U.S. patent application number 15/717230 was filed with the patent office on 2018-01-11 for polypeptides.
The applicant listed for this patent is VHSQUARED LIMITED. Invention is credited to Tim Carlton, Scott Crowe, Kevin Roberts, Mike West.
Application Number | 20180009881 15/717230 |
Document ID | / |
Family ID | 55661405 |
Filed Date | 2018-01-11 |
United States Patent
Application |
20180009881 |
Kind Code |
A1 |
Crowe; Scott ; et
al. |
January 11, 2018 |
POLYPEPTIDES
Abstract
There is provided inter alia a polypeptide comprising an
immunoglobulin chain variable domain comprising three
complementarity determining regions (CDR1-CDR3) and four framework
regions, wherein: (a) at least one lysine residue in CDR1, CDR2
and/or CDR3 has been substituted with at least one histidine
residue, and/or (b) at least one arginine residue in CDR1, CDR2
and/or CDR3 has been substituted with at least one histidine
residue; wherein the polypeptide has increased intestinal stability
relative to a corresponding polypeptide not having said histidine
substitutions.
Inventors: |
Crowe; Scott; (Cambridge,
GB) ; West; Mike; (Cambridge, GB) ; Roberts;
Kevin; (Cambridge, GB) ; Carlton; Tim;
(Cambridge, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
VHSQUARED LIMITED |
Cambridge |
|
GB |
|
|
Family ID: |
55661405 |
Appl. No.: |
15/717230 |
Filed: |
September 27, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
PCT/EP2016/057024 |
Mar 31, 2016 |
|
|
|
15717230 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 1/00 20180101; C07K
2317/94 20130101; C07K 2317/22 20130101; Y02A 50/30 20180101; C07K
2317/54 20130101; C07K 16/2866 20130101; C07K 16/241 20130101; A61P
37/06 20180101; A61P 29/00 20180101; C07K 2317/92 20130101; C07K
2317/622 20130101; Y02A 50/472 20180101; C07K 2317/35 20130101;
C07K 2317/567 20130101; A61K 2039/542 20130101; C07K 2317/569
20130101; C07K 2317/55 20130101; A61P 31/04 20180101; Y02A 50/484
20180101; C07K 16/1282 20130101; A61P 1/04 20180101; A61P 37/00
20180101; A61P 31/00 20180101; C07K 2317/565 20130101 |
International
Class: |
C07K 16/12 20060101
C07K016/12; C07K 16/24 20060101 C07K016/24; C07K 16/28 20060101
C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 31, 2015 |
EP |
15162115.8 |
Jan 21, 2016 |
EP |
16152320.4 |
Claims
1. A method of increasing the intestinal stability of a polypeptide
comprising an immunoglobulin chain variable domain, wherein the
immunoglobulin chain variable domain comprises three
complementarity determining regions (CDR1-CDR3) and four framework
regions, wherein the method comprises the step of substituting: (a)
at least one lysine residue in CDR1, CDR2 and/or CDR3 with at least
one histidine residue, and/or (b) at least one arginine residue in
CDR1, CDR2 and/or CDR3 with at least one histidine residue.
2. A method of making a polypeptide comprising an immunoglobulin
chain variable domain, wherein the immunoglobulin chain variable
domain comprises three complementarity determining regions
(CDR1-CDR3) and four framework regions, wherein the method
comprises the step of substituting: (a) at least one lysine residue
in CDR1, CDR2 and/or CDR3 with at least one histidine residue,
and/or (b) at least one arginine residue in CDR1, CDR2 and/or CDR3
with at least one histidine residue wherein the polypeptide has
increased intestinal stability relative to a corresponding
polypeptide not having said histidine substitutions.
3. The method according to claim 1, wherein the substitutions
increase the stability of the polypeptide in the intestinal tract,
such as in the small and/or large intestine, such as in the
duodenum, jejunum, ileum cecum, colon, rectum and/or anal canal,
relative to a corresponding polypeptide not having said histidine
substitutions.
4. The method according to claim 1, wherein the substitutions
increase the stability of the polypeptide in a model of the
intestinal tract, such as in the small and/or large intestine, such
as in the duodenum, jejunum, ileum cecum, colon, rectum and/or anal
canal, relative to a corresponding polypeptide not having said
histidine substitutions.
5. The method according to claim 4 wherein the model of the
intestinal tract is the Standard Human Faecal Supernatant
Intestinal Tract Model.
6. The method according to claim 5, wherein the stability of the
polypeptide is increased by at least 5%, more suitably 30%, more
suitably 50%, relative to a corresponding polypeptide not having
said histidine substitutions, after 1 hour incubation in the
Standard Human Faecal Supernatant Intestinal Tract Model.
7. The method according to claim 1, wherein the substitutions
increase the stability of the polypeptide to one or more proteases
produced in the small or large intestine, relative to a
corresponding polypeptide not having said histidine
substitutions.
8. The method according to claim 7, wherein the one or more
proteases are selected from the group consisting of trypsin,
chymotrypsin, MMPs, cathepsin, enteropeptidase, host inflammatory
proteases, proteases originating from gut commensal microflora
and/or pathogenic bacteria actively secreted and/or released by
lysis of microbial cells, and proteases originating from pathogenic
bacteria, such as C. difficile-specific proteases.
9. The method according to claim 1 wherein the potency of the
polypeptide is substantially the same as the potency of a
corresponding polypeptide not having said histidine
substitutions.
10. The method according to claim 9 wherein the EC50 of the
polypeptide is increased by no more than 400% relative to a
corresponding polypeptide not having said histidine
substitutions.
11. The method according to claim 1, wherein the substitutions are
introduced synthetically.
12. The method according to claim 11, wherein the substitutions are
introduced by a method selected from the group consisting of:
error-prone PCR, shuffling, oligonucleotide-directed mutagenesis,
assembly PCR, PCR mutagenesis, in vivo mutagenesis, cassette
mutagenesis, recursive ensemble mutagenesis, exponential ensemble
mutagenesis, site-specific mutagenesis, gene reassembly, Gene Site
Saturation Mutagenesis (GSSM), synthetic ligation reassembly (SLR),
recombination, recursive sequence recombination,
phosphothioate-modified DNA mutagenesis, uracil-containing template
mutagenesis, gapped duplex mutagenesis, point mismatch repair
mutagenesis, repair-deficient host strain mutagenesis, chemical
mutagenesis, radiogenic mutagenesis, deletion mutagenesis,
restriction-selection mutagenesis, restriction-purification
mutagenesis, ensemble mutagenesis, chimeric nucleic acid multi mer
creation, or a combination thereof.
13. The method according to claim 1, wherein the substitutions are
not introduced by V(D)J recombination or somatic mutation.
14. The method according to claim 1, wherein the at least one
lysine residue is present in a window defined as the second third
of CDR1 and/or the second third of CDR2 and/or the second third of
CDR3.
15. The method according to claim 1, wherein the at least one
arginine residue is present in a window defined as the second third
of CDR1 and/or the second third of CDR2 and/or the second third of
CDR3.
16. The method according to claim 1, wherein each lysine and/or
arginine residue in CDR1, CDR2 and/or CDR3 has been substituted
with at least one histidine residue each.
17. The method according to claim 1, wherein the polypeptide
consists of an immunoglobulin chain variable domain.
18. The method according to claim 1, wherein the polypeptide is
selected from the group consisting of an antibody, a modified
antibody containing additional antibody binding regions, an
antibody fragment such as an scFv, a Fab fragment, a F(ab')2
fragment and an immunoglobulin chain variable domain such as a VHH,
a VH, a VL, a V-NAR.
19. The method according to claim 1, wherein the polypeptide binds
to a target accessible via the intestinal tract.
20. The method according to claim 19, wherein the polypeptide binds
to a target within the intestinal tract.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation application of
PCT/EP2016/057024 filed Mar. 31, 2016 which claims priority from EP
15162115.8 filed Mar. 31, 2015 and EP 16152320.4 filed Jan. 21,
2016, the contents of each of which are incorporated herein by
reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to polypeptides comprising a
region which is capable of binding a target with high affinity,
especially those comprising immunoglobulin chain variable domains
(ICVD) as well as to constructs comprising said polypeptides and
pharmaceutical compositions comprising such polypeptides and
constructs. The polypeptides, constructs and pharmaceutical
compositions of the invention are all suitable for oral
administration. The present invention also relates to methods of
increasing the intestinal stability of a polypeptide comprising an
immunoglobulin chain variable domain, methods of making a
polypeptide comprising an immunoglobulin chain variable domain, and
methods which utilise such polypeptides, constructs comprising such
polypeptides, nucleic acids encoding such polypeptides, cDNA and
vectors comprising nucleic acids encoding such polypeptides, host
cells expressing or capable of expressing such polypeptides,
pharmaceutical compositions comprising such polypeptides and to
uses of such polypeptides.
BACKGROUND OF THE INVENTION
[0003] Pharmaceutical research and development is becoming
increasingly focussed on biopharmaceuticals such as therapeutic
polypeptides, including antibodies. Typically, therapeutic
polypeptides are administered either directly or indirectly into
the circulation, via a systemic route. However, many therapeutic
polypeptides would ideally be delivered via the oral route.
Delivering therapeutic polypeptides orally could provide the
following advantages: (a) direct targeting to the gastrointestinal
tract (GIT) for localised treatment of gastrointestinal diseases
(Jones and Martino 2015 Crit Rev Biotechnol 20:1-15), (b) the risk
of adverse immune reactions could be reduced due to the naturally
immuno-tolerant nature of the GIT, ensuring the long-term safety of
repeatedly ingesting therapeutic polypeptide materials, (c) without
the stringent regulatory requirements of manufacturing injectable
therapeutic polypeptides, production costs could be reduced and (d)
higher levels of patient acceptance and long term compliance could
be achieved (Shaji and Patole Indian J Pharm Sci 2008
70(3):269-277).
[0004] Many therapeutic polypeptides are, however, unstable in the
intestinal tract and therefore the beneficial effect obtained from
oral administration is generally limited (Bruno et al 2013 Ther
Deliv4(11):1443-1467). Consequently, oral dosage forms used for
conventional small molecule drugs have been employed for oral
polypeptide delivery. Various strategies currently under
investigation include formulation vehicles, use of enzyme
inhibitors, absorption enhancers and mucoadhesive polymers (Shaji
and Patole, ibid).
[0005] Alternative strategies involving modifications to the
therapeutic polypeptides themselves have also been employed, such
as the introduction of (additional) cysteine bridges. Hussack et al
2011 PLoS ONE 6(11):e28218 describe the introduction of additional
cysteine bridges into anti-TcdA VHHs. The effectiveness of these
additional cysteine bridges on increasing proteolytic stability was
highly dependent on the specific protease concerned and in some
circumstances these additional cysteine bridges were detrimental to
recombinant production levels. Similarly, Kim et al 2014 mAbs 6:1
219-235 engineered human VL domains with disulphide bridges, with
mixed results.
[0006] In theory, one could consider substituting specific amino
acids in a therapeutic polypeptide which are believed to be
responsible for low intestinal stability of the therapeutic
polypeptide, in order to enhance stability in the intestinal tract.
However, in the context of immunoglobulin chain variable domains,
single substitutions in amino acid sequence may detrimentally
impact binding capability. This is particularly relevant to the
complementarity determining regions (CDRs) of an immunoglobulin
chain variable domain, which are responsible for binding target
antigen. For example, regarding in particular CDR3 of a VHH, it is
known that " . . . inasmuch as the CDR3 amino acids either are in
direct contact with the antigen or maintain and influence the
conformation of the CDR3 amino acids that directly contact the
antigen, the CDR3 amino acids responsible for reduced stability
cannot be replaced without serious loss of affinity." (Muyldermans
Annu Rev Biochem 2013 82:775-797). This view is reinforced by, for
example, the finding that substitutions to a VHH targetting C.
jejuni flagella, including in particular an R to G substitution in
CDR2, caused a large decrease in binding capability of the VHH
(approaching control) (Hussack et al 2014 Protein Engineering,
Design & Selection 27(6):191-198).
[0007] There is a long-felt need therefore for polypeptides which
have increased intestinal stability, and for methods to increase
the intestinal stability of such polypeptides.
[0008] Polypeptides of the present invention may, in at least some
embodiments, have one or more of the following advantages compared
to substances of the prior art: [0009] (i) increased suitability
for oral administration; [0010] (ii) increased suitability for
local delivery to the intestinal tract following oral
administration; [0011] (iii) increased intestinal stability whilst
substantially maintaining binding affinity and/or potency; [0012]
(iv) increased stability in a model of the intestinal tract such as
the Standard Trypsin Intestinal Tract Model, the Standard Mouse
Small Intestinal Supernatant Intestinal Tract Model or the Standard
Human Faecal Supernatant Intestinal Tract Model, whilst maintaining
binding affinity and/or potency; [0013] (v) increased stability in
the presence of proteases, for example (a) in the presence of
proteases found in the small and/or large intestine and/or IBD
inflammatory proteases, for example trypsin, chymotrypsin, MMPs,
cathepsin, enteropeptidase, host inflammatory proteases and/or (b)
in the presence of proteases from gut commensal microflora and/or
pathogenic bacteria, actively secreted and/or released by lysis of
microbial cells found in the small and/or large intestine; [0014]
(vi) increased stability when expressed in a heterologous host such
as a yeast such as a yeast belonging to the genera Aspergillus,
Saccharomyces, Kluyveromyces, Hansenula or Pichia (by virtue of
increased resistance to yeast proteases); [0015] (vii) reduced risk
of adverse immune reactions; [0016] (viii) reduced production
costs; [0017] (ix) improved treatment and/or prevention of
intestinal infection or autoimmune and/or inflammatory diseases;
[0018] (x) improved patient acceptance and long term compliance;
[0019] (xi) improved yield during recombinant production; [0020]
(xii) improved bioactivity and/or biodistribution; [0021] (xiii)
reduced required dosage; [0022] (xiv) suitability for, and improved
properties for, use in a pharmaceutical; [0023] (xv) suitability
for, and improved properties for, use in a functional food.
SUMMARY OF THE INVENTION
[0024] The present inventors have produced surprisingly
advantageous polypeptides comprising immunoglobulin chain variable
domains, suitable for oral administration. These polypeptides are
particularly advantageous due to their increased intestinal
stability (i.e. increased stability in the intestinal tract). It
may be expected that these polypeptides have particular utility in
the prevention or treatment of diseases of the gastrointestinal
tract such as autoimmune and/or inflammatory disease such as
inflammatory bowel disease, or in the prevention or treatment of
infection from intestinal tract resident pathogenic microbe. Also
provided are methods of increasing the intestinal stability of a
polypeptide comprising an immunoglobulin chain variable domain and
methods of making a polypeptide comprising an immunoglobulin chain
variable domain having increased stability.
[0025] Accordingly, the present invention provides a polypeptide
comprising an immunoglobulin chain variable domain comprising three
complementarity determining regions (CDR1-CDR3) and four framework
regions, wherein: (a) at least one lysine residue in CDR1, CDR2
and/or CDR3 has been substituted with at least one histidine
residue, and/or (b) at least one arginine residue in CDR1, CDR2
and/or CDR3 has been substituted with at least one histidine
residue; wherein the polypeptide has increased intestinal stability
relative to a corresponding polypeptide not having said histidine
substitutions.
[0026] Also provided is a method of increasing the intestinal
stability of a polypeptide comprising an immunoglobulin chain
variable domain, wherein the immunoglobulin chain variable domain
comprises three complementarity determining regions (CDR1-CDR3) and
four framework regions, wherein the method comprises the step of
substituting: (a) at least one lysine residue in CDR1, CDR2 and/or
CDR3 with at least one histidine residue, and/or (b) at least one
arginine residue in CDR1, CDR2 and/or CDR3 with at least one
histidine residue.
[0027] Also provided is a method of making a polypeptide comprising
an immunoglobulin chain variable domain, wherein the immunoglobulin
chain variable domain comprises three complementarity determining
regions (CDR1-CDR3) and four framework regions, wherein the method
comprises the step of substituting: (a) at least one lysine residue
in CDR1, CDR2 and/or CDR3 with at least one histidine residue,
and/or (b) at least one arginine residue in CDR1, CDR2 and/or CDR3
with at least one histidine residue wherein the polypeptide has
increased intestinal stability relative to a corresponding
polypeptide not having said histidine substitutions.
[0028] Also provided is a polypeptide comprising a region which is
capable of binding a target with high affinity wherein: (a) at
least one lysine residue in the region has been substituted with at
least one histidine residue, and/or (b) at least one arginine
residue in the region has been substituted with at least one
histidine residue; wherein the polypeptide has increased intestinal
stability relative to a corresponding polypeptide not having said
histidine substitutions.
DESCRIPTION OF THE FIGURES
[0029] FIG. 1--Example TcdA dose-response curve on Vero cells
[0030] FIG. 2A--Potency of anti-TNF ICVDs Q65B1, ID8F-EV, ID43F and
ID44F (Experiment 1) against human TNF in the TNFR2/TNF
interference ELISA
[0031] FIG. 2B--Potency of anti-TNF ICVDs Q65B1 and ID8F-EV
(Experiment 2) against human TNF in the TNFR2/TNF interference
ELISA
[0032] FIG. 3A--Stability of anti-TNF ICVDs Q65B1, ID8F-EV, ID43F
and ID44F in mouse small intestinal supernatant after 6 hours
incubation
[0033] FIG. 3B--Stability of anti-TNF ICVDs Q65B1 and ID8F-EV in
human faecal and mouse small intestinal supernatant after 16 hour
incubation
[0034] FIG. 4--Potency of ICVDs ID32F and ID34F against human TNF
in the TNFR2/TNF interference ELISA
[0035] FIG. 5A--Stability of anti-TNF ICVDs ID32F and ID34F in
mouse small intestinal supernatant after 16 hours incubation
[0036] FIG. 5B--Stability of anti-TNF ICVDs ID32F and ID34F in
human faecal supernatant pool 4 after 16 hours incubation
[0037] FIG. 6A--TcdB 027 neutralisation by ID45B-ID50B in the Vero
cell cytotoxicity assay
[0038] FIG. 6B--Stability of anti-TcdB ICVDs ID45B-ID50B in human
faecal supernatant pool 4 after 30 minutes incubation, analysed by
western blot
[0039] FIG. 7--TcdB 027 neutralisation by ID2B, ID20B, ID21B and
ID22B in the Vero cell cytotoxicity assay
[0040] FIG. 8A--ID2B trypsin assay--stained polyacrylamide gel
[0041] FIG. 8B--ID20B and ID21B trypsin assays--stained
polyacrylamide gels
[0042] FIG. 8C--ID22B trypsin assay--stained polyacrylamide gel
[0043] FIG. 9--Stability of anti-TcdB ICVDs ID2B and ID21B in human
faecal supernatants after 1 hour incubation
[0044] FIG. 10A--TcdB 027 neutralisation by ID1B, ID24B, ID25B and
ID27B in the Vero cell cytotoxicity assay
[0045] FIG. 10B--Stability of anti-TcdB ICVDs ID1B, ID24B, ID25B
and ID27B in human faecal supernatant pool 2 after 1 hour
incubation
[0046] FIG. 11A--ID1B trypsin assay--stained polyacrylamide gel
[0047] FIG. 11B--ID24B and 25B trypsin assays--stained
polyacrylamide gels
[0048] FIG. 11C--ID27B trypsin assay--stained polyacrylamide
gel
[0049] FIG. 12A--TcdB 017 neutralisation by bihead constructs ID41B
and ID43B in the Vero cell cytotoxicity assay
[0050] FIG. 12B--Stability of anti-TcdB bihead constructs ID41B and
ID43B in C. difficile negative human faecal supernatant pool 2
after 4 hour incubation (three repeat ELISAs)
[0051] FIG. 12C--Stability of anti-TcdB bihead constructs ID41B and
ID43B in C. difficile negative human faecal supernatant pool 3
after 4 hour incubation (three repeat ELISAs)
[0052] FIG. 12D--Stability of anti-TcdB bihead constructs ID41B and
ID43B in C. difficile negative human faecal supernatant pool 4
after 4 hour incubation (three repeat ELISAs)
[0053] FIG. 13A--TcdA 087 neutralisation by ID17A and ID29A in the
Vero cell cytotoxicity assay
[0054] FIG. 13B--Stability of anti-TcdA bihead constructs ID17A and
ID29A in human faecal supernatants after 1 hour incubation
DESCRIPTION OF THE SEQUENCES
[0055] SEQ ID NO: 1--Polypeptide sequence of anti-TNF-alpha ICVD
Q65B1
[0056] SEQ ID NO: 2--Polypeptide sequence of anti-TNF-alpha ICVD
ID8F-EV (ID32F)
[0057] SEQ ID NO: 3--Polypeptide sequence of anti-TNF-alpha ICVD
ID43F
[0058] SEQ ID NO: 4--Polypeptide sequence of anti-TNF-alpha ICVD
ID44F
[0059] SEQ ID NO: 5--Polypeptide sequence of anti-TNF-alpha ICVD
ID34F
[0060] SEQ ID NO: 6--Polypeptide sequence of anti-TcdB ICVD
B10F1
[0061] SEQ ID NO: 7--Polypeptide sequence of anti-TcdB ICVD
Q31B1
[0062] SEQ ID NO: 8--Polypeptide sequence of anti-TcdB ICVD
ID1B
[0063] SEQ ID NO: 9--Polypeptide sequence of anti-TcdB ICVD
ID2B
[0064] SEQ ID NO: 10--Polypeptide sequence of anti-TcdB ICVD
ID20B
[0065] SEQ ID NO: 11--Polypeptide sequence of anti-TcdB ICVD
ID21B
[0066] SEQ ID NO: 12--Polypeptide sequence of anti-TcdB ICVD
ID22B
[0067] SEQ ID NO: 13--Polypeptide sequence of anti-TcdB ICVD
ID24B
[0068] SEQ ID NO: 14--Polypeptide sequence of anti-TcdB ICVD
ID25B
[0069] SEQ ID NO: 15--Polypeptide sequence of anti-TcdB ICVD
ID27B
[0070] SEQ ID NO: 16--Polypeptide sequence of anti-TcdB construct
ID41B
[0071] SEQ ID NO: 17--Polypeptide sequence of anti-TcdB construct
ID43B
[0072] SEQ ID NO: 18--Polypeptide sequence of anti-TcdB ICVD
ID45B
[0073] SEQ ID NO: 19--Polypeptide sequence of anti-TcdB ICVD
ID46B
[0074] SEQ ID NO: 20--Polypeptide sequence of anti-TcdB ICVD
ID47B
[0075] SEQ ID NO: 21--Polypeptide sequence of anti-TcdB ICVD
ID48B
[0076] SEQ ID NO: 22--Polypeptide sequence of anti-TcdB ICVD
ID49B
[0077] SEQ ID NO: 23--Polypeptide sequence of anti-TcdB ICVD
ID50B
[0078] SEQ ID NO: 24--Polypeptide sequence of anti-TcdA construct
ID17A
[0079] SEQ ID NO: 25--Polypeptide sequence of anti-TcdA construct
ID29A
[0080] SEQ ID NO: 26--Example CDR A
[0081] SEQ ID NO: 27--First third of Example CDR A
[0082] SEQ ID NO: 28--Second third of Example CDR A
[0083] SEQ ID NO: 29--Third third of Example CDR A
[0084] SEQ ID NO: 30--Example CDR B
[0085] SEQ ID NO: 31--Second third of Example CDR B
[0086] SEQ ID NO: 32--Polypeptide sequence of anti-IL-6R ICVD
7F6
[0087] SEQ ID NO: 33--Polypeptide sequence of anti-IL-6R ICVD
ID-3V
[0088] SEQ ID NO: 34--Polypeptide sequence of anti-IL-6R ICVD
5G9
[0089] SEQ ID NO: 35--Polypeptide sequence of anti-IL-6R ICVD
ID-54V
DETAILED DESCRIPTION OF THE INVENTION
[0090] Polypeptides, Antigen-Binding Polypeptides, Antibodies and
Antibody Fragments including Immunoglobulin Chain Variable Domains
(ICVD) such as the VH and VHH
[0091] Polypeptides are organic polymers consisting of a number of
amino acid residues bonded together in a chain. As used herein,
`polypeptide` is used interchangeably with `protein` and `peptide`.
