser Thr Ile Ser Leu Ser Gly Pro1 5
1018012PRTArtificialFragment of C. difficile toxin TcdB 180Cys Ser
Asn Leu Ile Val Lys Gln Ile Glu Asn Arg1 5
1018114PRTArtificialFragment of C. difficile toxin TcdB 181Thr Glu
Gln Glu Ile Asn Ser Leu Trp Ser Phe Asp Gln Ala1 5
1018225PRTArtificialFragment of C. difficile toxin TcdB 182Thr Glu
Gln Glu Ile Asn Ser Leu Trp Ser Phe Asp Pro Glu Ala Arg1 5 10 15Ser
Thr Ile Ser Leu Ser Gly Pro Cys 20 2518313PRTArtificialFragment of
C. difficile toxin TcdB 183Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu
Leu Leu Cys1 5 1018414PRTArtificialFragment of C. difficile toxin
TcdB 184Cys Ala Asn Gln Tyr Glu Val Arg Ile Asn Ser Glu Gly Arg1 5
1018515PRTArtificialFragment of C. difficile toxin TcdB 185Val Asn
Thr Leu Asn Ala Ala Phe Phe Ile Gln Ser Leu Ile Cys1 5 10
1518613PRTArtificialFragment of C. difficile toxin TcdB 186Tyr Ala
Gln Leu Phe Ser Thr Gly Leu Asn Thr Ile Cys1 5
1018716PRTArtificialFragment of C. difficile toxin TcdB 187Cys Ala
Gly Ile Ser Ala Gly Ile Pro Ser Leu Val Asn Asn Glu Leu1 5 10
1518816PRTArtificialFragment of C. difficile toxin TcdB 188Asp Asp
Leu Val Ile Ser Glu Ile Asp Phe Asn Asn Asn Ser Ile Cys1 5 10
1518910PRTArtificialFragment of C. difficile toxin TcdB 189Met Glu
Gly Gly Ser Gly His Thr Val Thr1 5 1019035PRTArtificialFragment of
C. difficile toxin TcdB 190Ala Val Asn Asp Thr Ile Asn Val Leu Pro
Thr Ile Thr Glu Gly Ile1 5 10 15Pro Ile Val Ser Thr Ile Leu Asp Gly
Ile Asn Leu Gly Ala Ala Ile 20 25 30Lys Glu Leu
3519120PRTArtificialFragment of C. difficile toxin TcdB 191Cys Gly
Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile1 5 10 15Glu
Gly Gln Ala 2019220PRTArtificialFragment of C. difficile toxin TcdB
192Cys Gly Tyr Lys Tyr Phe Ala Pro Ala Asn Thr Val Asn Asp Asn Ile1
5 10 15Tyr Gly Gln Ala 2019312PRTArtificialFragment of C. difficile
toxin TcdB 193Cys Lys Tyr Tyr Phe Asn Thr Asn Thr Ala Glu Ala1 5
1019412PRTArtificialFragment of C. difficile toxin TcdB 194Cys Lys
Tyr Tyr Phe Asp Glu Asp Thr Ala Glu Ala1 5 10
* * * * *
References