Polypeptides are said to be antigen-binding when they contain one
or more stretches of amino acid residues which form an
antigen-binding site, capable of binding to an epitope on a target
antigen with an affinity (suitably expressed as a Kd value, a Ka
value, a kon-rate and/or a koff-rate, as further described herein).
Antigen-binding polypeptides include polypeptides such as
antibodies, antibodies modified to comprise additional binding
regions, and antigen-binding fragments.
[0092] A polypeptide may comprise a region which is capable of
binding a target with high affinity (suitably expressed as a Kd
value, a Ka value, a k.sub.on-rate and/or a k.sub.off-rate, as
further described herein). Such polypeptides include DARPins (Binz
et al. Journal of Molecular Biology 332(2):489-503), Affimers.TM.,
Fynomers.TM., Centyrins, Nanofitins.RTM. and cyclic peptides.
[0093] A conventional antibody or immunoglobulin (Ig) is a protein
comprising four polypeptide chains: two heavy (H) chains and two
light (L) chains. Each chain is divided into a constant region and
a variable domain. The heavy chain variable domains are abbreviated
herein as VHC, and the light (L) chain variable domains are
abbreviated herein as VLC. These domains, domains related thereto
and domains derived therefrom, are referred to herein as
immunoglobulin chain variable domains. The VHC and VLC domains can
be further subdivided into regions of hypervariability, termed
"complementarity determining regions" ("CDRs"), interspersed with
regions that are more conserved, termed "framework regions"
("FRs"). The framework and complementarity determining regions have
been precisely defined (Kabat et al 1991 Sequences of Proteins of
Immunological Interest, Fifth Edition U.S. Department of Health and
Human Services, NIH Publication Number 91-3242, herein incorporated
by reference in its entirety). In a conventional antibody, each VHC
and VLC is composed of three CDRs and four FRs, arranged from
amino-terminus to carboxy-terminus in the following order: FR1,
CDR1, FR2, CDR2, FR3, CDR3, FR4. The conventional antibody tetramer
of two heavy immunoglobulin chains and two light immunoglobulin
chains is formed with the heavy and the light immunoglobulin chains
inter-connected by e.g. disulfide bonds, and the heavy chains
similarity connected. The heavy chain constant region includes
three domains, CH1, CH2 and CH3. The light chain constant region is
comprised of one domain, CL. The variable domain of the heavy
chains and the variable domain of the light chains are binding
domains that interact with an antigen. The constant regions of the
antibodies typically mediate the binding of the antibody to host
tissues or factors, including various cells of the immune system
(e.g. effector cells) and the first component (C1q) of the
classical complement system. The term antibody includes
immunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as
subtypes thereof), wherein the light chains of the immunoglobulin
may be kappa or lambda types. The overall structure of
immunoglobulin-gamma (IgG) antibodies assembled from two identical
heavy (H)-chain and two identical light (L)-chain polypeptides is
well established and highly conserved in mammals (Padlan 1994 Mol
Immunol 31:169-217).
[0094] An exception to conventional antibody structure is found in
sera of Camelidae. In addition to conventional antibodies, these
sera possess special IgG antibodies. These IgG antibodies, known as
heavy-chain antibodies (HCAbs), are devoid of the L chain
polypeptide and lack the first constant domain (CH1). At its
N-terminal region, the H chain of the homodimeric protein contains
a dedicated immunoglobulin chain variable domain, referred to as
the VHH, which serves to associate with its cognate antigen
(Muyldermans 2013 Annu Rev Biochem 82:775-797, Hamers-Casterman et
al 1993 Nature 363(6428):446-448, Muyldermans et al 1994 Protein
Eng 7(9):1129-1135, herein incorporated by reference in their
entirety).
[0095] The total number of amino acid residues in a VHH or VH may
be in the region of 105-140, is suitably 108-130, and is most
suitably 110-125.
[0096] An antigen-binding fragment (or "'antibody fragment",
"immunoglobulin fragment" or "antigen-binding polypeptide") as used
herein refers to a portion of an antibody that specifically binds
to a target (e.g. a molecule in which one or more immunoglobulin
chains is not full length, but which specifically binds to a
target). An antigen-binding fragment comprises an immunoglobulin
chain variable domain. Examples of binding fragments encompassed
within the term antigen-binding fragment include:
[0097] (i) a Fab fragment (a monovalent fragment consisting of the
VLC, VHC, CL and CH1 domains);
[0098] (ii) a F(ab')2 fragment (a bivalent fragment comprising two
Fab fragments linked by a disulfide bridge at the hinge
region);
[0099] (iii) a Fd fragment (consisting of the VHC and CH1
domains);
[0100] (iv) a Fv fragment (consisting of the VLC and VHC domains of
a single arm of an antibody);
[0101] (v) an scFv fragment (consisting of VLC and VHC domains
joined, using recombinant methods, by a synthetic linker that
enables them to be made as a single protein chain in which the VLC
and VHC regions pair to form monovalent molecules);
[0102] (vi) a VH (an immunoglobulin chain variable domain
consisting of a VHC domain (Ward et al Nature 1989
341:544-546);
[0103] (vii) a VL (an immunoglobulin chain variable domain
consisting of a VLC domain);
[0104] (viii) a V-NAR (an immunoglobulin chain variable domain
consisting of a VHC domain from chondrichthyes IgNAR (Roux et al
1998 Proc Natl Acad Sci USA 95:11804-11809 and Griffiths et al 2013
Antibodies 2:66-81, herein incorporated by reference in their
entirety)
[0105] (ix) a VHH.
[0106] Suitably the polypeptide of the invention consists of an
immunoglobulin chain variable domain. Suitably the polypeptide of
the invention is an antibody, a modified antibody containing
additional antibody binding regions or an antibody fragment such as
a VHH, a VH, a VL, a V-NAR, scFv, a Fab fragment or a F(ab').sub.2
fragment
[0107] Polypeptides of the invention may for example be obtained by
preparing a nucleic acid encoding the polypeptide using techniques
for nucleic acid synthesis, followed by expression of the nucleic
acid thus obtained (as detailed further herein).
[0108] The examples provided herein relate to immunoglobulin chain
variable domains per se. The principles of the invention disclosed
herein are, however, equally applicable to at least any polypeptide
comprising an immunoglobulin chain variable domain, such as
antibodies and antibody fragments. For example, the immunoglobulin
chain variable domains disclosed herein may be incorporated into a
polypeptide such as a full length antibody. Such an approach is
demonstrated by McCoy et al Retrovirology 2014 11:83, who provide
an anti-HIV VHH engineered as a fusion with a human Fc region
(including hinge, CH2 and CH3 domains), expressed as a dimer
construct.
[0109] Polypeptide and Polynucleotide Sequences
[0110] As used herein, numbering of polypeptide sequences and
definitions of CDRs and FRs are as defined according to the Kabat
system (Kabat et al, ibid). A "corresponding" amino acid residue
between a first and second polypeptide sequence is an amino acid
residue in a first sequence which shares the same position
according to the Kabat system with an amino acid residue in a
second sequence, whilst the amino acid residue in the second
sequence may differ in identity from the first. Suitably
corresponding residues will share the same number (and letter) if
the framework and CDRs are the same length according to Kabat
definition. Alignment can be achieved manually or by using, for
example, a known computer algorithm for sequence alignment such as
NCBI BLAST v2.0 (BLASTP or BLASTN) using standard settings. Two or
more polypeptides are `corresponding` if they share the same
sequence but for any changes specified.
TABLE-US-00001 The Kabat numbering system applied to ICVD Q65B1
Region FR1 FR1 FR1 FR1 FR1 FR1 FR1 FR1 FR1 FR1 FR1 Residue # 1 2 3
4 5 6 7 8 9 10 11 Q65B1 E V Q L V E S G G G L Kabat H1 H2 H3 H4 H5
H6 H7 H8 H9 H10 H11 numbering Region FR1 FR1 FR1 FR1 FR1 FR1 FR1
FR1 FR1 FR1 Residue # 12 13 14 15 16 17 18 19 20 21 Q65B1 V Q P G G
S L K L S Kabat H12 H13 H14 H15 H16 H17 H18 H19 H20 H21 numbering
Region FR1 FR1 FR1 FR1 FR1 FR1 FR1 FR1 FR1 CDR1 CDR1 Residue # 22
23 24 25 26 27 28 29 30 31 32 Q65B1 C A A S G F D F S S H Kabat H22
H23 H24 H25 H26 H27 H28 H29 H30 H31 H32 numbering Region CDR1 CDR1
CDR1 FR2 FR2 FR2 FR2 FR2 FR2 FR2 Residue # 33 34 35 36 37 38 39 40
41 42 Q65B1 W M Y W V R Q A P G Kabat H33 H34 H35 H36 H37 H38 H39
H40 H41 H42 numbering Region FR2 FR2 FR2 FR2 FR2 FR2 FR2 CDR2 CDR2
CDR2 CDR2 Residue # 43 44 45 46 47 48 49 50 51 52 53 Q65B1 K E L E
W L S E I N T Kabat H43 H44 H45 H46 H47 H48 H49 H50 H51 H52 H52A
numbering Region CDR2 CDR2 CDR2 CDR2 CDR2 CDR2 CDR2 CDR2 CDR2 CDR2
Residue # 54 55 56 57 58 59 60 61 62 63 Q65B1 N G L I T K Y G D S
Kabat H53 H54 H55 H56 H57 H58 H59 H60 H61 H62 numbering Region CDR2
CDR2 CDR2 FR3 FR3 FR3 FR3 FR3 FR3 FR3 FR3 Residue # 64 65 66 67 68
69 70 71 72 73 74 Q65B1 V K G R F T V S R N N Kabat H63 H64 H65 H66
H67 H68 H69 H70 H71 H72 H73 numbering Region FR3 FR3 FR3 FR3 FR3
FR3 FR3 FR3 FR3 FR3 Residue # 75 76 77 78 79 80 81 82 83 84 Q65B1 A
A N K M Y L E L T Kabat H74 H75 H76 H77 H78 H79 H80 H81 H82 H82A
numbering Region FR3 FR3 FR3 FR3 FR3 FR3 FR3 FR3 FR3 FR3 FR3
Residue # 85 86 87 88 89 90 91 92 93 94 95 Q65B1 R L E P E D T A L
Y Y Kabat H82B H82C H83 H84 H85 H86 H87 H88 H89 H90 H91 numbering
Region FR3 FR3 FR3 CDR3 CDR3 CDR3 CDR3 CDR3 CDR3 FR4 Residue # 96
97 98 99 100 101 102 103 104 105 Q65B1 C A R N Q K G L N K Kabat
H92 H93 H94 H95 H96 H97 H98 H101 H102 H103 numbering Region FR4 FR4
FR4 FR4 FR4 FR4 FR4 FR4 FR4 FR4 Residue # 106 107 108 109 110 111
112 113 114 115 Q65B1 G Q G T Q V T V S S Kabat H104 H105 H106 H107
H108 H109 H110 H111 H112 H113 numbering
TABLE-US-00002 The Kabat characterisation system applied to ICVD
and ICVD construct sequences CDRs 1, 2 and 3 are the first, second
and third underlined portions of each ICVD or construct. FRs 1, 2,
3 and 4 are the first, second, third and fourth portions joining
the CDRs of each ICVD. The linker is also shown in the case of
biheads. Substitutions relative to unmodified comparators are shown
italicised and emboldened. Substitution descriptions in brackets
are referred-to by N-to-C-terminal numbering (as opposed to Kabat
numbering). Anti-TNF-alpha (SEQ ID NO: 1) Q65B1
EVQLVESGGGLVQPGGSLKLSCAASGFDFS SHWMY WVRQAPGKELEWLS
EINTNGLITKYGDSVKG RFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQKGLN
KGQGTQVTVSS (SEQ ID NO: 2) ID32F/ID8F-EV
EVQLVESGGGLVQPGGSLKLSCAASGFDFS SHWMY WVRQAPGKELEWLS EINTNGLIT
YGDSVKG RFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQKGLN KGQGTQVTVSS (SEQ ID
NO: 3) ID43F EVQLVESGGGLVQPGGSLKLSCAASGFDFS SHWMY WVRQAPGKELEWLS
EINTNGLIT YGDSVKG RFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQKGLN
KGQGTQVTVSS (SEQ ID NO: 4) ID44F EVQLVESGGGLVQPGGSLKLSCAASGFDFS
SHWMY WVRQAPGKELEWLS EINTNGLIT YGDSVKG
RFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQKGLN KGQGTQVTVSS (SEQ ID NO: 5)
ID34F EVQLVESGGGLVQPGGSLKLSCAASGFDFS SHWMY WVRQAPGKELEWLS EINTNGLIT
YGDSVKG RFTVSRNNAANKMYLELTRLEPEDTALYYCAR NQ GLN KGQGTQVTVSS
Anti-TcdB B10F1 (unmodified) (SEQ ID NO: 6)
QVQLQESGGGLVQAGGSLRLSCAASGRTFS SYYMG WFRQAPGKEREFVA
AINGSGGNRISADSVKG RFTISRDNAKNTVYLQLNSLKPEDTAVYYCAA SLTYYGRSARYDY
WGQGTQVTVSS Q31B1 (unmodified) (SEQ ID NO: 7)
EVQLVESGGGLVQAGDSLRLSCAASGRTLS SYTMG WFRQAPEKEREFVA
GSSRDGRTNYYANSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA HTTSGVPVRERSYAY
WGQGTQVTVSS ID1B (B10F1 with Q1D and R27A) (SEQ ID NO: 8)
DVQLQESGGGLVQAGGSLRLSCAASGATFS SYYMG WFRQAPGKEREFVA
AINGSGGNRISADSVKG RFTISRDNAKNTVYLQLNSLKPEDTAVYYCAA SLTYYGRSARYDY
WGQGTQVTVSS ID2B (Q31B1 with E1D, V5Q and R27A) (SEQ ID NO: 9)
DVQLQESGGGLVQAGDSLRLSCAASGATLS SYTMG WFRQAPEKEREFVA
GSSRDGRTNYYANSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA HTTSGVPVRERSYAY
WGQGTQVTVSS ID20B (ID2B with M34I, R53H, R.56H) (SEQ ID NO: 10)
DVQLQESGGGLVQAGDSLRLSCAASGATLS SYTIG WFRQAPEKEREFVA GSS DG
TNYYANSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA HTTSGVPVRERSYAY
WGQGTQVTVSS ID21B (ID2B with M34I, R107H) (SEQ ID NO: 11)
DVQLQESGGGLVQAGDSLRLSCAASGATLS SYTIG WFRQAPEKEREFVA
GSSRDGRTNYYANSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA HTTSGVP ERSYAY
WGQGTQVTVSS ID22B (ID2B with M34I, R109H) (SEQ ID NO: 12)
DVQLQESGGGLVQAGDSLRLSCAASGATLS SYTIG WFRQAPEKEREFVA
GSSRDGRTNYYANSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA HTTSGVPRE SYAY
WGQGTQVTVSS ID24B (ID1B with M34I, R58H) (SEQ ID NO: 13)
DVQLQESGGGLVQAGGSLRLSCAASGATFS SYYIG WFRQAPGKEREFVA AINGSGGN
ISADSVKG RFTISRDNAKNTVYLQLNSLKPEDTAVYYCAA SLTYYGRSARYDY WGQGTQVTVSS
ID25B (ID1B with M34I, R108H) (SEQ ID NO: 14)
DVQLQESGGGLVQAGGSLRLSCAASGATFS SYYIG WFRQAPGKEREFVA
AINGSGGNRISADSVKG RFTISRDNAKNTVYLQLNSLKPEDTAVYYCAA SLTYYGRSA YDY
WGQGTQVTVSS ID27B (ID1B with M34I, R105H) (SEQ ID NO: 15)
DVQLQESGGGLVQAGGSLRLSCAASGATFS SYYIG WFRQAPGKEREFVA
AINGSGGNRISADSVKG RFTISRDNAKNTVYLQLNSLKPEDTAVYYCAA SLTYYG SARYDY
WGQGTQVTVSS ID41B ((ID2B with R107H) x (ID1B with R105H), with
[G.sub.4S].sub.4 linker) (SEQ ID NO: 16)
DVQLQESGGGLVQAGDSLRLSCAASGATLS SYTMG WFRQAPEKEREFVA
GSSRDGRTNYYANSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA HTTSGVPV ERSYAY
WGQGTQVTVSS GGGGSGGGGSGGGGSGGGGS DVQLQESGGGLVQAGGSLRLSCAASGATFS
SYYMG WFRQAPGKEREFVA AINGSGGNRISADSVKG
RFTISRDNAKNTVYLQLNSLKPEDTAVYYCAA SLTYYG SARYDY WGQGTQVTVSS ID43B
((ID2B with R108H) x (ID1B with R105H), with [G.sub.4S].sub.4
linker) (SEQ ID NO: 17) DVQLQESGGGLVQAGDSLRLSCAASGATLS SYTMG
WFRQAPEKEREFVA GSSRDGRTNYYANSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA
HTTSGVPV ERSYAY WGQGTQVTVSS GGGGSGGGGSGGGGSGGGGS
DVQLQESGGGLVQAGGSLRLSCAASGATFS SYYMG WFRQAPGKEREFVA
AINGSGGNRISADSVKG RFTISRDNAKNTVYLQLNSLKPEDTAVYYCAA SLTYYG SA YDY
WGQGTQVTVSS ID45B (ID2B with D1E and Q5V, wild type R107) (SEQ ID
NO: 18) EVQLVESGGGLVQAGDSLRLSCAASGATLS SYTMG WFRQAPEKEREFVA
GSSRDGRTNYYANSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA HTTSGVPVRERSYAY
WGQGTQVTVSS ID46B (ID45B with R107H) (SEQ ID NO: 19)
EVQLVESGGGLVQAGDSLRLSCAASGATLS SYTMG WFRQAPEKEREFVA
GSSRDGRTNYYANSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA HTTSGVPV ERSYAY
WGQGTQVTVSS ID47B (ID45B with R107A) (SEQ ID NO: 20)
EVQLVESGGGLVQAGDSLRLSCAASGATLS SYTMG WFRQAPEKEREFVA
GSSRDGRTNYYANSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA HTTSGVPV ERSYAY
WGQGTQVTVSS ID48B (ID45B with R107Q) (SEQ ID NO: 21)
EVQLVESGGGLVQAGDSLRLSCAASGATLS SYTMG WFRQAPEKEREFVA
GSSRDGRTNYYANSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA HTTSGVPV ERSYAY
WGQGTQVTVSS ID49B (ID45B with R107F) (SEQ ID NO: 22)
EVQLVESGGGLVQAGDSLRLSCAASGATLS SYTMG WFRQAPEKEREFVA
GSSRDGRTNYYANSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA HTTSGVPV ERSYAY
WGQGTQVTVSS ID50B (ID45B with R107W) (SEQ ID NO: 23)
EVQLVESGGGLVQAGDSLRLSCAASGATLS SYTMG WFRQAPEKEREFVA
GSSRDGRTNYYANSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA HTTSGVPV ERSYAY
WGQGTQVTVSS Anti-TcdA ID17A (SEQ ID NO: 24)
DVQLQESGGGLVQAGGSLRLSCAASGATSD VYAMG WFRQVPGKEREFVA
TINRSGSDSYYADSVKG RFTISRDNAKNTVYLQMNSLKPEETAVYYCAA
SRSDCIGYGCRRVSQDY WGQGTQVTVSS GGGGSGGGGSGGGGSGGGGS
DVQLQESGGGLVQAGGSLRLSCVISGMDFS HKPAG WFRQAPGKEREFVA
SITTRASTHYADSVKG RFTISRDNAKNTVYLEMNSLKPEDTAVYYCNS EYY WGQGTQVTVSS
ID29A (ID17A with R109H) (SEQ ID NO: 25)
DVQLQESGGGLVQAGGSLRLSCAASGATSD VYAMG WFRQVPGKEREFVA
TINRSGSDSYYADSVKG RFTISRDNAKNTVYLQMNSLKPEETAVYYCAA SRSDCIGYGC
RVSQDY WGQGTQVTVSS GGGGSGGGGSGGGGSGGGGS DVQLQESGGGLVQAGGSLRLSCVISG
MDFS HKPAG WFRQAPGKEREFVA SITTRASTHYADSVKG
RFTISRDNAKNTVYLEMNSLKPEDTAVYYCNS EYY WGQGTQVTVSS Anti-IL-6R 7F6
(SEQ ID NO: 32) EVQLVESGGGLVQAGGSTRLTCLASGSISS INVIG WYRQAPGKQRELVA
MIGRGEGANYGDFAKG RFTISRDNSKNTVYLQMNSLKPEDTAVYYCYA DYEDRDSPFNGS
WGQGTQVTVSS ID-3V (7F6 with R102H) (SEQ ID NO: 33)
EVQLVESGGGLVQAGGSTRLTCLASGSISS INVIG WYRQAPGKQRELVA
MIGRGEGANYGDFAKG RFTISRDNSKNTVYLQMNSLKPEDTAVYYCYA DYEDHDSPFNGS
WGQGTQVTVSS 5G9 (SEQ ID NO: 34) EVQLVESGGGLVQAGGSTRLTCKASGSIFNINS
INVMA WYRQAPGKQRELVA IIGKGGGTNYADFVKG
RFTISRDAAKNTVNLQMNSLKPEDTAVYYCYA DYEDRDSPFNAS WGQGTQVTVSS ID-54V
(5G9 with R105H) (SEQ ID NO: 35) EVQLVESGGGLVQAGGSTRLTCKASGSIFNINS
INVMA WYRQAPGKQRELVA IIGKGGGTNYADFVKG RFTISRDAAKNTVN
LQMNSLKPEDTAVYYCYA DYEDHDSPFNAS WGQGTQVTVSS
[0111] Suitably at least one, such as two, such as three arginine
and/or lysine residues in the CDRs of a polypeptide of the
invention are substituted with a histidine residue. Suitably one
arginine and/or lysine residue is substituted. Suitably the
substitutions are made in at least one, such as at least two, such
as three CDRs. Suitably 1 to 3, such as 1 to 2, such as 1
substitution(s) are made in all three, two or one CDR(s) of a
polypeptide of the invention. Suitably no more than three, such as
no more than 2 lysine and/or arginine residues are substituted.
[0112] Suitably each lysine and/or arginine residue in CDR1, CDR2
and/or CDR3 of a polypeptide of the invention has been substituted
with at least one, more suitably one, histidine residue each.
[0113] Suitably each CDR of a polypeptide of the invention which
includes a substitution is no shorter than 3, more suitably no
shorter than 4, more suitably no shorter than 5, more suitably no
shorter than 6, more suitably no shorter than 7, more suitably no
shorter than 8, more suitably no shorter than 9, more suitably no
shorter than 10, more suitably no shorter than 11, more suitably no
shorter than 12, more suitably no shorter than 13 amino acids.
[0114] Suitably each CDR of a polypeptide of the invention which
includes a substitution is no longer than 35, more suitably no
longer than 30, more suitably no longer than 25, more suitably no
longer than 23, more suitably no longer than 21, more suitably no
longer than 20, more suitably no longer than 19, more suitably no
longer than 18, more suitably no longer than 17 amino acids.
[0115] Suitably the polypeptide of the invention is no longer than
2000, more suitably no longer than 1500, more suitably no longer
than 1200, more suitably no longer than 900, more suitably no
longer than 700, more suitably no longer than 600, more suitably no
longer than 500, more suitably no longer than 400, more suitably no
longer than 300, more suitably no longer than 250, more suitably no
longer than 200, more suitably no longer than 150 amino acids.
[0116] Windows Defined Within CDRs
[0117] The residues within a CDR may be considered to belong to a
particular fraction of that CDR. For example, a CDR consisting of
fifteen amino acids (ARNECDQGHILKMFP, SEQ ID NO: 26) can be
considered to consist of three thirds: a first third (a window
consisting of ARNEC, SEQ ID NO: 27), a second third (a window
consisting of DQGHI, SEQ ID NO: 28) and a third third (a window
consisting of LKMFP, SEQ ID NO: 29). Similarly, this CDR can be
considered to consist of five fifths: a first fifth (a window
consisting of ARN), a second fifth (a window consisting of ECD), a
third fifth (a window consisting of QGH), a fourth fifth (a window
consisting of ILK) and a fifth fifth (a window consisting of MFP).
The numbering of the fractions of a CDR is from N- to C-terminus.
If a CDR consists of a number of residues such that division into
fractions would result in a non-whole number of residues residing
in each fraction (such as sevenths of a CDR consisting of
ARNECDQGHILKMFP, SEQ ID NO: 26) then (a) if the CDR consists of an
odd number of residues, then the number of residues in the central
fraction (e.g. the second third or the third fifth, etc) is rounded
up to the nearest odd number or (b) if the CDR consists of an even
number of residues, then the number of residues in the central
fraction is rounded up and to the nearest even number. For example,
the fourth seventh of a CDR consisting of ARNECDQGHILKMFP is the
window consisting of QGH and the second third of a CDR consisting
of ARNECDQG (SEQ ID NO: 30) is the window consisting of NECD (SEQ
ID NO: 31).
[0118] Suitably the at least one lysine and/or arginine residue is
present in a window defined as the second third of CDR1 and/or the
second third of CDR2 and/or the second third of CDR3 and/or the
third fifth of CDR1 and/or the third fifth of CDR2 and/or the third
fifth of CDR3 and/or the fourth seventh of CDR1 and/or the fourth
seventh of CDR2 and/or the fourth seventh of CDR3.
[0119] According to a specific embodiment, a polypeptide according
to the invention does not have an amino acid sequence which is
exactly the same as (i.e. shares 100% sequence identity with) the
amino acid sequence of a naturally occurring polypeptide.
[0120] In one embodiment there is provided a polypeptide comprising
an immunoglobulin chain variable domain comprising three
complementarity determining regions (CDR1-CDR3) and four framework
regions, having: (a) at least one histidine residue in place of at
least one lysine residue in CDR1, CDR2 and/or CDR3, and/or (b) at
least one histidine residue in place of at least one arginine
residue in CDR1, CDR2 and/or CDR3; wherein the polypeptide has
increased intestinal stability relative to a corresponding
progenitor polypeptide not having said histidine substitutions.
[0121] A progenitor polypeptide is suitably a polypeptide which has
not undergone the inventive histidine substitutions disclosed
herein. Suitably the corresponding progenitor polypeptide is the
`wild type` polypeptide (for example an antibody) which was
directly produced by an animal, for example by V(D)J recombination
and somatic mutation (such as a llama, such as following
immunisation), and which may have optionally undergone further
synthetic modifications, before undergoing the inventive histidine
substitutions disclosed herein.
[0122] Specificity, Affinity and Avidity
[0123] Specificity refers to the number of different types of
antigens or antigenic determinants to which a particular
antigen-binding polypeptide can bind. The specificity of an
antigen-binding polypeptide is the ability of the antigen-binding
polypeptide to recognise a particular antigen as a unique molecular
entity and distinguish it from another.
[0124] Affinity, represented by the equilibrium constant for the
dissociation of an antigen with an antigen-binding polypeptide
(Kd), is a measure of the binding strength between an antigenic
determinant and an antigen-binding site on an antigen-binding
polypeptide: the lesser the value of the Kd, the stronger the
binding strength between an antigenic determinant and the
antigen-binding polypeptide (alternatively, the affinity can also
be expressed as the affinity constant (Ka), which is 1/Kd).
Affinity can be determined by known methods, depending on the
specific antigen of interest.
[0125] Avidity is the measure of the strength of binding between an
antigen-binding polypeptide and the pertinent antigen. Avidity is
related to both the affinity between an antigenic determinant and
its antigen-binding site on the antigen-binding polypeptide and the
number of pertinent binding sites present on the antigen-binding
polypeptide.
[0126] Suitably, polypeptides of the invention bind to their target
with a dissociation constant (Kd) of 10.sup.-6 to 10.sup.-12 M,
more suitably 10.sup.-7 to 10.sup.-12 M, more suitably 10.sup.-8 to
10.sup.-12 M and more suitably 10.sup.-9 to 10.sup.-12 M.
[0127] Any Kd value less than 10.sup.-6 is considered to indicate
specific binding. Specific binding of an antigen-binding
polypeptide to an antigen or antigenic determinant can be
determined in any suitable known manner, including, for example,
Scatchard analysis and/or competitive binding assays, such as
radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich
competition assays, and the different variants thereof known in the
art.
[0128] Potency, Inhibition and Neutralisation
[0129] Potency is a measure of the activity of a therapeutic agent
expressed in terms of the amount required to produce an effect of
given intensity. A highly potent agent evokes a greater response at
low concentrations compared to an agent of lower potency that
evokes a smaller response at low concentrations. Potency is a
function of affinity and efficacy. Efficacy refers to the ability
of therapeutic agent to produce a biological response upon binding
to a target ligand and the quantitative magnitude of this response.
The term half maximal effective concentration (EC50) refers to the
concentration of a therapeutic agent which causes a response
halfway between the baseline and maximum after a specified exposure
time. The therapeutic agent may cause inhibition or stimulation. It
is commonly used, and is used herein, as a measure of potency.
[0130] A neutralising polypeptide for the purposes of the invention
is a polypeptide which binds to an agent (such as TNF-alpha)
inhibiting the binding of the agent to one or more of its cognate
receptors (such as TNFR1 and TNFR2), as measured by ELISA.
Alternatively, or in addition, a neutralising polypeptide for the
purposes of the invention is a polypeptide which defends a cell
from the effects of an agent (such as TNF-alpha) by, for example,
inhibiting the biological effect of the agent. For example, a
neutralising polypeptide for the purposes of the invention is a
polypeptide which defends a cell from the effects of a toxin (such
as Clostridium Difficile Toxin A or B--"TcdA/TcdB") by, for
example, inhibiting the biological effect of the toxin.
Alternatively, or in addition, a neutralising polypeptide for the
purposes of the invention is a polypeptide which binds to IL-6R
(and therefore the IL-6R/IL-6 complex), inhibiting binding of the
IL-6R/IL-6 complex to gp130, as measured by ELISA.
[0131] The effectiveness (e.g. neutralising ability) of a
therapeutic agent can be ascertained using a potency assay. A
particularly suitable potency assay is the measurement of Vero cell
viability using Alamar Blue (Fields and Lancaster American
Biotechnology Laboratory 1993 11(4):48-50). Using a range of known
concentrations of a toxin, this assay can be performed to ascertain
the ability of a therapeutic polypeptide to neutralise the effects
of the toxin by producing a dose-response curve and/or by
ascertaining the half maximal effective concentration (EC50) of the
therapeutic polypeptide. This Vero Cell Cytotoxicity Standard Assay
is used herein and detailed further in the Examples section
below.
[0132] Another particularly suitable potency assay is the Standard
TNFR2/TNF Interference ELISA Assay (detailed further in the
Examples section below), which tests the effectiveness of a
therapeutic agent in blocking TNF-alpha binding to TNFR2, in
respect of a range of known concentrations of agent, producing a
dose-response curve and/or by ascertaining the half maximal
effective concentration (EC50) of the therapeutic polypeptide.
[0133] Another particularly suitable potency assay is the Standard
gp130 ELISA Assay (detailed further in the Examples section below),
which tests the effectiveness of a therapeutic agent in blocking
the sIL-6/IL-6R complex binding to gp130, in respect of a range of
known concentrations of agent, producing a dose-response curve
and/or by ascertaining the half maximal effective concentration
(EC50) of the therapeutic polypeptide.
[0134] Suitably the potency of the polypeptide of the invention is
substantially the same as the potency of a corresponding
polypeptide not having histidine substitutions of the
invention.
[0135] Suitably, the polypeptide of the invention or the
polypeptide of the methods of the invention inhibits binding of a
binding agent to a binding partner, such as TNF-alpha to TNFR2 in
the Standard TNF/TNFR2 Interference ELISA Assay, with an EC50 of
300 nM or less, more suitably 200 nM or less, more suitably 100 nM
or less, more suitably 80 nM or less, more suitably 60 nM or less,
more suitably 40 nM or less, more suitably 20 nM or less, more
suitably 10 nM or less, more suitably 5 nM or less.
[0136] Suitably, the EC50 of the polypeptide of the invention or
the polypeptide of the methods of the invention is increased by no
more than 300 pM, more suitably no more than 200 pM, more suitably
no more than 100 pM, more suitably no more than 50 pM, more
suitably no more than 25 pM, more suitably no more than 10 pM, more
suitably no more than 5 pM, relative to a corresponding polypeptide
not having histidine substitutions of the invention, such as in
inhibiting binding of TNF-alpha to TNFR2 in the Standard TNF/TNFR2
Interference ELISA Assay.
[0137] Suitably, the EC50 of the polypeptide of the invention or
the polypeptide of the methods of the invention is increased by no
more than 500%, more suitably 400%, more suitably 300%, more
suitably 200%, more suitably 100%, more suitably 70%, more suitably
60%, more suitably 50%, more suitably 40%, more suitably 30%, more
suitably 25%, more suitably 20%, more suitably 15%, more suitably
10%, more suitably 5%, more suitably 2%, more suitably 1%, relative
to a corresponding polypeptide not having histidine substitutions
of the invention, such as in inhibiting binding of TNF-alpha to
TNFR2 in the Standard TNF/TNFR2 Interference ELISA Assay.
[0138] Suitably the polypeptide of the invention or the polypeptide
of the methods of the invention neutralizes the cytotoxicity of a
toxin, such as TcdA or TcdB, in the Vero Cell Cytotoxicity Standard
Assay with an EC50 of 100 nM or less, more suitably 80 nM or less,
more suitably 60 nM or less, more suitably 40 nM or less, more
suitably 30 nM or less, more suitably 20 nM or less, more suitably
10 nM or less, more suitably 9 nM or less, more suitably 8 nM or
less, more suitably 7 nM or less, jmore suitably 6 nM or less more
suitably 5 nM or less, more suitably 4 nM or less, more suitably 3
nM or less, more suitably 2 nM or less, more suitably 1 nM or
less.
[0139] Suitably, the EC50 of the polypeptide of the invention or
the polypeptide of the methods of the invention is increased by no
more than 200 nM, more suitably 150 nM, more suitably 100 nM, more
suitably 80 nM, more suitably 60 nM, more suitably 40 nM, more
suitably 20 nM, more suitably 10 nM, more suitably 5 nM, relative
to a corresponding polypeptide not having histidine substitutions
of the invention, in neutralising the cytotoxicity of a toxin, such
as TcdA or TcdB, in the Vero Cell Cytotoxicity Standard Assay.
[0140] Suitably, the EC50 of the polypeptide of the invention or
the polypeptide of the methods of the invention is increased by no
more than 500%, more suitably 400%, more suitably 300%, more
suitably 200%, more suitably 100%, more suitably 70%, more suitably
60%, more suitably 50%, more suitably 40%, more suitably 30%, more
suitably 25%, more suitably 20%, more suitably 15%, more suitably
10%, more suitably 5%, more suitably 2%, more suitably 1%, relative
to a corresponding polypeptide not having histidine substitutions
of the invention, in neutralising the cytotoxicity of a toxin, such
as TcdA or TcdB, in the Vero Cell Cytotoxicity Standard Assay.
[0141] Suitably, the polypeptide of the invention or the
polypeptide of the methods of the invention inhibits binding of a
binding agent to a binding partner, such the sIL-6/IL-6R complex
binding to gp130 in the Standard gp130 ELISA Assay, with an EC50 of
300 nM or less, more suitably 200 nM or less, more suitably 100 nM
or less, more suitably 80 nM or less, more suitably 60 nM or less,
more suitably 40 nM or less, more suitably 20 nM or less, more
suitably 10 nM or less, more suitably 5 nM or less, more suitably 1
nM or less, more suitably 0.5 nM or less, more suitably 0.3 nM or
less, more suitably 0.2 nM or less, more suitably 0.15 nM or
less.
[0142] Suitably, the EC50 of the polypeptide of the invention or
the polypeptide of the methods of the invention is increased by no
more than 300 pM, more suitably no more than 200 pM, more suitably
no more than 100 pM, more suitably no more than 80 pM, more
suitably no more than 70 pM, more suitably no more than 60 pM, more
suitably no more than 50 pM, more suitably no more than 25 pM, more
suitably no more than 20 pM, more suitably no more than 15 pM, more
suitably no more than 10 pM, more suitably no more than 5 pM,
relative to a corresponding polypeptide not having histidine
substitutions of the invention, such as in inhibiting binding of a
binding agent to a binding partner, such the sIL-6/IL-6R complex
binding to gp130 in the Standard gp130 ELISA Assay.
[0143] Suitably, the EC50 of the polypeptide of the invention or
the polypeptide of the methods of the invention is increased by no
more than 600%, more suitably no more than 500%, more suitably
400%, more suitably 300%, more suitably 200%, more suitably 100%,
more suitably 70%, more suitably 60%, more suitably 50%, more
suitably 40%, more suitably 30%, more suitably 25%, more suitably
20%, more suitably 15%, more suitably 10%, more suitably 5%, more
suitably 2%, more suitably 1%, relative to a corresponding
polypeptide not having histidine substitutions of the invention,
such the sIL-6/IL-6R complex binding to gp130 in the Standard gp130
ELISA Assay.
[0144] Substitutions may be made to a polypeptide with the
objective of introducing pH sensitivity, for example to
significantly reduce the affinity of an antibody for an antigen
upon entry of the antibody into the acidic endosome. However, the
substitutions of the present invention suitably do not invoke
substantial pH sensitivity. Suitably the substitutions to the
polypeptide of the invention or the substitutions to the
polypeptide of the methods of the invention are not for engineering
pH dependency of target binding. Suitably the affinity of the
polypeptide of the invention or the polypeptide of the methods of
the invention remains substantially the same at any pH from 3 to 9,
more suitably any pH from 4 to 8.
[0145] The Gastrointestinal Tract (GIT) and Digestive Enzymes
[0146] The GIT is an organ system responsible for consuming and
digesting foodstuffs, absorbing nutrients, and expelling waste. In
humans and other mammals, the GIT consists of the mouth,
oesophagus, stomach, small intestine (duodenum, jejunum and ileum)
and large intestine (cecum, colon, rectum and anal canal). The
intestinal tract, as opposed to the gastrointestinal tract,
consists of only the small intestine and the large intestine.
Various pathogens may colonise, and various diseases may manifest
in, different areas of the gastrointestinal tract.
[0147] The different parts of the gastrointestinal tract each
contain a complex mixture of digestive enzymes. These digestive
enzymes include proteases, lipases, amylases and nucleases.
Proteases include serine proteases, threonine proteases, cysteine
proteases, aspartate proteases, glutamic acid proteases and
metalloproteases. Proteases are involved in digesting polypeptide
chains into shorter fragments by splitting the peptide bonds that
link amino acid residues (proteolysis). Some detach the terminal
amino acids from the protein chain (exopeptidases); others attack
internal peptide bonds of a protein (endopeptidases). The
intestinal tract comprises a vast array of different proteases.
[0148] Proteolysis in the intestinal tract can be highly
promiscuous such that a wide range of protein substrates are
hydrolysed by the wide variety of proteases present. This is the
case for proteases which cleave the wide array of ingested
polypeptides in the intestinal tract into smaller polypeptide
fragments.
[0149] Suitably the substitutions made to the polypeptide of the
invention or to the polypeptide of the methods of the invention
increase the stability of the polypeptide to one or more proteases
present in the small or large intestine, relative to a
corresponding polypeptide not having histidine substitutions of the
invention. Suitably the proteases include proteases originating
from intestinal microbiota or pathogenic bacteria, for example
wherein the proteases are cell membrane-attached proteases,
secreted proteases and/or proteases released on cell lysis.
Suitably the one or more proteases are selected from the group
consisting of trypsin, chymotrypsin, host inflammatory proteases,
proteases originating from microbiota and proteases originating
from pathogenic bacteria such as C. difficile-specific proteases.
Suitably the intestinal tract is a mammalian intestinal tract, such
as a human, simian, murine, bovine, ovine, canine, feline, equine
or porcine intestinal tract.
[0150] Suitably the substitutions made to the polypeptide of the
invention, or substitutions made to the polypeptide of the methods
of the invention, increase the stability of the polypeptide in the
intestinal tract, or in a model of the intestinal tract, such as in
the small and/or large intestine, such as in the duodenum, jejunum,
ileum cecum, colon, rectum and/or anal canal, relative to a
corresponding polypeptide not having histidine substitutions of the
invention. Suitably the model of the intestinal tract is the
Standard Human Faecal Supernatant Intestinal Tract Model, the
Standard Mouse Small Intestinal Supernatant Intestinal Tract Model,
or the Standard Trypsin Intestinal Tract Model.
[0151] Suitably at least 20%, more suitably at least 25%, more
suitably at least 30%, more suitably at least 35%, more suitably at
least 40%, more suitably at least 50%, more suitably at least 60%
of the polypeptide of the invention or the polypeptide of the
methods of the invention remains viable, as determined for example
by the Standard TNFR2/TNF Interference ELISA Assay when the ICVD is
an anti-TNF-alpha ICVD or the Standard Toxin ELISA Assay when the
ICVD is an anti-toxin ICVD, after 6 or 16 hours incubation in the
Standard Mouse Small Intestinal Supernatant Intestinal Tract
Model.
[0152] Suitably the stability of a polypeptide of the invention or
the polypeptide of the methods of the invention, as determined for
example by the Standard TNFR2/TNF Interference ELISA Assay when the
ICVD is an anti-TNF-alpha ICVD or the Standard Toxin ELISA Assay
when the ICVD is an anti-toxin ICVD, is increased by at least 1%,
more suitably 2%, more suitably 3%, more suitably 5%, more suitably
7%, more suitably 10%, more suitably 15%, more suitably 20%, more
suitably 30%, more suitably 40%, more suitably 50%, more suitably
60%, more suitably 70%, relative to a corresponding polypeptide not
having histidine substitutions of the invention, after 6 or 16
hours incubation in the Standard Mouse Small Intestinal Supernatant
Intestinal Tract Model.
[0153] Suitably at least 20%, more suitably at least 25%, more
suitably at least 30%, more suitably at least 35%, more suitably at
least 40%, more suitably at least 50%, more suitably at least 60%,
more suitably at least 70%, more suitably at least 80%, more
suitably at least 90% of the polypeptide of the invention or the
polypeptide of the methods of the invention remains viable, as
determined for example by the Standard TNFR2/TNF Interference ELISA
Assay when the ICVD is an anti-TNF-alpha ICVD, the Standard Toxin
ELISA Assay when the ICVD is an anti-toxin ICVD or the Standard
Western Blot Stability Assay after 30 minutes, 1 hour, 4 hours or
16 hours incubation in the Standard Human Faecal Supernatant
Intestinal Tract Model.
[0154] Suitably the stability of a polypeptide of the invention or
the polypeptide of the methods of the invention, as determined for
example by the Standard TNFR2/TNF Interference ELISA Assay when the
ICVD is an anti-TNF-alpha ICVD, the Standard Toxin ELISA Assay when
the ICVD is an anti-toxin ICVD or the Standard Western Blot
Stability Assay, is increased by at least 1%, more suitably 2%,
more suitably 3%, more suitably 5%, more suitably 7%, more suitably
10%, more suitably 15%, more suitably 20%, more suitably 25%, more
suitably 30%, more suitably 40%, more suitably 50%, more suitably
60%, more suitably 70%, relative to a corresponding polypeptide not
having histidine substitutions of the invention, after 30 minutes,
1 hour, 4 hours or 16 hours incubation in the Standard Human Faecal
Supernatant Intestinal Tract Model.
[0155] Suitably at least 5%, more suitably at least 10%, more
suitably at least at least 20%, more suitably at least 25%, more
suitably at least 30%, more suitably at least 35%, more suitably at
least 40%, more suitably at least 50%, more suitably at least 60%
of the polypeptide of the invention or the polypeptide of the
methods of the invention remains viable, as determined for example
by the Standard gp130 ELISA Assay when the ICVD is an anti-IL-6R
ICVD, after 4 hours incubation in the Standard Mouse Small
Intestinal Supernatant Intestinal Tract Model.
[0156] Suitably the stability of a polypeptide of the invention or
the polypeptide of the methods of the invention, as determined for
example by the Standard gp130 ELISA Assay when the ICVD is an
anti-IL-6R ICVD, is increased by at least 1%, more suitably 2%,
more suitably 3%, more suitably 5%, more suitably 7%, more suitably
10%, more suitably 15%, more suitably 20%, more suitably 30%, more
suitably 40%, more suitably 50%, more suitably 60%, more suitably
70%, relative to a corresponding polypeptide not having histidine
substitutions of the invention, after 4 hours incubation in the
Standard Mouse Small Intestinal Supernatant Intestinal Tract
Model.
[0157] Suitably at least 20%, more suitably at least 25%, more
suitably at least 30%, more suitably at least 35%, more suitably at
least 40%, more suitably at least 50%, more suitably at least 60%,
more suitably at least 70%, more suitably at least 80%, more
suitably at least 90% of the polypeptide of the invention or the
polypeptide of the methods of the invention remains viable, as
determined for example by the Standard gp130 ELISA Assay when the
ICVD is an anti-IL-6R ICVD after 16 hours incubation in the
Standard Human Faecal Supernatant Intestinal Tract Model.
[0158] Suitably the stability of a polypeptide of the invention or
the polypeptide of the methods of the invention, as determined for
example by the Standard gp130 ELISA Assay when the ICVD is an
anti-IL-6R ICVD, is increased by at least 1%, more suitably 2%,
more suitably 3%, more suitably 5%, more suitably 7%, more suitably
10%, more suitably 15%, more suitably 20%, more suitably 25%, more
suitably 30%, more suitably 40%, more suitably 50%, more suitably
60%, more suitably 70%, relative to a corresponding polypeptide not
having histidine substitutions of the invention, after 16 hours
incubation in the Standard Human Faecal Supernatant Intestinal
Tract Model.
[0159] The percentage of `viable` ICVD remaining after incubation
refers to the proportion of intact ICVD (for example in the
Standard Western Blot Stability Assay), or the proportion of
functional ICVD (for example in the Standard TNFR2/TNF Interference
ELISA Assay when the ICVD is an anti-TNF-alpha ICVD or Standard
Toxin ELISA Assay when the ICVD is an anti-toxin ICVD).
Alternatively, or in addition, the percentage of `viable` ICVD
remaining after incubation refers to the proportion of intact ICVD
(for example in the Standard Western Blot Stability Assay), or the
proportion of functional ICVD (for example in the Standard gp130
ELISA Assay when the ICVD is an anti-IL-6R ICVD).
[0160] Diseases of the Gastrointestinal Tract
[0161] Diseases of the gastrointestinal tract refer to diseases
involving the gastrointestinal tract, namely the oesophagus,
stomach, small intestine (duodenum, jejunum and ileum) and large
intestine (cecum, colon, rectum and anal canal). The polypeptide of
the invention or the polypeptide of the methods of the invention
may be used in the treatment or prevention of such diseases.
Suitably the polypeptide of the invention or the polypeptide of the
methods of the invention is used in local and/or topical treatment
or prevention of such diseases.
[0162] Exemplary Diseases of the Gastrointestinal Tract are
Described Below.
[0163] Autoimmune Diseases and/or Inflammatory Diseases of the
Gastrointestinal Tract
[0164] Autoimmune diseases develop when the immune system responds
adversely to normal body tissues. Autoimmune disorders may result
in damage to body tissues, abnormal organ growth and/or changes in
organ function. The disorder may affect only one organ or tissue
type or may affect multiple organs and tissues. Organs and tissues
commonly affected by autoimmune disorders include blood components
such as red blood cells, blood vessels, connective tissues,
endocrine glands such as the thyroid or pancreas, muscles, joints
and skin. An inflammatory disease is a disease characterised by
inflammation. Many inflammatory diseases are autoimmune diseases
and vice-versa.
[0165] The chronic inflammatory bowel diseases (IBDs) Crohn's
disease and ulcerative colitis, which afflict both children and
adults, are examples of autoimmune and inflammatory diseases of the
gastrointestinal tract (Hendrickson et al 2002 Clin Microbiol Rev
15(1):79-94, herein incorporated by reference in its entirety).
Ulcerative colitis is defined as a condition where the inflammatory
response and morphologic changes remain confined to the colon. The
rectum is involved in 95% of patients. Inflammation is largely
limited to the mucosa and consists of continuous involvement of
variable severity with ulceration, edema, and hemorrhage along the
length of the colon (Hendrickson et al 2002 Clin. Microbiol Rev
15(1):79-94, herein incorporated by reference in its entirety).
Ulcerative colitis is usually manifested by the presence of blood
and mucus mixed with stool, along with lower abdominal cramping
which is most severe during the passage of bowel movements.
Clinically, the presence of diarrhoea with blood and mucus
differentiates ulcerative colitis from irritable bowel syndrome, in
which blood is absent. Unlike ulcerative colitis, the presentation
of Crohn's disease is usually subtle, which leads to a later
diagnosis. Factors such as the location, extent, and severity of
involvement determine the extent of symptoms. Patients who have
ileocolonic involvement usually have postprandial abdominal pain,
with tenderness in the right lower quadrant and an occasional
inflammatory mass.
[0166] Suitably the composition of the invention is for use in the
treatment of an autoimmune and/or inflammatory disease of the
gastrointestinal tract, suitably selected from the list consisting
of Crohn's disease, ulcerative colitis, irritable bowel syndrome,
diabetes type II, glomerulonephritis, autoimmune hepatitis,
Sjogren's syndrome, coeliac disease and drug- or radiation-induced
mucositis (most suitably Crohn's disease).
[0167] Infection of the Gastrointestinal Tract
[0168] Viral, bacterial, parasitic and other pathogenic infections
can occur in the gastrointestinal tract. These may be confined to
the gastrointestinal tract or initiated in the gastrointestinal
tract before spreading to other parts of the body. The polypeptide
of the invention may be used for the treatment or prevention of
bacterial infection including infection by common bacterial
gastrointestinal tract pathogens including Escherichia coli,
Salmonella, Campylobacter, Vibrio cholerae, Shigella, Clostridium
perfringens, Clostridium difficile, Bacillus cereus, Vibrio
parahaemolyticus and Yersinia enerocolitica. The polypeptide of the
invention may be used for the treatment or prevention of viral
infection including common viral gastrointestinal tract pathogens
which include rotavirus, norovirus and small round viruses.
Suitably the polypeptide of the invention is for use in the
treatment or prevention of nosocomial infection. Suitably the
polypeptide of the invention is for use in the treatment or
prevention of C. difficile infection.
[0169] Suitably, the polypeptide of the invention binds to a target
accessible via the intestinal tract, such as a target within the
intestinal tract. Suitably the target is a deleterious agent
originating from an intestinal tract resident pathogenic microbe.
Suitably the target is a target originating from host microbiota
which may induce pathogenesis, a host cell, host derived
inflammatory mediators or a protein involved in disease
pathogenesis. Suitably the target is selected from the group
consisting of: TNF-alpha, C. difficile toxin A, or C. difficile
toxin B. Alternatively the target is selected from the group
consisting of: IL-6R, TNF-alpha, C. difficile toxin A, or C.
difficile toxin B.
[0170] Linkers and Multimers
[0171] A construct according to the invention comprises multiple
polypeptides and therefore may suitably be multivalent. Such a
construct may comprise at least two identical polypeptides
according to the invention. A construct consisting of two identical
polypeptides according to the invention is a "homobihead". In one
aspect of the invention there is provided a construct comprising a
polypeptide of the invention. In a further aspect there is provided
a construct comprising two or more (possibly identical)
polypeptides of the invention.
[0172] Alternatively, a construct may comprise at least two
polypeptides which are different, but are both still polypeptides
according to the invention (a "heterobihead").
[0173] Alternatively, such a construct may comprise (a) at least
one polypeptide according to the invention and (b) at least one
polypeptide such as an antibody or antigen-binding fragment
thereof, which is not a polypeptide of the invention (also a
"heterobihead"). The at least one polypeptide of (b) may bind
TNF-alpha, TcdA or TcdB (for example via a different epitope to
that of (a)), or alternatively may bind to another target
altogether. Suitably the different polypeptide (b) binds to, for
example, another pro inflammatory cytokine or chemokine or their
respective receptors, other inflammatory mediators or
immunologically relevant ligands involved in human pathological
processes.
[0174] Constructs can be multivalent and/or multispecific. A
multivalent construct (such as a bivalent construct) comprises two
or more binding polypeptides therefore presents two or more sites
at which attachment to one or more antigens can occur. An example
of a multivalent construct could be a homobihead or a heterobihead.
A multispecific construct (such as a bispecific construct)
comprises two or more different binding polypeptides which present
two or more sites at which either (a) attachment to two or more
different antigens can occur or (b) attachment to two or more
different epitopes on the same antigen can occur. An example of a
multispecific construct could be a heterobihead. A multispecific
construct is multivalent.
[0175] Suitably, the polypeptides comprised within the construct
are antibody fragments. More suitably, the polypeptides comprised
within the construct are selected from the list consisting of: a
VHH, a VH, a VL, a V-NAR, scFv, a Fab fragment or a F(ab')2
fragment. More suitably, the polypeptides comprised within the
construct are VHHs.
[0176] The polypeptides of the invention can be linked to each
other directly (i.e. without use of a linker) or via a linker. The
linker is suitably a polypeptide and will be selected so as to
allow binding of the polypeptides to their epitopes. If used for
therapeutic purposes, the linker is suitably non-immunogenic in the
subject to which the polypeptides are administered. Suitably the
polypeptides are all connected by linkers. Suitably the linker is
of the format (G.sub.4S).sub.x. Most suitably.times.is 6.
[0177] Therapeutic use and Delivery
[0178] Suitably the polypeptide of the invention is for use as a
medicament, delivered by oral administration, suitably for use in
the treatment or prevention of diseases of the gastrointestinal
tract (see supra). The polypeptide of the invention or the
polypeptide of the methods of the invention may also be used in the
treatment or prevention of other medical conditions by oral
administration such as metabolic disorders, such as obesity. In one
embodiment, the polypeptide of the invention is intended to have
local effect in the intestinal tract. In one embodiment, the
polypeptide of the invention or the polypeptide of the methods of
the invention is not for use in the treatment or prevention of
diseases by delivery into the circulation in therapeutically
effective quantities.
[0179] In one aspect of the invention there is provided a method of
treating diseases of the gastrointestinal tract comprising
administering to a person in need thereof a therapeutically
effective amount of the inventive polypeptide.
[0180] A therapeutically effective amount of a polypeptide is an
amount which is effective, upon single or multiple dose
administration to a subject, in neutralising the biological effects
of a chosen target to a significant extent in a subject. A
therapeutically effective amount may vary according to factors such
as the disease state, age, sex, and weight of the individual, and
the ability of the polypeptide to elicit a desired response in the
individual. A therapeutically effective amount is also one in which
any toxic or detrimental effects of the polypeptide are outweighed
by the therapeutically beneficial effects. The polypeptide of the
invention can be incorporated into pharmaceutical compositions
suitable for oral administration to a subject. The polypeptide of
the invention can be in the form of a pharmaceutically acceptable
salt.
[0181] In one aspect of the invention, there is provided a
pharmaceutical composition comprising a polypeptide of the
invention and one or more pharmaceutically acceptable diluents or
carriers.
[0182] A pharmaceutical composition of the invention may be
formulated for oral delivery. The pharmaceutical compositions of
the invention may be in a variety of forms. These include, for
example, liquid, semi-solid and solid dosage forms, such as liquid
solutions, dispersions or suspensions, tablets, pills and powders.
Solid dosage forms are preferred. The pharmaceutical composition
may comprise a pharmaceutically acceptable excipient, and suitably
may be used in the form of ingestible tablets, buccal tablets,
troches, capsules, elixirs, suspensions, syrups, wafers, and the
like.
[0183] Typically, the composition of the invention or
pharmaceutical composition of the invention comprises a polypeptide
of the invention and a pharmaceutically acceptable excipient such
as a carrier. Examples of pharmaceutically acceptable carriers
include one or more of water, saline, phosphate buffered saline,
dextrose, glycerol, ethanol and the like, as well as combinations
thereof. Pharmaceutically acceptable carriers may further comprise
minor amounts of auxiliary substances such as wetting or
emulsifying agents, preservatives or buffers, which enhance the
shelf life or effectiveness of the polypeptide of the invention.
Pharmaceutical compositions may include antiadherents, binders,
coatings, disintegrants, flavours, colours, lubricants, sorbents,
preservatives, sweeteners, freeze dry excipients (including
lyoprotectants) or compression aids. Suitably, the polypeptide of
the invention is lyophilised before being incorporated into a
pharmaceutical composition.
[0184] A polypeptide of the invention may also be provided with an
enteric coating. An enteric coating is a polymer barrier applied on
oral medication which protects the polypeptide from the low pH of
the stomach. Materials used for enteric coatings include fatty
acids, waxes, shellac, plastics, and plant fibers. Suitable enteric
coating components include methyl acrylate-methacrylic acid
copolymers, cellulose acetate succinate, hydroxy propyl methyl
cellulose phthalate, hydroxy propyl methyl cellulose acetate
succinate (hypromellose acetate succinate), polyvinyl acetate
phthalate (PVAP), methyl methacrylate-methacrylic acid copolymers,
sodium alginate and stearic acid. Suitable enteric coatings include
pH-dependent release polymers. These are polymers which are
insoluble at the highly acidic pH found in the stomach, but which
dissolve rapidly at a less acidic pH. Thus, suitably, the enteric
coating will not dissolve in the acidic juices of the stomach (pH
.about.3), but will do so in the higher pH environment present in
the small intestine (pH above 6) or in the colon (pH above 7.0).
The pH-dependent release polymer is selected such that the
polypeptide of the invention will be released at about the time
that the dosage reaches the target region of the intestinal
tract.
[0185] The composition of the invention may be formulated in a
buffer, in order to stabilise the pH of the composition, at a
concentration between 5-50, or more suitably 15-40 or more suitably
25-30 g/litre. Examples of suitable buffer components include
physiological salts such as sodium citrate and/or citric acid.
Suitably buffers contain 100-200, more suitably 125-175 mM
physiological salts such as sodium chloride. Suitably the buffer is
selected to have a pKa close to the pH of the composition or the
physiological pH of the patient.
[0186] Exemplary polypeptide concentrations in a pharmaceutical
composition may range from about 10 ng/mL to about 200 mg/mL, such
as about 50 ng/mL to about 100 mg/mL, such as about 1 ug/mL to
about 80 mg/mL, such as about 10 .mu.g/mL to about 50 mg/mL, such
as about 50 ug/mL to about 30 mg/mL, such as about 100 ug/mL to
about 20 mg/mL, or about 1 mg/mL to about 200 mg/ml or from about
50 mg/mL to about 200 mg/mL, or from about 150 mg/mL to about 200
mg/mL.
[0187] An aqueous formulation of the polypeptide of the invention
may be prepared in a pH-buffered solution, e.g., at pH ranging from
about 4.0 to about 7.0, or from about 5.0 to about 6.0, or
alternatively about 5.5. Examples of suitable buffers include
phosphate-, histidine-, citrate-, succinate-, acetate-buffers and
other organic acid buffers. The buffer concentration can be from
about 1 mM to about 100 mM, or from about 5 mM to about 50 mM,
depending, for example, on the buffer and the desired tonicity of
the formulation.
[0188] The tonicity of the pharmaceutical composition may be
altered by including a tonicity modifier. Such tonicity modifiers
can be charged or uncharged chemical species. Typical uncharged
tonicity modifiers include sugars or sugar alcohols or other
polyols, preferably trehalose, sucrose, mannitol, glycerol,
1,2-propanediol, raffinose, sorbitol or lactitol (especially
trehalose, mannitol, glycerol or 1,2-propanediol). Typical charged
tonicity modifiers include salts such as a combination of sodium,
potassium or calcium ions, with chloride, sulfate, carbonate,
sulfite, nitrate, lactate, succinate, acetate or maleate ions
(especially sodium chloride or sodium sulphate); or amino acids
such as arginine or histidine. Suitably, the aqueous formulation is
isotonic, although hypertonic or hypotonic solutions may be
suitable. The term "isotonic" denotes a solution having the same
tonicity as some other solution with which it is compared, such as
physiological salt solution or serum. Tonicity agents may be used
in an amount of about 5 mM to about 350 mM, e.g., in an amount of 1
mM to 500 nM. Suitably, at least one isotonic agent is included in
the composition.
[0189] A surfactant may also be added to the pharmaceutical
composition to reduce aggregation of the formulated polypeptide
and/or minimize the formation of particulates in the formulation
and/or reduce adsorption. Exemplary surfactants include
polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene
alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers (Triton-X),
polyoxyethylene-polyoxypropylene copolymer (Poloxamer, Pluronic),
and sodium dodecyl sulfate (SDS). Examples of suitable
polyoxyethylenesorbitan-fatty acid esters are polysorbate 20, and
polysorbate 80. Exemplary concentrations of surfactant may range
from about 0.001% to about 10% w/v.
[0190] A lyoprotectant may also be added in order to protect the
polypeptide of the invention against destabilizing conditions
during the lyophilization process. For example, known
lyoprotectants include sugars (including glucose, sucrose, mannose
and trehalose); polyols (including mannitol, sorbitol and
glycerol); and amino acids (including alanine, glycine and glutamic
acid). Lyoprotectants can be included in an amount of about 10 mM
to 500 mM.
[0191] The dosage ranges for administration of the pharmaceutical
composition of the invention are those to produce the desired
therapeutic effect. The dosage range required depends on the
precise nature of the pharmaceutical composition, the target region
of the intestinal tract, the nature of the formulation, the age of
the patient, the nature, extent or severity of the patient's
condition, contraindications, if any, and the judgement of the
attending physician. Variations in these dosage levels can be
adjusted using standard empirical routines for optimisation.
[0192] The increased intestinal stability of a polypeptide of the
invention means that a lower dose may be delivered orally than
would otherwise need to be delivered orally in the case of a
corresponding polypeptide not having histidine substitutions of the
invention.
[0193] Suitable daily dosages of a polypeptide of the invention or
pharmaceutical composition of the invention are in the range of 50
ng-50 mg per kg, such as 50 ug-40 mg per kg, such as 5-30 mg per kg
of (e.g. human) body weight, such as less than 25, such as less
than 20, such as less than 15, such as less than 10 mg, such as
less than 50 ug, such as less than 50 ng per kg of body weight. The
unit dose will typically will be in the region of 250-2000 mg per
dose, such as from less than 1000 mg, such as less than 700 mg,
such as less than 400 mg, such as less than 100 mg, such as less
than 100 ug, such as less than 50 ug, such as less than 10 ug, such
as less than 100 ng, such as less than Song.
[0194] A dose may be administered daily or more frequently, for
example 2, 3 or 4 times per day or less frequently for example
every other day, once per week, once per fortnight or once per
month.
[0195] Treatment of diseases also embraces treatment of
exacerbations thereof and also embraces treatment of patients in
remission from disease symptoms to prevent relapse of disease
symptoms.
[0196] Combination Therapy
[0197] A pharmaceutical composition of the invention may also
comprise one or more active agents (e.g. active agents suitable for
treating diseases such as those mentioned herein). It is within the
scope of the invention to use the pharmaceutical composition of the
invention in therapeutic methods for the treatment of bacterial
infection, autoimmune and/or inflammatory diseases as an adjunct
to, or in conjunction with, other established therapies normally
used in the treatment of bacterial, autoimmune and/or inflammatory
diseases.
[0198] For the treatment of inflammatory bowel disease (such as
Crohn's disease or ulcerative colitis), possible combinations
include combinations with, for example, one or more active agents
selected from the list comprising: 5-aminosalicylic acid, or a
prodrug thereof (such as sulfasalazine, olsalazine or bisalazide);
corticosteroids (e.g. prednisolone, methylprednisolone, or
budesonide); immunosuppressants (e.g. cyclosporin, tacrolimus,
methotrexate, azathioprine or 6-mercaptopurine); anti-TNF-alpha
antibodies (e.g., infliximab, adalimumab, certolizumab pegol or
golimumab); anti-IL12/IL23 antibodies (e.g., ustekinumab);
anti-IL-6R antibodies or small molecule IL12/IL23 inhibitors (e.g.,
apilimod); Anti-alpha-4-beta-7 antibodies (e.g., vedolizumab);
MAdCAM-1 blockers (e.g., PF-00547659); antibodies against the cell
adhesion molecule alpha-4-integrin (e.g., natalizumab); antibodies
against the IL2 receptor alpha subunit (e.g., daclizumab or
basiliximab); JAK3 inhibitors (e.g., tofacitinib or R348); Syk
inhibitors and prodrugs thereof (e.g., fostamatinib and R-406);
Phosphodiesterase-4 inhibitors (e.g., tetomilast); HMPL-004;
probiotics; Dersalazine; semapimod/CPSI-2364; and protein kinase C
inhibitors (e.g. AEB-071). The most suitable combination agents are
infliximab, adalimumab, certolizumab pegol or golimumab.
[0199] For the treatment of bacterial infections, such as
Clostridium difficile infection, possible combinations include
combinations with, for example, one or more active agents selected
from the list comprising C. difficile toxoid vaccine, ampicillin,
amoxicillin, vancomycin, metronidazole, fidaxomicin, linezolid,
nitazoxanide, rifaximin, ramoplanin, difimicin, clindamycin,
cephalosporins (such as second and third generation
cephalosporins), fluoroquinolones (such as gatifloxacin or
moxifloxacin), macrolides (such as erythromycin, clarithromycin,
azithromycin), penicillins, aminoglycosides,
trimethoprim-sulfamethoxazole, chloramphenicol, tetracycline,
imipenem, meropenem, antibacterial agents, bactericides, or
bacteriostats. Possible combinations also include combinations with
one or more active agents which are probiotics, for example
Saccharomyces boulardii or Lactobacillus rhamnosus GG.
[0200] Hence another aspect of the invention provides a
pharmaceutical composition of the invention in combination with one
or more further active agents, for example one or more active
agents described above. In a further aspect of the invention, the
pharmaceutical composition or polypeptide is administered
sequentially, simultaneously or separately with at least one active
agent selected from the list above.
[0201] Similarly, another aspect of the invention provides a
combination product comprising:
[0202] (A) a pharmaceutical composition of the present invention;
and
[0203] (B) one or more other active agents,
[0204] wherein each of components (A) and (B) is formulated in
admixture with a pharmaceutically-acceptable adjuvant, diluent or
carrier. In this aspect of the invention, the combination product
may be either a single (combination) formulation or a kit-of-parts.
Thus, this aspect of the invention encompasses a combination
formulation including a pharmaceutical composition of the present
invention and another therapeutic agent, in admixture with a
pharmaceutically acceptable adjuvant, diluent or carrier.
[0205] The invention also encompasses a kit of parts comprising
components:
[0206] (i) a pharmaceutical composition of the present invention in
admixture with a pharmaceutically acceptable adjuvant, diluent or
carrier; and
[0207] (ii) a formulation including one or more other active
agents, in admixture with a pharmaceutically-acceptable adjuvant,
diluent or carrier, which components (i) and (ii) are each provided
in a form that is suitable for administration in conjunction with
the other.
[0208] Component (i) of the kit of parts is thus component (A)
above in admixture with a pharmaceutically acceptable adjuvant,
diluent or carrier. Similarly, component (ii) is component (B)
above in admixture with a pharmaceutically acceptable adjuvant,
diluent or carrier. The one or more other active agents (i.e.
component (B) above) may be, for example, any of the agents
mentioned above in connection with the treatment of bacterial
infection such as Clostridium difficile infection, autoimmune
and/or inflammatory diseases such as IBD (e.g. Crohn's disease
and/or ulcerative colitis). If component (B) is more than one
further active agent, these further active agents can be formulated
with each other or formulated with component (A) or they may be
formulated separately. In one embodiment component (B) is one other
therapeutic agent. In another embodiment component (B) is two other
therapeutic agents. The combination product (either a combined
preparation or kit-of-parts) of this aspect of the invention may be
used in the treatment or prevention of an autoimmune disease (e.g.
the autoimmune diseases mentioned herein).
[0209] Vectors and Hosts
[0210] The term "vector, as used herein, is intended to refer to a
nucleic acid molecule capable of transporting another nucleic acid
to which it has been linked. One type of vector is a plasmid, which
refers to a circular double stranded DNA loop into which additional
DNA segments may be ligated. Another type of vector is a viral
vector, wherein additional DNA segments may be ligated into the
viral genome. Certain vectors are capable of autonomous replication
in a host cell into which they are introduced (e.g., bacterial
vectors having a bacterial origin of replication and episomal
mammalian and yeast vectors). Other vectors (e.g. non-episomal
mammalian vectors) can be integrated into the genome of a host cell
upon introduction into the host cell, and thereby are replicated
along with the host genome. Moreover, certain vectors are capable
of directing the expression of genes to which they are operatively
linked. Such vectors are referred to herein as "recombinant
expression vectors" (or simply, "expression vectors"). In general,
expression vectors of utility in recombinant DNA techniques are
often in the form of plasmids. In the present specification,
"plasmid" and vector" may be used interchangeably as the plasmid is
the most commonly used form of vector. However, the invention is
intended to include such other forms of expression vectors, such as
viral vectors (e.g. replication defective retroviruses.
adenoviruses and adeno-associated viruses), which serve equivalent
functions, and also bacteriophage and phagemid systems. The
invention also relates to nucleotide sequences that encode
polypeptides of the invention. The term "recombinant host cell" (or
simply "host cell"), as used herein, is intended to refer to a cell
into which a recombinant expression vector has been introduced.
Such terms are intended to refer not only to the particular subject
cell but to the progeny of such a cell.
[0211] In one aspect of the invention there is provided a
polynucleotide encoding a polypeptide of the invention. In a
further aspect of the invention there is provided a vector
comprising the polynucleotide or cDNA comprising said
polynucleotide. In a further aspect of the invention there is
provided a host cell transformed with said vector, which is capable
of expressing the polypeptide of the invention. Suitably the host
cell is a mammalian cell, a plant cell, a yeast cell such as a
yeast cell belonging to the genera Aspergillus, Saccharomyces,
Kluyveromyces, Hansenula or Pichia, such as S. cerevisiae or P.
Pastoris; or a bacterial cell such as E. coli.
[0212] Preparative Methods
[0213] Polypeptides of the invention can be obtained and
manipulated using the techniques disclosed for example in Green and
Sambrook 2012 Molecular Cloning: A Laboratory Manual 4.sup.th
Edition Cold Spring Harbour Laboratory Press. Suitably the
substitutions made to the polypeptide of the invention, or
substitutions made in the methods of the invention, are introduced
synthetically. Suitably, the substitutions are not introduced by
V(D)J recombination or somatic mutation.
[0214] In particular, artificial gene synthesis may be used to
produce a polypeptide according to the invention (Nambiar et al
1984 Science 223:1299-1301, Sakamar and Khorana 1988 Nucl. Acids
Res 14:6361-6372, Wells et al 1985 Gene 34:315-323 and Grundstrom
et al 1985 Nucl. Acids Res 13:3305-3316, herein incorporated by
reference in their entirety). A gene encoding a polypeptide of the
invention can be synthetically produced by, for example,
solid-phase DNA synthesis. Entire genes may be synthesized de novo,
without the need for precursor template DNA. To obtain the desired
oligonucleotide, the building blocks are sequentially coupled to
the growing oligonucleotide chain in the order required by the
sequence of the product. Upon the completion of the chain assembly,
the product is released from the solid phase to solution,
deprotected, and collected. Products can be isolated by
high-performance liquid chromatography (HPLC) to obtain the desired
oligonucleotides in high purity (Verma and Eckstein 1998 Annu Rev
Biochem 67:99-134).
[0215] The constructs of the invention may be fused genetically at
the DNA level i.e. a polynucleotide construct which encodes the
complete construct comprising one or more polypeptides. One way of
joining multiple polypeptides via the genetic route is by linking
the polypeptide coding sequences via a labile peptide linker coding
sequence. For example, the carboxy-terminal end of the first
polypeptide may be linked to the amino-terminal end of the next
polypeptide via a labile peptide linker coding sequence. This
linking mode can be extended in order to link polypeptides for the
construction of tri-, tetra-, etc. functional constructs. A method
for producing multivalent (such as bivalent) VHH polypeptide
constructs is disclosed in WO96/34103 (herein incorporated by
reference in its entirety).
[0216] Mutations can be made to the DNA or cDNA that encode
polypeptides which are silent as to the amino acid sequence of the
polypeptide, but which provide preferred codons for translation in
a particular host. The preferred codons for translation of a
nucleic acid in, e.g., E. coli and S. cerevisiae, are known.
[0217] Mutation of polypeptides can be achieved for example by
substitutions, additions or deletions to a nucleic acid encoding
the polypeptide. A substitution is the replacement of a residue
with a different residue in the same, corresponding location. The
substitutions, additions or deletions to a nucleic acid encoding
the polypeptide can be introduced by many synthetic methods,
including for example error-prone PCR, shuffling,
oligonucleotide-directed mutagenesis, assembly PCR, PCR
mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive
ensemble mutagenesis, exponential ensemble mutagenesis,
site-specific mutagenesis (Ling et al 1997 Anal Biochem
254(2):157-178, herein incorporated by reference in its entirety),
gene reassembly, Gene Site Saturation Mutagenesis (GSSM), synthetic
ligation reassembly (SLR) or a combination of these methods. The
modifications, additions or deletions to a nucleic acid can also be
introduced by a method comprising recombination, recursive sequence
recombination, phosphothioate-modified DNA mutagenesis,
uracil-containing template mutagenesis, gapped duplex mutagenesis,
point mismatch repair mutagenesis, repair-deficient host strain
mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion
mutagenesis, restriction-selection mutagenesis,
restriction-purification mutagenesis, ensemble mutagenesis,
chimeric nucleic acid multimer creation, or a combination
thereof.
[0218] Expression of polypeptides comprising immunoglobulin chain
variable domains such as VHs and VHHs can be achieved using a
suitable expression vector such as a prokaryotic cell such as
bacteria, for example E. coli (for example according to the
protocols disclosed in WO94/04678 and WO96/34103, which are
incorporated herein by reference). Expression of immunoglobulin
chain variable domains such as VHs and VHHs can also be achieved
using eukaryotic cells, for example insect cells, CHO cells, Vero
cells or suitably yeast cells such as yeasts belonging to the
genera Aspergillus, Saccharomyces, Kuyveromyces, Hansenula or
Pichia. Suitably S. cerevisiae is used (for example according to
the protocols disclosed in WO94/025591, which is incorporated
herein by reference).
[0219] Suitably, a polypeptide of the invention can be produced in
a fungus such as a yeast (for example, S. cerevisiae) comprising
growth of the fungus on a medium comprising a carbon source wherein
50-100 wt % of said carbon source is ethanol, according to the
methods disclosed in WO02/48382.
[0220] Clauses
[0221] A set of clauses defining the invention and its preferred
aspects is as follows: [0222] 1. A polypeptide comprising an
immunoglobulin chain variable domain comprising three
complementarity determining regions (CDR1-CDR3) and four framework
regions, wherein: [0223] (a) at least one lysine residue in CDR1,
CDR2 and/or CDR3 has been substituted with at least one histidine
residue, and/or [0224] (b) at least one arginine residue in CDR1,
CDR2 and/or CDR3 has been substituted with at least one histidine
residue; [0225] wherein the polypeptide has increased intestinal
stability relative to a corresponding polypeptide not having said
histidine substitutions. [0226] 2. A method of increasing the
intestinal stability of a polypeptide comprising an immunoglobulin
chain variable domain, wherein the immunoglobulin chain variable
domain comprises three complementarity determining regions
(CDR1-CDR3) and four framework regions, wherein the method
comprises the step of substituting: [0227] (a) at least one lysine
residue in CDR1, CDR2 and/or CDR3 with at least one histidine
residue, and/or [0228] (b) at least one arginine residue in CDR1,
CDR2 and/or CDR3 with at least one histidine residue. [0229] 3. A
method of making a polypeptide comprising an immunoglobulin chain
variable domain, wherein the immunoglobulin chain variable domain
comprises three complementarity determining regions (CDR1-CDR3) and
four framework regions, wherein the method comprises the step of
substituting: [0230] (a) at least one lysine residue in CDR1, CDR2
and/or CDR3 with at least one histidine residue, and/or [0231] (b)
at least one arginine residue in CDR1, CDR2 and/or CDR3 with at
least one histidine residue [0232] wherein the polypeptide has
increased intestinal stability relative to a corresponding
polypeptide not having said histidine substitutions.
[0233] 4. The polypeptide, method of increasing the intestinal
stability of a polypeptide or method of making a polypeptide
according to any one of clauses 1 to 3, wherein the substitutions
increase the stability of the polypeptide in the intestinal tract,
such as in the small and/or large intestine, such as in the
duodenum, jejunum, ileum cecum, colon, rectum and/or anal canal,
relative to a corresponding polypeptide not having said histidine
substitutions.
[0234] 5. The polypeptide, method of increasing the intestinal
stability of a polypeptide or method of making a polypeptide
according to any one of clauses 1 to 4, wherein the substitutions
increase the stability of the polypeptide in a model of the
intestinal tract, such as in the small and/or large intestine, such
as in the duodenum, jejunum, ileum cecum, colon, rectum and/or anal
canal, relative to a corresponding polypeptide not having said
histidine substitutions.
[0235] 6. The polypeptide, method of increasing the intestinal
stability of a polypeptide or method of making a polypeptide
according to clause 5 wherein the model of the intestinal tract is
the Standard Human Faecal Supernatant Intestinal Tract Model.
[0236] 7. The polypeptide, method of increasing the intestinal
stability of a polypeptide or method of making a polypeptide
according to clause 6, wherein the stability of the polypeptide, as
determined by the Standard TNFR2/TNF Interference ELISA Assay when
the immunoglobulin chain variable domain is an anti-TNF-alpha
immunoglobulin chain variable domain, or the Standard gp130 ELISA
Assay when the immunoglobulin chain variable domain is an
anti-IL-6R immunoglobulin chain variable domain, is increased by at
least 1%, more suitably 5%, more suitably 10%, relative to a
corresponding polypeptide not having said histidine substitutions,
after 16 hours incubation in the Standard Human Faecal Supernatant
Intestinal Tract Model. [0237] 8. The polypeptide, method of
increasing the intestinal stability of a polypeptide or method of
making a polypeptide according to any one of clauses 1 to 7,
wherein the substitutions increase the stability of the polypeptide
to one or more proteases produced in the small or large intestine,
relative to a corresponding polypeptide not having said histidine
substitutions. [0238] 9. The polypeptide, method of increasing the
intestinal stability of a polypeptide or method of making a
polypeptide according to any one of clauses 1 to 8 wherein the
potency of the polypeptide is substantially the same as the potency
of a corresponding polypeptide not having said histidine
substitutions. [0239] 10. The polypeptide, method of increasing the
intestinal stability of a polypeptide or method of making a
polypeptide according to any one of clauses 1 to 9, wherein the at
least one lysine and/or arginine residue is present in a window
defined as the second third of CDR1 and/or the second third of CDR2
and/or the second third of CDR3. [0240] 11. The polypeptide, method
of increasing the intestinal stability of a polypeptide or method
of making a polypeptide according to clause 11, wherein each lysine
and/or arginine residue in CDR1, CDR2 and/or CDR3 has been
substituted with one histidine residue each. [0241] 12. The
polypeptide, method of increasing the intestinal stability of a
polypeptide or method of making a polypeptide according to any one
of clauses 1 to 12, wherein the polypeptide is an antibody, a
modified antibody containing additional antibody binding regions or
an antibody fragment such as an scFv, a Fab fragment, a F(ab')2
fragment or an immunoglobulin chain variable domain such as a VHH,
a VH, a VL, a V-NAR. [0242] 13. The polypeptide, method of
increasing the intestinal stability of a polypeptide or method of
making a polypeptide according to any one of clauses 1 to 12,
wherein the polypeptide binds to a target accessible via the
intestinal tract. [0243] 14. A pharmaceutical composition
comprising the polypeptide or construct according to any one of
clauses 1 to 13 for use as a medicament for oral administration.
[0244] 15. The pharmaceutical composition according to clause 14,
wherein the composition is presented in enterically coated
form.
Further Clauses
[0245] A set of further clauses defining the invention and its
preferred aspects is as follows. The features recited in claims 4
to 61 recited below optionally apply mutatis mutandis to these
further clauses 1 to 3. [0246] 1. A polypeptide comprising a region
which is capable of binding a target with high affinity, wherein:
[0247] (a) at least one lysine residue in the region has been
substituted with at least one histidine residue, and/or [0248] (b)
at least one arginine residue in the region has been substituted
with at least one histidine residue; [0249] wherein the polypeptide
has increased intestinal stability relative to a corresponding
polypeptide not having said histidine substitutions. [0250] 2. A
method of increasing the intestinal stability of a polypeptide
comprising a region which is capable of binding a target with high
affinity, wherein the method comprises the step of substituting:
[0251] (a) at least one lysine residue in the region with at least
one histidine residue, and/or [0252] (b) at least one arginine
residue in the region with at least one histidine residue. [0253]
3. A method of making a polypeptide comprising a region which is
capable of binding a target with high affinity, wherein the method
comprises the step of substituting: [0254] (a) at least one lysine
residue in the region with at least one histidine residue, and/or
[0255] (b) at least one arginine residue in the region with at
least one histidine residue, [0256] wherein the polypeptide has
increased intestinal stability relative to a corresponding
polypeptide not having said histidine substitutions.
[0257] The present invention will now be further described by means
of the following non-limiting examples.
EXAMPLES
Example 1
Standard Intestinal Tract Models, Standard Intestinal Stability
Assays and Standard Potency Assays
[0258] The intestinal stability and potency of a polypeptide
comprising an immunoglobulin chain variable domain can be assayed
using the following methods. The methods below refer to
[0259] ICVDs, but are equally applicable to any polypeptide which
comprises an ICVD, such as an antibody.
[0260] 1.1 Standard Intestinal Tract Models
[0261] Ex vivo samples from human faeces and mouse small intestine
samples are highly relevant matrices for estimation of stability in
the human intestinal tract. Such samples contain native
host-produced, and associated microbial-produced, proteases along
with any chaotropic agents or surfactants that may influence ICVD
stability in the presence of proteases. The enzymatic cleavage
sites of at least some proteases present in the small intestine
from murine and human origin are well characterised and conserved
between the two species. Murine small intestinal supernatants were
found to be a particularly stringent challenge in terms of total
protease activity by comparison to small intestinal samples from
pigs and clinically-derived human lavage samples of the small
intestine.
[0262] The intestinal tract models detailed below, which utilise ex
vivo samples from human faeces and mouse small intestine, therefore
allow one to assay the stability of a polypeptide comprising an
ICVD in an environment which is highly representative of the
conditions of the intestinal tract. The percentage of viable ICVD
remaining after incubation is assessed after incubation in an
intestinal tract model using an appropriate assay such as the
Standard Western Blot Stability Assay (for assaying proportions of
intact ICVD) or the Standard TNFR2/TNF Interference ELISA Assay or
Standard Toxin ELISA Assay (both for assaying proportions of
functional ICVD).
[0263] Note that from the point of sampling from mouse or human up
to the point of use in an ICVD stability assay, all
faecal/intestinal samples, slurries and supernatants should be kept
chilled on ice or manipulations such as centrifugation carried out
at 4.degree. C. Once generated, supernatant samples may be frozen
at -80.degree. C. and thawed once (or twice) before use. Repeat
freeze-thawing is likely to result in loss of protease stability.
Prolonged storage (>1 year) at -80.degree. C. does not appear to
reduce total protease activity. However, slurries and supernatants
should be monitored on a case-by-case basis over time.
[0264] 1.1.1 The Standard Human Faecal Supernatant Intestinal Tract
Model
[0265] Faecal Supernatant Pool Production
[0266] To generate supernatants for stability testing, 1.times. PBS
is added to faecal samples at a ratio of 1 or 2 mLs 1.times.PBS per
gram of faeces. The samples are then vortexed to homogeneity. The
resulting material is referred to as a faecal slurry (in the case
of a very limited number of particularly firm samples used in the
examples below, it was necessary to add 3 mLs 1.times.PBS per gram
faeces in order to generate a homogenous faecal slurry). To
generate supernatants for testing, slurries are centrifuged at 4.5
k rpm or 13.5 k rpm (4.degree. C.) for 1-5 minutes to remove the
bulk of the solid material and all cellular material. The
supernatant from the first spin is then re-centrifuged at 13.5 k
rpm (4.degree. C.) for 5 minutes, leaving only the soluble
fraction, including proteases. Supernatants from multiple
individuals are pooled together such that each pool represents the
combined protease output from the faeces of multiple
individuals.
[0267] For the purposes of the worked examples below,
hospital-derived human faecal samples were obtained (and the
presence of C. difficile in the samples was established), before
supernatant pools were then generated as described above. The pools
were characterised according to Table 1.
TABLE-US-00003 TABLE 1 ID Individuals per pool Clostridium
difficile status Pool 2 2 Toxin Negative by Vero Cell Cytotox Assay
Pool 3 5 Toxin Positive by Vero Cell Cytotox Assay Pool4 5 Toxin
Negative by Vero Cell Cytotox Assay
[0268] Performing the Assay
[0269] Prepare 20.times. protease inhibitor solution by adding 1
tab of Sigmafast Protease Inhibitor Cocktail (Sigma S8830,
containing AEBSF (4-(2-Aminoethyl) benzenesulfonyl fluoride,
Bestatin, E-64, Pepstatin A, Phosphoramidon, Leupeptin, Aprotinin)
to 5 mL protease stop buffer (1.times.PBS, 2% BSA, 5 mM EDTA). This
solution may be stored for 2 weeks at 2-8 degrees C. On the day of
the assay, briefly vortex the supernatant matrix to ensure
homogeneity. Prepare all reactions on ice and keep chilled until
the assay is first incubated.
[0270] Prepare 2.times. protease stop solution by diluting
20.times. protease inhibitor solution in protease stop buffer and
adding PMSF to a concentration of 1 mM in the 2.times. stop buffer
( 1/100 dilution of 0.1 M solution Sigma 93482). Keep this solution
chilled on ice at all times before use.
[0271] Prepare ICVD (or antibody) solutions at 250 .mu.g/mL in 0.1%
BSA. On ice, in thin-walled PCR tubes or plates, dilute the 250
.mu.g/mL ICVD into the supernatant matrix to give a final ICVD
concentration (at time zero) of 20 .mu.g/mL. Mix the resulting
solution on ice by pipetting, ensuring the solution does not warm
up. Once homogenous, immediately remove one volume of the sample
matrix plus 20 .mu.g/mL ICVD and mix with an equal volume of
2.times. protease stop solution. Mix the stopped matrix solution on
ice and immediately freeze at -80 degrees C. This is the time zero
sample. Incubate the remaining test matrix sample plus 20 .mu.g/mL
ICVD at 37 degrees C. in a PCR machine or similar apparatus. At the
required timepoints repeat the procedure above to generate stopped
supernatant samples for comparison to the time zero sample. In
addition, generate a protease-stopped matrix control that does not
contain ICVD by adding one volume of matrix sample (containing no
ICVD) from time zero with an equal volume of 2.times. protease stop
solution. This will be used as a control in downstream analysis to
assess the effect of the matrix on, for example, ELISAs or western
blotting profiles.
[0272] Following measurement using the Standard Western Blot
Stability Assay, the Standard TNFR2/TNF Interference ELISA Assay or
the Standard Toxin ELISA Assay, the amount of viable ICVD remaining
after incubation in a matrix sample at a given timepoint is divided
by the amount present at the zero timepoint. The resulting figure
is then multiplied by 100 to give % stability. In the case of the
Standard Western Blot Stability Assay, this provides proportion of
intact ICVD. In the case of the Standard TNFR2/TNF Interference
ELISA Assay or the Standard Toxin ELISA Assay, this provides the
proportion of functional ICVD.
[0273] 1.1.2 The Standard Mouse Small Intestinal Supernatant
Intestinal Tract Model
[0274] Faecal Supernatant Pool Production
[0275] C57BL/6 (`black 6`) mice are sacrificed. The small
intestine, including the full duodenum, jejunum and ileum are
excised from the body cavity of mice carefully so as to minimise
unnecessary tissue damage. The solid contents of the small
intestine are collected and the internal surface of the small
intestine flushed with 1 mL 0.9% saline (to preserve the native pH
of the intestinal contents). The 1 mL intestinal washout solution
and intestinal contents samples are then mixed together and
homogenised fully by vortexing to generate a small intestinal
slurry. To generate supernatants for testing, slurries are
centrifuged at 13.5 k rpm (4.degree. C.) for 2 minutes to remove
the bulk of the solid material and all cellular material. The
supernatant from the first spin are then re-centrifuged at 13.5 k
rpm (4.degree. C.) for 5 minutes, leaving only the soluble
fraction, including proteases. Supernatants from multiple mice (5
on average per pool) are mixed together such that each pool
represents the combined protease output from the small intestine of
multiple mice.
[0276] In the examples below, it was found that different pools of
mouse small intestinal supernatant used over time demonstrated
similar proteolytic activity.
[0277] Performing the Assay
[0278] The supernatants are used in the same manner as described
above under The Standard Human Faecal Supernatant Intestinal Tract
Model under `Performing the Assay`.
[0279] 1.2 The Standard Western Blot Stability Assay
[0280] For Assessment of Percentage Viable ICVD Remaining after
Incubation in an Intestinal Tract Model
[0281] Preparation of Samples for SDS-PAGE (Under Reducing
Conditions): [0282] 1) Prepare sample buffer for reducing SDS-PAGE:
Add reducing agent 0.5M Dithiothreitol (DTT) (Novex NP0004) to
Novex 4.times. LDS sample buffer (NP0007) in a ratio of 1:9. For
example, add 10 .mu.L 0.5M DTT to 90 .mu.L of 4.times. sample
buffer. The resulting solution will be referred to as `4.times.
load dye` from this point forward. [0283] 2) 1.times. load dye may
be prepared by diluting the 4.times. load dye stock 1:3 with
sterile H.sub.2O. [0284] 3) Add 15 .mu.L of each ICVD-containing
experimental sample in digestive matrix, from time zero or 30
minutes, to 5 .mu.L 4.times. load dye. Aim to load a final amount
of 100-200 ng
[0285] ICVD from the stopped zero timepoint. Match the volume of
sample from the 30 minute timepoint to the volume added for the
zero timepoint, so that any loss/degradation of ICVD over time is
evident by eye on the final blot (the same applies for other
timepoints such as 15 minute, 1 hour, 2 hour, etc, which may also
be used). If possible, include un-treated standards of the test
ICVD (at 100 and 10 ng) to confirm that the transfer and detection
systems are performing correctly. [0286] 4) Heat all samples
containing ICVD to 95.degree. C. for 5-10 minutes (treat all
samples equally) to denature the proteins and coat them with LDS
present in the load dye. Allow the samples to cool, spin them down
briefly in a centrifuge to collect all of the liquid. [0287] 5)
Prepare a suitable reference ladder that can be visualised
following blotting (Super Signal MW protein Ladder (Pierce)). Add
6.5 .mu.L of protein ladder+13 .mu.L 1.times. load dye. Note that
the reference ladder does not need to be heated before gel loading
(see supplier's instructions).
[0288] Electrophoresis
[0289] Use a Novex 10% Bis-Tris gel (NP0302Box) in combination with
1.times. SDS-MES running buffer (Novex NP0002-02) to visualise
ICVDs by SDS-PAGE. [0290] 1) Prepare a 1.times. SDS-MES solution
(from Novex NP0002-02, 20.times. stock) and assemble a Novex 10%
Bis-Tris gel in an appropriate electrophoresis tank. [0291] 2) Load
15 .mu.L of the samples prepared above per lane of the gel using
gel loading pipette tips. [0292] 3) Run the gel at 200V until the
dye front reaches the edge of the gel, but no further.
[0293] Blotting [0294] 1) Following electrophoresis, transfer
proteins onto nitrocellulose membranes (IB3010, Invitrogen) using
an iblot semi-dry transfer apparatus (Invitrogen, 7 minute semi-dry
transfer program 3). [0295] 2) Block the membrane by incubating
with 25 mL block solution (1% BSA, 2% Marvel, 0.05% Tween20,
1.times.PBS pH7.4) for 2 hours rocking gently at room temperature.
[0296] 3) For the primary detection antibody, prepare a 1/1000
dilution of pAb 1952 Rabbit .alpha.-VHH (raised at Eurogentech
using a VHH immunogen--another pAb rabbit .alpha.-ICVD, such as a
pAb rabbit .alpha.-VH, could also be used) in block solution (1%
BSA, 2% Marvel, 0.05% Tween20, 1.times.PBS pH7.4). Incubate the
blot with 25 mL of this solution rocking gently at 4.degree. C.
overnight. [0297] 4) The following day, place the blot into 25 mL
PBST (1.times.PBS, 0.1% Tween20) and incubate on a rocker for 5
minutes at room temperature. Repeat this procedure 5 times, each
time using a fresh volume of PBST to wash off any non-specifically
bound primary antibody. Complete 6 washes in total. [0298] 5) For
the secondary detection antibody, prepare HRP-conjugated pAb Swine
.alpha.-Rabbit (Dako, P0217) at a dilution of 1/1000 in block
solution. Add normal goat serum (Dako) to this solution to a final
concentration of 1% (for example 500 .mu.L goat serum in 50 mL of
secondary antibody solution). Incubate the blot with 25 mL of this
solution for 2 hours rocking gently at room temperature. [0299] 6)
Place the blot into 25 mL PBST (1.times.PBS, 0.1% Tween 20) and
incubate on a rocker for 5 minutes. Repeat this procedure 5 times,
each time using a fresh volume of PBST, to wash off any
non-specifically bound secondary antibody. Complete 6 washes in
total. [0300] 7) To develop the blot, incubate with 2 mL
SuperSignal West Pico Chemiluminescent (ECL, Pierce 34087) for 1-2
minutes, ensuring that the full surface of the blot is covered in
substrate [0301] 8) Visualise the ICVD present on the blot using an
ImageQuant LAS4000 machine or equivalent, 5-10 minutes exposure.
Vary the exposure time used to obtain the best ICVD signal. Band
densities are determined using ImageQuant TL software or
equivalent.
[0302] The amount of viable ICVD in a matrix sample at a given
timepoint is divided by the amount present at the zero timepoint.
The resulting figure is then multiplied by 100 to give %
stability.
[0303] 1.3 The Standard Toxin ELISA Assay
[0304] For Assessing the Potency of an Anti-TcdA or Anti-TcdB ICVD
and for Assessment of Percentage Viable Anti-TcdA or Anti-TcdB ICVD
Remaining after Incubation in an Intestinal Tract Model.
[0305] Materials: [0306] 96-well, Flat-Bottomed, Nunc Maxisorp
Immunoplates [0307] Recombinant, N-terminally His 10-tagged,
Clostridium difficile TcdB cell binding domain (CBD-B) from strain
R20291 (ribotype 027) in 1.times.PBS. This protein was cloned,
expressed from E. coli, and His-tag purified by FPLC. [0308]
Purified, full-length Clostridium difficile toxin A from strain
VP110463 (ribotype 087). Bacteria grown in static, anaerobic
cultures and secreted TcdA purified by FPLC ion exchange
chromatography. [0309] Anti-VHH Polyclonal Rabbit antibody: 6CP
(equivalent anti-ICVD, such as anti-VH polyclonal rabbit antibodies
could also be used). [0310] Swine anti-rabbit polyclonal
immunoglobulins--HRP conjugated (Dako, P0217) [0311] Supersensitive
TMB for ELISA: Sigma (T4444) [0312] 0.5M Sulphuric Acid [0313]
Block buffer: 1% BSA in 1.times. PBS (pH 7.2-7.5). [0314] Block
buffer plus 2.times. Protease inhibitor (1% BSA in 1.times. PBS, pH
7.3-7.5, 2.times. protease inhibitor cocktail, 2.5 mM EDTA, 0.5 mM
PMSF). [0315] PBST: 1.times.PBS plus 0.05% Tween 20.
[0316] Block buffer plus 2.times. Protease inhibitor is used as the
assay diluent to prepare ICVD solutions prior to addition to the
ELISA plate, when the ICVD sample is present in a digestive matrix
such as mouse small intestinal supernatant or human faecal
supernatant that may otherwise interfere with the performance of
the ELISA. 1/200 dilution of 0.1 M PMSF solution Sigma 93482 can be
used to achieve 0.5 mM PMSF. EDTA must also be added to a final
concentration of 2.5 mM. Sigmafast protease Inhibitor cocktail
(Sigma S8830, contains AEBSF (4-(2-Aminoethyl) benzenesulfonyl
fluoride, Bestatin, E-64, Pepstatin A, Phosphoramidon, Leupeptin,
Aprotinin) is used in this buffer. A stock of 20.times. protease
inhibitor solution can be made by adding 1 tab of Sigmafast
Protease Inhibitor Cocktail (Sigma S8830) to 5 mL protease stop
buffer (1.times.PBS, 2% BSA, 5 mM EDTA). This solution may be
stored for 2 weeks at 2-8.degree. C. and diluted into block buffer
on the day of the ELISA.
[0317] Anti-TcdA ICVD Detection by ELISA
[0318] This assay is designed to test anti-TcdA specific ICVDs for
their ability to bind to Clostridium difficile toxin A bound to an
ELISA plate. The plate coating toxin for this assay is full-length
TcdA VP110463 (087).
[0319] Method: [0320] 1. Dilute C. difficile TcdA in 1.times.PBS to
make a 2 .mu.g/mL coating solution. Add 50 .mu.L of this per well
of a Nunc Maxisorp plate, seal the plate and incubate overnight at
2-8.degree. C. Do not prepare large numbers of plates (over 3) with
the same stock of 2 .mu.g/mL solution TcdA. [0321] 2. Wash the
plate.times.4 with 380 .mu.L PBST with a plate washer. Tap the
plate out to ensure minimal residue is left. [0322] 3. Add 200
.mu.L per well of block buffer, seal and leave to incubate at room
temperature for at least an hour shaking. Plates can also be left
to block overnight at 2-8.degree. C. if necessary. [0323] 4.
Prepare a serial dilution series of ICVD reference standard using
block buffer, or block buffer plus 2.times. Protease inhibitor if
the main assay samples are from a digestive matrix, as a diluent.
The dilution range should be adjusted based on the binding of each
ICVD tested such that it covers the full assay signal range, from
the background signal to saturation, with the linear range
well-defined. Prepare a sufficient volume of each dilution to plate
50 .mu.L in triplicate [0324] 5. Prepare appropriate dilutions of
ICVD-containing samples to be tested in block buffer, or block
buffer plus 2.times. protease inhibitor if the samples are from a
digestive matrix, as a diluent. Prepare dilutions such that their
estimated concentration will fall in the linear range of assay
detection. The dilution range should be adjusted based on the
binding of each ICVD tested. These dilutions should also be made
serially in a microplate such that there is sufficient volume for
triplicate 50 .mu.L replicates on the final ELISA plate. Include an
assay blank (no ICVD). For digest analysis ELISAs, include a
protease inhibitor-stopped time zero matrix control (containing no
ICVD) to check for background signal in the assay. This should be
diluted in Block buffer plus 2.times. protease inhibitor and should
match the top concentration of matrix that contains an ICVD sample
tested on the plate. Keep samples chilled during preparation if
they are prepared from a digestive matrix. Prepare enough of each
sample to add to the plate in triplicate at 50 .mu.L/well [0325] 6.
Remove the Block buffer on the ELISA plate to waste, tap out any
residual onto a paper towel and add 50 .mu.L of diluted sample to
each well. Include 1) no matrix, no ICVD (blank wells) and 2)
Matrix only (no ICVD)wells. Seal the plate and incubate at room
temp, shaking for 2 hours. [0326] 7. Wash .times.4 as per step 2.
[0327] 8. Add 50 .mu.L per well of rabbit anti-VHH PAb diluted to
1/2000 in block buffer, seal the plate and incubate at room
temperature, shaking, for 1 hour. [0328] 9. Wash .times.4 as per
step 2. [0329] 10. Add 50 .mu.L per well of Swine anti-rabbit-HRP
diluted to 1/2000 using Block buffer, seal the plate and incubate
at room temperature, shaking, for 1 hour. [0330] 11. Wash .times.4
as per step 2. [0331] 12. Add 100 .mu.L per well of TMB, seal the
plate and incubate at room temperature for no longer than 30
minutes, shaking. The plate should be covered with silver foil as
TMB is light sensitive. [0332] 13. Add 50 .mu.L of 0.5 M sulphuric
acid to each well and read the plate at 450 nm. [0333] 14. Use the
ICVD standard calibration curve to interpolate unknown sample
concentrations using GraphPad Prism software (or equivalent).
[0334] Anti-TcdB ICVD Detection by ELISA
[0335] This assay is designed to test anti-TcdB specific ICVDs for
their ability to bind to Clostridium difficile TcdB Cell Binding
Domain (CBD-B) bound to an ELISA plate. It is critical to check
before running this assay that the ICVD being tested does not bind
elsewhere on TcdB, otherwise no signal will be observed.
[0336] Method: [0337] 1. Dilute C. difficile CBD-B (027) in PBS to
make a coating solution of 0.5-1 .mu.g/mL. Add 50 .mu.L of this per
well of a Nunc Maxisorp plate, seal with film and incubate
overnight at 2-8.degree. C. Do not prepare large numbers of plates
(over 3) with the same stock of 0.5-1 .mu.g/mL CBD-B solution.
[0338] 2. Add 200 .mu.L per well of block buffer, seal and leave to
incubate at room temperature for at least and hour shaking. Plates
can also be left to block overnight at 2-8.degree. C. if necessary.
[0339] 3. Prepare a serial dilution series of ICVD reference
standard using block buffer, or block buffer plus 2.times. Protease
inhibitor if the main assay samples are from a digestive matrix, as
a diluent. The dilution range should be adjusted based on the
binding of each ICVD tested such that it covers the full assay
signal range, from the background signal to saturation, with the
linear range well-defined. Prepare a sufficient volume of each
dilution to plate 50 .mu.L in triplicate [0340] 4. Prepare
appropriate dilutions of ICVD-containing samples to be tested in
block buffer, or block buffer plus 2.times. protease inhibitor if
the samples are from a digestive matrix, as a diluent. Prepare
dilutions such that their estimated concentration will fall in the
linear range of assay detection. The dilution range should be
adjusted based on the binding of each ICVD tested. These dilutions
should also be made serially in a microplate such that there is
sufficient volume for triplicate 50 .mu.L replicates on the final
ELISA plate.
[0341] Include an assay blank (no ICVD). For digest analysis
ELISAs, include a protease inhibitor -stopped time zero matrix
control (containing no ICVD) to check for background signal in the
assay. This should be diluted in Block buffer plus 2.times.
protease inhibitor and should match the top concentration of matrix
that contains an ICVD sample tested on the plate. Keep samples
chilled during preparation if they are prepared from a digestive
matrix. Prepare enough of each sample to add to the plate in
triplicate at 50 .mu.L/well [0342] 5. Remove the Block buffer on
the ELISA plate to waste, tap out any residual onto a paper towel
and add 50 .mu.L of sample dilution to each well. Include 1) no
matrix, no ICVD (blank wells) and 2) Matrix only (no ICVD) wells.
Seal the plate and incubate at room temp, shaking for 2 hours.
[0343] 6. Wash .times.4 as per step 2. [0344] 7. Add 50 .mu.L per
well of rabbit anti-VHH pAb (or other ICVD equivalent) diluted to
1/2000 in block buffer, seal the plate and incubate at room
temperature, shaking, for 1 hour. [0345] 8. Wash .times.4 as per
step 2. [0346] 9. Add 50 .mu.L per well of Swine anti-rabbit-HRP
diluted to 1/2000 using Block buffer, seal the plate and incubate
at room temperature, shaking, for 1 hour. [0347] 10. Wash .times.4
as per step 2. [0348] 11. Add 100 .mu.L per well of TMB, seal the
plate and incubate at room temperature for no longer than 30
minutes, shaking. The plate should be covered with silver foil as
TMB is light sensitive. [0349] 12. Add 50 .mu.L of 0.5 M sulphuric
acid to each well and read the plate at 450 nm. [0350] 13. Use the
ICVD standard calibration curve to interpolate unknown sample
concentrations using GraphPad Prism software (or equivalent).
[0351] 1.4 The Standard TNFR2/TNF Interference ELISA Assay
[0352] For assessing the potency of an anti-TNF ICVD and for
assessment of percentage viable anti-TNF ICVD remaining after
incubation in an intestinal tract model
[0353] 1. Principle
[0354] This assay detects binding of recombinant human TNF to the
fusion protein, Enbrel (etanercept). This protein is comprised of
soluble TNRF2 bound to the Fc region of human IgG, and can be used
for capture of TNF.alpha.. This interaction can be competed for by
anti-TNF ICVDs, causing reduced binding of TNF.alpha. to Enbrel.
Bound TNF is then detected by an anti-hTNF.alpha. antibody.
Therefore, high signal in this ELISA represents a low concentration
of anti-TNF ICVD, and vice versa. Due to an overnight incubation
step with the primary detection antibody, this assay usually takes
approximately one and a half days to complete.
[0355] 2. Materials
[0356] Solutions required: [0357] 0.5 M Sulphuric acid
(H.sub.2SO.sub.4) [0358] 1.times. PBS [0359] PBST (1.times. PBS,
0.05% Tween 20) [0360] Block buffer (1% BSA in 1.times. PBS, pH
7.3-7.5) [0361] Block buffer plus 2.times. Protease inhibitor (1%
BSA in 1.times. PBS, pH 7.3-7.5, 2.times. protease inhibitor
cocktail, 2.5 mM EDTA, 0.5 mM PMSF).
[0362] Block buffer plus 2.times. Protease inhibitor is used as the
assay diluent to prepare ICVD and TNF solutions, prior to mixing
and addition to the ELISA plate, when the ICVD sample is present in
a digestive matrix such as mouse small intestinal supernatant or
human faecal supernatant that may otherwise interfere with the
performance of the ELISA. 1/200 dilution of 0.1 M PMSF solution
Sigma 93482 can be used to achieve 0.5 mM PMSF. EDTA must also be
added to a final concentration of 2.5 mM. Sigmafast protease
Inhibitor cocktail (Sigma S8830, contains AEBSF (4-(2-Aminoethyl)
benzenesulfonyl fluoride, Bestatin, E-64, Pepstatin A,
Phosphoramidon, Leupeptin, Aprotinin) is used in this buffer. A
stock of 20.times. protease inhibitor solution can be made by
adding 1 tab of Sigmafast Protease Inhibitor Cocktail (Sigma S8830)
to 5 mL protease stop buffer (1.times.PBS, 2% BSA, 5 mM EDTA). This
solution may be stored for 2 weeks at 2-8.degree. C. and diluted
into block buffer on the day of the ELISA.
[0363] Reagents required: [0364] Enbrel stock of known
concentration (e.g. 2 mg/ml in PBS) [0365] Recombinant human TNF
stock of known concentration (Life Technologies, Cat No PHC 3015)
made up at 10.mu.g/ml in 1% BSA in PBS and kept at -80.degree. C.
in small (.ltoreq.20 .mu.l) aliquots [0366] Anti TNF.alpha. ICVD
standard of known concentration [0367] Rabbit anti human TNF.alpha.
antibody (Peprotech, 500-P31ABt, 300 .mu.g/ml) [0368] ExtrAvidin
HRP (Sigma, E2886) [0369] TMB substrate (Microwell Peroxidase
substrate System 2-C, KPL, 50-70-00)
[0370] 3. Procedure
[0371] Preparation:
[0372] Determine number of plates required for the assay. Coat
Maxisorb 96-well ELISA plate (Nunc) with 50 .mu.l/well 1 .mu.g/ml
Enbrel in 1.times. PBS. Shake plate briefly, seal and incubate at
4.degree. C. overnight.
[0373] Assay: [0374] 1. Wash the ELISA plate using a plate washer
(4.times..about.380 .mu.l PBST). Bang the plate on towel to remove
residual liquid. [0375] 2. Apply 200 .mu.l/well block buffer. Seal
and incubate on a rotary plate shaker for .gtoreq.1 hour. [0376] 3.
Prepare a serial dilution series of ICVD reference standards
between 0.04 nM and 10 nM in minimum final volumes of 100 .mu.l
using block buffer, or Block buffer plus 2.times. Protease
inhibitor if the main assay samples are from a digestive matrix, as
a diluent. The dilution range should be adjusted based on the
potency of each ICVD tested. Example shown in Table 2.
TABLE-US-00004 [0376] TABLE 2 Minimum volume of Volume to Volume 10
nM [Final be diluent in Dilution ICVD needed Dilution solutions],
transferred, each well factor (ul) number (pM) (ul) (ul) 2.545 280
1 10000.0 110 170 2 3928.571 3 1543.367 4 606.323 5 238.198 6
93.578 7 36.763
[0377] 4. Prepare appropriate dilutions of ICVD-containing samples
to be tested in block buffer, or block buffer plus 2.times.
Protease inhibitor if the samples are from a digestive matrix, as a
diluent. Prepare a serial dilution series. The dilution range
should be adjusted based on the potency of each ICVD tested such
that it covers the full assay signal range, from the background
signal to saturation, with the linear range well-defined. These
dilutions should also be made serially in a microplate such that
there is sufficient volume for triplicate 50 .mu.L replicates on
the final ELISA plate. For digest analysis ELISAs, include a
protease inhibitor-stopped time zero matrix control (containing no
ICVD). This should be diluted in Block buffer plus 2.times.
Protease inhibitor and should match the top concentration of matrix
that contains an ICVD sample tested on the plate. Keep samples
chilled during preparation if they are prepared from a digestive
matrix. [0378] 5. Prepare a 5 ng/ml solution of hrTNF.alpha. in
block buffer, or Block buffer plus 2.times. Protease inhibitor if
the assay samples are from a digestive matrix. [0379] 6. In a
separate 96-well plate, fill the blank well (for example, well H1)
with block buffer or Block buffer plus 2.times. Protease inhibitor.
Fill remaining relevant wells with 85 .mu.l TNF solution. [0380] 7.
Mix together 85 .mu.l of each ICVD dilution from the preparation
plate with 85 .mu.l hrTNF.alpha. solution in the second plate.
Include one well containing block buffer, or Block buffer plus
2.times. Protease inhibitor only (blank well). Include another well
where hrTNF.alpha. is diluted with block buffer, or Block buffer
plus 2.times. Protease inhibitor only (TNF only control well).
Include a well where hrTNF.alpha. is diluted with `stopped`
digestive matrix, as described above. Seal, and incubate on a
rotary plate shaker for 1 hour. [0381] 8. Wash blocked ELISA plate
as in step 1. [0382] 9. Transfer 50 .mu.l ICVD-TNF mixtures (plus
appropriate controls; 1) no TNF, no ICVD, 2) TNF, but no ICVD 3)
TNF plus `stopped` digestive matrix, no ICVD) to washed ELISA plate
in triplicate. Seal and incubate on a rotary plate shaker for 2
hours. [0383] 10. Wash blocked ELISA plate as in step 1. [0384] 11.
Prepare 5 ml/plate 1/1000 dilution of anti human TNF.alpha.
antibody (Peprotech, P31A) made up in block buffer. Add 50
.mu.l/well, seal, shake on rotary plate shaker briefly, then
incubate in cold room/fridge (4.degree. C.) overnight. Note: This
step can be reduced to 2h on the plate shaker at RT, but the signal
will be reduced with consequent reduction in sensitivity. [0385]
12. Wash blocked ELISA plate as in step 1. [0386] 13. Prepare 5
ml/plate 1/1000 dilution of ExtrAvidin-linked HRP (Sigma, E2886).
Add 50 .mu.l/well, seal and incubate on a rotary plate shaker for
.gtoreq.30 min. [0387] 14. Wash blocked ELISA plate as in step 1.
[0388] 15. Prepare 10 ml/plate TMB substrate (1:1 ratio of
substrate A and B). Add 100 .mu.l/well, seal and incubate on a
rotary plate shaker .ltoreq.30 mins. Shield from light. [0389] 16.
Stop reaction with 50 .mu.l/well 0.5 M H.sub.2SO.sub.4. [0390] 17.
Read plate at 450 nm. [0391] 18. Use the ICVD standard calibration
curve to interpolate unknown sample concentrations using GraphPad
Prism software (or equivalent).
[0392] In Step 6, equal volumes of diluted ICVD and TNF.alpha. are
mixed before addition to the ELISA plate. This step effectively
dilutes by twofold the concentrations of ICVD and TNF.alpha..
Therefore, the final concentration of TNF.alpha. on the plate will
be 2.5 ng/ml and the final concentration of the ICVD standard curve
will be from 0.02 nM to 5 nM. This dilution should be accounted for
when estimating appropriate sample dilution factors. The TMB
substrate reaction may progress quickly. The colour of the plate
should be checked periodically, and if a very bright blue colour
appears before 30 mins, the reaction should be stopped since very
high absorbance can lead to high background. Appropriate controls
should include triplicate wells of: BSA only, no ICVD (i.e. 2.5
ng/ml TNF.alpha. only), and if desired, no TNF.alpha. (i.e. 5 nM
ICVD only). For digestion analysis ELISAs, a no-ICVD matrix sample
that has been stopped by the addition of 2.times. protease stop
solution should be added to TNF. The lowest dilution (or highest
concentration) of the background matrix in the control should match
the lowest dilution (or highest concentration) of digestive matrix
in the highest ICVD concentration mixed with TNF/applied to the
plate.
[0393] 1.5 The Vero Cell Cytotoxicity Standard Assay
[0394] For assessing the potency of an anti-toxin ICVD
[0395] Culture and Maintenance of Vero Cells Prior to Use
[0396] Routine subculture of Vero cells can be achieved as follows:
[0397] 1. Once a flask of cells has grown to full confluence,
aspirate all cell culture medium and apply 2 ml 1.times. trypsin
(dissolved in 0.02% EDTA, Sigma E8008). Once the trypsin has been
applied work quickly to prevent loss of cells during washing. 2.
Wash the first trypsin application over the surface of the cells
and then fully aspirate to remove all traces of cell culture medium
(any traces of serum from the medium will inhibit trypsin
activity). [0398] 3. Apply 2 ml of trypsin and wash over the
surface of the cells. [0399] 4. Remove approximately 1.5-1.7 ml of
trypsin from the flask. [0400] 5. Tilt the flask so that the
remaining 300-500 .mu.L cover the Vero cells on the surface of the
plate. [0401] 6. Incubate the cells at 37.degree. C. 5% CO.sub.2
for 10-12 minutes. [0402] 7. To stop trypsin activity add 10 ml
Vero cell medium. [0403] 8. Resuspend the cells by gently jetting
the suspension against the bottom of the flask with a pipette until
the medium becomes cloudy (indicating dissipation of cell clumps).
3-4 times should be sufficient. Avoid excessive pipetting as this
may harm the cells. [0404] 9. Add 0.2 to 0.5 ml of the cell
suspension to 25-30 ml fresh Vero cell medium in a 75 cm.sup.2 cell
culture flask (Corning). Incubate the flask at 37.degree. C. 5%
CO.sub.2 to allow growth of the cells to full confluence. This
should occur in 3-5 days, depending on the inoculum volume and cell
count. To obtain finer control over the process, cells may be
enumerated using a haemocytometer, as outlined below, and added at
a fixed inoculum to the medium. Once in a confluent state the cell
monolayer should remain healthy for another 1-2 days without medium
replacement. To prolong the life of the confluent monolayer for use
it is often helpful to refresh 1/3-1/2 of the culture medium (do
not replace all the medium as it will have been conditioned with
cytokines from the growing Veros). The cells should be split before
rounding and detachment starts to occur.
[0405] Preparing Plates for the Assay (Day-1)
[0406] Ideally, plates should be prepared the day before use in the
cytotoxicity assay. However, plates may also be prepared on the day
of use if necessary. If the latter is the case, prepare plates in
the morning (for use in the afternoon) and ensure that at least 3
hours are allowed for cell attachment to the microplate prior to
use. A fully confluent flask of Vero cells should be used to make
the cell suspension for plating. [0407] 1. Add 150 .mu.l sterile
H.sub.2O to the inter-well spaces and 300 .mu.l to the top and
bottom row of a 96-well flat bottomed microplate. This ensures that
the cultured cells are hydrated during growth in the microplate.
[0408] 2. Trypsinise and resuspend (in 10 ml Vero cell culture
medium) a confluent flask of Vero cells, as described above. [0409]
3. Enumerate the cells using a haemocytometer and light microscope
(take four independent counts and use the mean, for example using
the four grid corners of a single haemocytometer slide). If there
is any concern about cell viability following trypsinisation add
Trypan blue dye to the cells before enumeration (1:1 v/v) and
multiply the viable cell count .times.2. [0410] 4. Dilute the cells
to 5.times.10.sup.4cells/ml in the required volume (allow 8 ml per
assay plate) of Vero cell culture medium. [0411] 5. Using a
multichannel pipette, dispense 100 .mu.l of the cell suspension
into each well. This is equivalent to 5000 cells/well. If multiple
plates are being prepared keep swirling and/or pipetting the cell
suspension between consecutive platings to ensure that the cells
are evenly distributed. [0412] 6. Centrifuge the microplate at
1,000 rpm for 2 minutes at room temperature to fix the cells evenly
in place across the bottom of the plate. Spin 2 .mu.lates maximum
in each arm of the centrifuge to avoid the arms tipping inward and
spilling the inter-well water. [0413] 7. Visually confirm that cell
distribution and number are as expected using a light microscope.
[0414] 8. Incubate plates at 37.degree. C. 5% CO.sub.2.
[0415] Setting up the Assay (Day 0)
[0416] Note: All solutions described in this section are prepared
in Vero cell culture medium. You should calculate the required
final volume of toxin and ICVD to cover the number of
plates/combinations before starting the assay. Mix all solutions
well (by vortexing and/or multiple inversions) between dilution
steps. [0417] 1. Prepare the required volume of toxin at double
(2.times.) the final assay concentration. The assay concentration
required should be determined beforehand (see preliminary work,
below). [0418] 2. Prepare the test ICVDs at double (2.times.) the
top concentration to be tested in the assay. Aim for a top
concentration of ICVD that will demonstrate a clear dose-response
toxin neutralisation relationship in the assay (see example graph,
below). [0419] 3. Prepare 10 serial dilutions (including the
undiluted top concentration) of the 2.times. ICVD stock in a
dilution trough. Typically, a 1/3 dilution produces a useful data
range. [0420] 4. Use a 96-well round-bottom microplate to prepare
mixed solutions before addition to the plates containing Vero
cells. [0421] 5. In triplicate, prepare solutions of medium only,
toxin only (1.times. dilution) and Triton-X100 (0.01%) controls and
add each to empty plate wells. [0422] 6. Attach 10 .mu.l pipette
tips to the central 6 rows of an 8-channel aspirator. Carefully
remove all medium (around 100 .mu.l per well) from the Vero cell
microplate prepared on Day 0. [0423] 7. Using a multichannel
pipette, add 100 .mu.l from one row of the preparation plate to the
cells on the assay plate. Repeat this twice to fill the two
adjacent rows on the assay plate (3 replicate rows in total):
[0424] 8. Once plate feeding is complete incubate at 37.degree. C.
for 3 days.
[0425] Processing the Assay (Day 3) [0426] 1. Observe the plates
under a light microscope. Check for confluent growth in the medium
only control wells and a good toxin response in the toxin-only
control well. [0427] 2. Using a multichannel pipette, in the dark,
add 10 .mu.l Alamar blue reagent (light sensitive) to each well.
[0428] 3. Shake the plate for 30 seconds to ensure mixing of the
Alamar blue into the culture medium. [0429] 4. Incubate the plate
for 1 hr 30 minutes at 37.degree. C. 5% CO.sub.2 [0430] 5.
Following incubation, in the dark, add 50 .mu.l 3% SDS. [0431] 6.
Read the plate using a plate reader (such as Fluostar Omega),
excitation filter 544, emission filter 590, bottom optic. Set the
blank (against which the data will be corrected) to the three plate
wells treated with Triton X100. [0432] 7. Calculate the mean of
three replicates for each treatment on the plate. Calculate % toxin
neutralisation values using the formula: % Neutralisation=(ICVD
treatment-toxin control)*100/(medium control-toxin control).
[0433] Preliminary Work: Determining the Optimal Amount of Toxin to
Use in the Main Neutralisation Assay
[0434] For ease of interpretation in the main assay, the
appropriate concentration of toxin to use should be determined
beforehand by conducting a toxin dose-response experiment on Vero
cells. Prepare 10 serial dilutions of toxin in a 12 well dilution
trough. Use the remaining two wells for 0.01% Triton and a medium
only control. Prepare a minimum of 330 .mu.L of each solution in
the dilution trough (this allows three replicates at 100 .mu.l
each). If there is no indication of how potent the toxin
preparation is in advance, choose a broad dilution range for the
preliminary experiment. This can be repeated over a finer
concentration range, if necessary. Apply these solutions to Vero
cells in a flat-bottomed microplate, incubate and process the plate
as described above.
[0435] To assay an ICVD, or full antibody, for neutralisating
activity against a given concentration of toxin, the minimum
concentration of each toxin preparation capable of inducing the
maximum reduction in cell viability is selected. An exemplary toxin
dose-response curve on Vero cells is provided in FIG. 1. The
horizontal bar indicates toxin concentrations suitable for use in
the main neutralisation assay.
[0436] 1.6 The Standard gp130 ELISA Assay
[0437] For Assessing the Potency of an Anti-IL-6R ICVD
[0438] The objective of this assay is to measure the potency of
anti-IL-6R ICVDs by measuring interference in the binding to gp130
of a sIL-6/IL-6R complex. This assay detects binding of
hIL-6R/hIL-6 complexes to recombinant human gp130. This interaction
can be competitively inhibited by anti-IL-6R ICVDs, causing reduced
binding of hIL-6R-hIL-6 complexes to gp130. Therefore, high signal
in this ELISA represents a low concentration of anti-IL-6R ICVD,
and vice versa.
[0439] Materials
[0440] Solutions Required:
[0441] 1.times. PBS
[0442] PBST (1.times. PBS, 0.05% Tween 20)
[0443] Block buffer (1% BSA in 1.times.PBS, pH 7.3-7.5)
[0444] 0.5 M Sulphuric acid (H.sub.2SO.sub.4)
[0445] Reagents Required:
[0446] Recombinant soluble human gp130 at known concentration
[0447] ICVD stock of known concentration
[0448] Recombinant soluble human IL-6 at known concentration
[0449] Recombinant soluble human IL-6R at known concentration
[0450] Biotinylated goat anti-IL-6R polyclonal antibody (R&D
systems BAF227); resuspended at 250 ug/ml in sterile PBS.
[0451] ExtrAvidin-Peroxidase (Sigma E2886)
[0452] TMB substrate (Microwell Peroxidase substrate System 2-C,
KPL, 50-70-00)
[0453] Procedure
[0454] Preparation: [0455] 1. Determine number of plates required
for the assay. [0456] 2. Prepare the relevant volume (up to 3
.mu.lates at a time) of 0.2 .mu.g/ml recombinant soluble human
gp130 in PBS with 5 ug/mL BSA in 1.times.PBS. [0457] 3. Working
quickly, dispense 50 .mu.l/well into Maxisorp 96-well ELISA plates
(Nunc), loading a maximum of 3 .mu.lates in one batch. [0458] 4.
Shake plate briefly, seal and incubate at 4.degree. C.
overnight.
[0459] Assay: [0460] 1. Wash the ELISA plate using a plate washer
(4.times..about.380 .mu.l PBST). Bang the plate on towel to remove
residual liquid. [0461] 2. Apply 200 .mu.l/well block buffer. Seal
and incubate on a rotary plate shaker for .gtoreq.1 hour. [0462] 3.
Prepare a dilution series of ICVD standards between 0.004 nM to 80
nM in minimum final volumes of 70 .mu.l using block buffer as a
diluent. [0463] 4. Prepare appropriate dilutions of samples to be
tested in block buffer, such that their estimated final
concentration on the plate will fall in the range of 0.001 nM to
250 nM ICVD. [0464] 5. Prepare a 40 ng/ml IL-6R solution in block
buffer. [0465] 6. In a separate 96-well plate, mix together 50
.mu.l of each ICVD dilution with 50 .mu.l IL-6R solution. In each
dilution series include one well with no ICVD. Incubate for 1 hour
on a rotary plate shaker. [0466] 7. Prepare a 100 ng/ml IL-6
solution in block buffer. [0467] 8. In a further additional 96-well
plate, mix together 85 .mu.l ICVD-IL-6R mixture from step 6 with 85
.mu.l IL-6 solution prepared in step 7. Include wells containing
block buffer only, such that the following controls are applied to
each plate: IL-6 only, and no ICVD (IL-6+IL-6R only). Incubate for
10 minutes on rotary plate shaker. [0468] 9. Wash blocked ELISA
plate as in step 1. [0469] 10. Transfer 50 .mu.l of the mixtures
prepared in step 8 to the washed ELISA plate in triplicate. Seal
and incubate on a rotary plate shaker for 2 hours. [0470] 11. Wash
blocked ELISA plate as in step 1. [0471] 12. Prepare 5.2 ml/plate
125 ug/mL of BAF227 anti-hIL-6R antibody made up in block buffer.
Add 50 .mu.l/well, seal, shake briefly, and incubate for 1 hour at
room temperature or overnight at 4.degree. C. [0472] 13. Wash
blocked ELISA plate as in step 1. [0473] 14. Prepare 5.2 ml/plate
of 1/1,000- 1/3000 dilution of Extravidin in block buffer. Add 50
.mu.l/well, seal, and incubate on a rotary shaker for 30 mins.
[0474] 15. Wash blocked ELISA plate as in step 1. [0475] 16.
Prepare 10 ml/plate TMB substrate (1:1 ratio of substrate A and B).
Add 100 .mu.l/well, seal and incubate on a rotary plate shaker
until a mid blue colour evolves in the lowest dilution wells or up
to a maximum of 30 mins. Shield from light. [0476] 17. Stop
reaction with 50 .mu.l/well 0.5 M H.sub.2SO.sub.4. [0477] 18. Read
plate at 450 nm. [0478] 19. Use standard curve to interpolate
concentrations of active ICVD. Raw OD450 values are adjusted with
readings taken from blank control wells. Standard curves are
plotted using appropriate software (e.g. Graphpad Prism using
Log(inhibitor) vs. response--variable slope (four parameters)).
ICVD concentrations in the test samples are calculated in the
software using the standard curve.
[0479] For Assessment of Percentage Viable Anti-IL-6R ICVD
Remaining After Incubation in an Intestinal Tract Mdel
[0480] The objective of this assay is to measure the remaining
concentration of active anti-IL-6R ICVDs which have previously been
incubated in the presence of proteolytic material, such as mouse
small intestinal supernatant or human faecal extract, thereby
elucidating the impact on the ICVD of any proteolysis which may
have taken place during incubation and therefore the proteolytic
stability of the anti-IL-6R ICVDs. This assay detects binding of
hIL-6R/hIL-6 complexes to recombinant human gp130. This interaction
can be competitively inhibited by anti-IL-6R ICVDs, causing reduced
binding of hIL-6R-hIL-6 complexes to gp130. Therefore, high signal
in this ELISA represents a low concentration or low affinity of
anti-IL-6R ICVD remaining active, and vice versa. The % survival is
the percentage concentration of active ICVD, interpolated using the
standard curve, maintained between a sample before and after
digestion.
[0481] Materials
[0482] Solutions required:
[0483] 1.times. PBS
[0484] 1% BSA in PBS
[0485] PBST (1.times. PBS, 0.05% Tween 20)
[0486] Block buffer (1% BSA in 1.times.PBS, pH 7.3-7.5)
[0487] Assay buffer (1% BSA, 2.times. protease inhibitor* in
1.times. PBS)
[0488] 0.5 M Sulphuric acid (H.sub.2SO.sub.4)
[0489] *2.times. protease inhibitor=1 tablet per 50 ml buffer
[0490] Reagents required:
[0491] Recombinant soluble human gp130 at known concentration
[0492] SigmaFast protease inhibitor tablets (S8820)
[0493] ICVD stock of known concentration
[0494] Soluble human IL-6 at known concentration
[0495] Soluble human IL-6R at known concentration
[0496] Biotinylated goat anti-IL-6R polyclonal antibody (R&D
systems BAF227); resuspended at 250 ug/ml in sterile PBS.
[0497] ExtrAvidin-Peroxidase (Sigma E2886)
[0498] TMB substrate (Microwell Peroxidase substrate System 2-C,
KPL, 50-70-00)
[0499] Procedure
[0500] Preparation: [0501] 1. Determine number of plates required
for the assay. [0502] 2. Prepare the relevant volume (up to 3
.mu.lates at a time) of 0.2 .mu.g/ml recombinant soluble human
gp130 in PBS+5 .mu.g/ml BSA. [0503] 3. Working quickly, dispense 50
.mu.l/well into Maxisorp 96-well ELISA plates (Nunc), loading a
maximum of 4 .mu.lates in one batch. [0504] 4. Shake plate briefly,
seal and incubate at 4.degree. C. overnight.
[0505] Assay:
[0506] 1. Wash the ELISA plate using a plate washer
(4.times..about.380 .mu.l PBST). Bang the plate on towel to remove
residual liquid.
[0507] 2. Apply 200 .mu.l/well block buffer. Seal and incubate on a
rotary plate shaker for .gtoreq.1 hour.
[0508] 3. Prepare a dilution series of ICVD standards between 0.004
nM to 1000 nM in minimum final volumes of 70 .mu.l using assay
buffer as a diluent.
[0509] 4. Prepare appropriate dilutions of samples to be tested in
assay buffer, such that their estimated final concentration on the
plate will fall in the range of 0.001 nM to 250 nM ICVD. Ensure
that samples containing GI/faecal material are kept on ice as much
as possible.
[0510] 5. Prepare a 400 ng/ml IL-6 solution in assay buffer.
[0511] 6. Prepare a 40 ng/ml IL-6R solution in assay buffer.
[0512] 7. In a separate 96-well plate, mix together 50 .mu.l of
each ICVD dilution with 50 .mu.l IL-6 solution. In each dilution
series include one well with no ICVD.
[0513] 8. In a further additional 96-well plate, mix together 85
.mu.l ICVD-IL-6 mixture from step 7 with 85 .mu.l IL-6R solution
prepared in step 6. Include wells containing assay buffer only,
such that the following controls are applied to each plate: IL-6
only, and no ICVD (IL-6+IL-6R only). Incubate for 5 minutes on
rotary plate shaker.
[0514] 9. Wash blocked ELISA plate as in step 1.
[0515] 10. Transfer 50 .mu.l of the mixtures prepared in step 8 to
the washed ELISA plate in triplicate. Seal and incubate on a rotary
plate shaker for 2 hours.
[0516] 11. Wash blocked ELISA plate as in step 1.
[0517] 12. Prepare 5 ml/plate 125 ng/mL of BAF227 anti-hIL-6R
antibody made up in block buffer. Add 50 .mu.l/well, seal, shake
briefly, and incubate for 1 hour at room temperature or overnight
at 4.degree. C.
[0518] 13. Wash blocked ELISA plate as in step 1.
[0519] 14. Prepare 5 ml/plate 1/1000-1/3000 dilution of Extravidin
in block buffer. Add 50 .mu.l/well, seal, and incubate on a rotary
shaker <30 mins
[0520] 15. Wash blocked ELISA plate as in step 1.
[0521] 16. Prepare 10 ml/plate TMB substrate (1:1 ratio of
substrate A and B). Add 100 .mu.l/well, seal and incubate on a
rotary plate shaker until a mid blue colour evolves in the lowest
dilution wells or up to a maximum of 30 mins. Shield from
light.
[0522] 17. Stop reaction with 50 .mu.l/well 0.5 M
H.sub.2SO.sub.4.
[0523] 18. Read plate at 450 nm.
[0524] 19. Use standard curve to interpolate concentrations of
active ICVD. Raw OD450 values are adjusted with readings taken from
blank control wells. Standard curves are plotted using appropriate
software (e.g. Graphpad Prism using Log(inhibitor) vs.
response--variable slope (four parameters)). ICVD concentrations in
the test samples are calculated in the software using the standard
curve. The active ICVD concentration in the test sample is
expressed as a % of that in the 0 h sample to give % survival.
Example 2
Substitution of a Lysine Residue with Alanine, Histidine or
Glutamine in CDR2 of an Anti-TNF-alpha ICVD
[0525] Q65B1 is an anti-TNF-alpha ICVD isolated, cloned and
purified from a llama immunised with soluble human recombinant
TNF-alpha. Residue K59 of the Q65B1 polypeptide sequence was
substituted with alanine, histidine or glutamine and the impact of
each substitution on intestinal tract stability and potency was
tested.
[0526] DNA encoding each ICVD was cloned into vector pMEK222,
expressed, and purified from the periplasm of E. coli (either by
Talon or Nickel NTA column). All ICVDs tested here carry an
identical C-terminal Flag-His6 tag.
[0527] Residue K59 resides in CDR2 of Q65B1. Q65B1 with a K59A
substitution is labelled "ID43F", Q65B1 with a K59H substitution is
labelled "ID8F-EV", and Q65B1 with a K59Q substitution is labelled
"ID44F".
[0528] 2.1.1 Potency--Standard TNFR2/TNF Interference ELISA
Assay--Experiment 1
[0529] Dose-response curves of each ICVD were generated using the
Standard TNFR2/TNF Interference ELISA Assay, which were used to
generate EC50 values (FIG. 2A and Table 3).
TABLE-US-00005 TABLE 3 Construct Substitution EC50 (pM) Q65B1 None
(K59) 98.4 ID8F-EV K59H 139.3 ID43F K59A 602.6 ID44F K59Q
245.47
[0530] 2.1.2 Potency--Standard TNFR2/TNF Interference ELISA
Assay--Experiment 2
[0531] In a repeat experiment, dose-response curves of Q65B1 and
ID8F-EV were generated again using the Standard TNFR2/TNF
Interference ELISA assay (FIG. 2B).
[0532] 2.2.1 Intestinal Stability--Standard Mouse Small Intestinal
Supernatant Intestinal Tract Model--Experiment 1
[0533] ICVDs were digested in mouse small intestinal material for 6
hours according to the Standard Mouse Small Intestinal Supernatant
Intestinal Tract Model. Percentage stability of ICVDs was
calculated using the Standard TNFR2/TNF Interference ELISA Assay.
The results are shown in FIG. 3A.
[0534] 2.2.2 Intestinal Stability--Standard Mouse Small Intestinal
Supernatant Intestinal Tract Model--Experiment 2
[0535] Q65B1 and ID8F-EV were digested in mouse small intestinal
material for 16 hours according to the Standard Mouse Small
Intestinal Supernatant Intestinal Tract Model. Percentage stability
of ICVDs were calculated using the Standard TNFR2/TNF Interference
ELISA Assay. The results are shown on the right hand side of FIG.
3B.
[0536] 2.2.3 Intestinal Stability--Standard Human Faecal
Supernatant Intestinal Tract Model
[0537] Q65B1 and ID8F-EV were digested for 16 hours in human faecal
supernatant according to the the Standard Human Faecal Supernatant
Intestinal Tract Model. Percentage stability of ICVDs were
calculated using the Standard TNFR2/TNF Interference ELISA Assay.
The results are shown on the left hand side of FIG. 3B.
[0538] 2.3 Conclusion
[0539] K59A and K59Q reduced potency compared to K59 and K59H (see
FIG. 2A, ID43F and ID44F vs Q65B1 and ID8F-EV, respectively). It
can be seen from FIGS. 2A and 2B that any observed variation in the
potency of ID8F-EV (K59H) relative to Q65B1 (K59) may be down to
experimental variation and that these ICVDs have substantially the
same potency.
[0540] K59A and K59Q reduced stability in mouse small intestinal
material after 6 hours incubation, compared to K59 (see FIG. 3A,
ID43F and ID44F vs Q65B1, respectively) and compared to K59H (see
FIG. 3A, ID8F-EV).
[0541] K59H increased stability in mouse small intestinal material
after 6 hours incubation and after 16 hours incubation, compared to
K59 (see FIG. 3A and FIG. 3B, ID8F-EV vs Q65B1). ID8F-EV and Q65B1
were undifferentiated in stability after 16 hours incubation in
this human faecal supernatant assay (FIG. 3B).
[0542] The stability increases of K59H were achieved without
significantly compromising potency.
Example 3
Substitution of a Lysine Residue with a Histidine Residue in both
CDR2 and CDR3 of an anti-TNF-alpha ICVD
[0543] Both residues K59 and K101 of Q65B1 were substituted with
histidine (making "ID34F"). Residue K59 resides in CDR2 of Q65B1
and residue K101 resides in CDR3 of Q65B1. DNA encoding ID34F was
cloned and expressed in yeast.
[0544] Q65B1 substituted with a K59H residue (as in Example 2) was
produced again, having the same sequence as ID8F-EV described
above. However, on this occasion DNA encoding this ICVD was cloned
and expressed in yeast (therefore lacking the C-terminal Flag-His6
tag) and is therefore labelled "ID32F" in this example.
[0545] 3.1 Potency--Standard TNFR2/TNF Interference ELISA Assay
[0546] Dose-response curves of each ICVD were generated using the
Standard TNFR2/TNF Interference ELISA Assay. A concentration range
of 0-3 nM was used (FIG. 4).
[0547] 3.2.1 Intestinal stability--Standard Mouse Small Intestinal
Supernatant Intestinal Tract Model
[0548] ICVDs were digested for 16 hours in mouse small intestinal
material according to the Standard Mouse Small Intestinal
Supernatant Intestinal Tract Model. Percentage stability of ICVDs
was calculated using the Standard TNFR2/TNF Interference ELISA
Assay. The results are shown in FIG. 5A.
[0549] 3.2.2 Intestinal stability--Standard Human Faecal
Supernatant Intestinal Tract Model
[0550] ICVDs were digested for 16 hours in human faecal supernatant
according to the Standard Human Faecal Supernatant Intestinal Tract
Model. Percentage stability of ICVDs was calculated using the
Standard TNFR2/TNF Interference ELISA Assay. The results are shown
in FIG. 5B.
[0551] 3.3 Conclusion
[0552] The additional K101H substitution in CDR3 of ID34F further
increased intestinal stability of the ICVD according to both the
Standard Mouse Small Intestinal Supernatant Intestinal Tract Model
(FIG. 5A) and the Standard Human Faecal Supernatant Intestinal
Tract Model (FIG. 5B), without significantly impacting potency
(FIG. 4).
Example 4
Substitution of an Arginine Residue with an Alanine, Histidine,
Glutamine, Phenylalanine or Tryptophan Residue in CDR3 of an
Anti-TcdB ICVD
[0553] ID45B is a modified anti-TcdB ICVD derived from a progenitor
ICVD (Q31B1). Q31B1 was isolated, cloned and purified from a llama
immunised with TcdB toxoids prepared by formalin inactivation of
purified TcdB. Residue R107 of the ID45B polypeptide sequence was
substituted with alanine, histidine, glutamine, phenylalanine or
tryptophan and the impact of each substitution on intestinal
stability and potency was tested.
[0554] DNA encoding each ICVD was cloned into vector pMEK222,
expressed, and purified from the periplasm of E. coli (either by
Talon or Nickel NTA column). All ICVDs tested here carry an
identical C-terminal Flag-His6 tag.
[0555] Residue R107 resides in CDR3 of ID45B. The substituted ICVDs
were labelled according to Table 4.
TABLE-US-00006 TABLE 4 ICVD Substitution ID45B None (R107) ID46B
R107H ID47B R107A ID48B R107Q ID49B R107F ID50B R107W
[0556] 4.1 Potency--Vero Cell Cytotoxicity Standard Assay
[0557] Dose-response curves of each ICVD were generated using the
Vero Cell Cytotoxicity Standard Assay (FIG. 6A).
[0558] 4.2 Intestinal Stability--Standard Human Faecal Supernatant
Intestinal Tract Model
[0559] ICVDs were digested for 30 minutes in human faecal
supernatant pool 4 according to the Standard Human Faecal
Supernatant Intestinal Tract Model. Percentage survival of ICVDs
was calculated using the Standard Western Blot Stability Assay. The
results are shown in FIG. 6B.
[0560] 4.3 Conclusion
[0561] All substitutions reduced potency relative to
`unsubstituted` ID45B. However, R107H and R107F substitutions
(ID46B and ID49B) resulted in only a minor potency reduction,
whilst R107A, R107Q and R107W substitutions (ID47B, ID48B and
ID50B) resulted in substantial potency reduction (FIG. 6A).
[0562] Whilst both R107H and R107F substitutions resulted in a
similar minor potency reduction, R107H resulted in the highest
intestinal stability increase of all substitutions tested (see FIG.
6B, ID46B--an approximate 35% increase in recovery compared to 0
mins, compared to ID45B R107). The R107F substitution, in contrast,
resulted in an approximate 10% decrease compared to R107 (FIG. 6B,
ID49B).
[0563] R107H provided the largest increase in stability, with only
a minor impact on potency.
Example 5
Substitution of Multiple Arginine Residues with Histidine Residues
in CDR2 of Anti-TcdB ICVD ID2B, and the Impact of Substitution
Position within CDR3 of ID2B
[0564] ID2B is a modified anti-TcdB ICVD derived from a progenitor
ICVD (Q31B1). Residues R53 and R56 in CDR2 of the ID2B polypeptide
sequence were both substituted with histidine residues (making
"ID20B"). Independently, residues R107 and R109 in CDR3 of the ID2B
polypeptide sequence were each substituted with a histidine residue
(the sole R107H substitution making "ID21B" and the sole R109H
substitution making "ID22B"). These ICVDs are summarised in Table
5. The impact of these substitutions on trypsin stability,
intestinal stability and potency was tested.
TABLE-US-00007 TABLE 5 ICVD Substitution(s) ID2B None ID20B R53H
and R56H (both in CDR2) M34I ID21B R107H (in CDR3) M34I ID22B R109H
(in CDR3) M34I
[0565] DNA encoding ID2B was cloned into vector pMEK222, expressed,
and purified from the periplasm of E. coli. ID2B carries a
C-terminal Flag-His6 tag. DNA encoding ID20B, ID21B and ID22B was
cloned and expressed in yeast.
[0566] 5.1 Potency--Vero Cell Cytotoxicity Standard Assay
[0567] Dose-response curves of each ICVD were generated using TcdB
from the 027 C. difficile ribotype in the Vero Cell Cytotoxicity
Standard Assay (FIG. 7).
[0568] 5.2.1 The Standard Trypsin Intestinal Tract Model
[0569] The ICVDs were assayed for trypsin stability. A buffered (10
mM acetic acid, pH 3.2, containing 0.01% thimerosal) aqueous
suspension of TPCK-treated Trypsin-agarose beads (trypsin from
bovine pancreas; T4019; Sigma Aldrich) is used for the assay. The
beads are washed 3 times with water (250 .mu.l beads+1.25 ml water)
followed by washing 5 times with Trypsin buffer (TRYP buffer; 1 mM
Tris-HCl, 20 mM CaCl2 [pH 8.0]). Finally, the resin is resuspended
in TRYP buffer as a 50% (v/v) suspension.
[0570] 100 .mu.l of a 2 mg/ml construct solution is mixed with 225
.mu.l 50% (v/v) immobilized TPCK-treated Trypsin in TRYP buffer.
After time intervals of 0, 10, 15, 30, 45 and 60 minutes of
incubation at 37.degree. C. in a shaker, samples are taken as
follows: resin is pelleted by a 1 min centrifugation step at
500.times.g, and a 40 .mu.l sample is taken from the supernatant
and mixed with 2.times. sample loading buffer (such as Laemmli
buffer). The remaining suspension is mixed again, and put back at
37.degree. C. in the shaker.
[0571] For analysis, 15 .mu.l of each sample is mixed with 5 .mu.l
4.times. loading dye, boiled for 10 mins and 15 .mu.l is loaded per
lane on a polyacrylamide gel (such as NuPAGE 10% acrylamide
Bis-Tris gel). Gels are run in SDS-MES buffer at 200 V for 35 mins.
Gels are fixed in 40% methanol, 7% acetic acid for 30 mins and
stained in colloidal Coomassie Brilliant Blue stain overnight. Gels
are destained in water before imaging (such as using ImageQuant
LAS4000 with 7 secs exposure) (FIGS. 8A-C). The quantity of intact
constructs relative to cleaved constituent polypeptides can be
assessed by comparing the corresponding bands in each time point
lane. Asterisks and # in the electrophoresis gel figures indicate
bands containing cleaved fragments.
[0572] 5.2.2 Intestinal Stability--Standard Human Faecal
Supernatant Intestinal Tract Model
[0573] ID2B and ID21B were digested for 1 hour in Faecal Pools 3
and 4 (FIG. 9) according to the Standard Human Faecal Supernatant
Intestinal Tract Model. Percentage stability of ICVDs was
calculated using the Standard Toxin ELISA Assay.
[0574] 5.3 Conclusion
[0575] The single CDR3 substitutions resulted in a minor reduction
in potency (FIG. 7, ID21B and ID22B), whilst the double CDR2
substitution resulted in a more pronounced reduction in potency
(FIG. 7, ID20B).
[0576] Due to the presence of the His-tag in ID2B, the results from
the electrophoresis gel in FIG. 8A are unclear. The more central
R107H substitution (FIG. 8B, ID21B) provided a greater trypsin
stability increase than the more peripheral R109H substitution
(FIG. 8C, ID22B). This indicates that such substitutions may be
more stabilising when made in a central `window` of a CDR.
[0577] The faecal supernatant stability of ID21B (R107H) was
substantially increased in both pool 3 (C. diff positive patient
faeces) and pool 4 (C. diff negative patent faeces) compared to
unsubstituted ID2B (FIG. 9).
Example 6
Substitution of an Arginine Residue with a Histidine Residue in
CDR2 of anti-TcdB ICVD ID1B, and the Impact of Substitution
Position within CDR3 of ID1B
[0578] ID1B is a modified anti-TcdB ICVD derived from a progenitor
ICVD (B10F1). B10F1 was isolated, cloned and purified from a llama
immunised with 100 ug of TcdB toxoids prepared by formalin
inactivation of purified TcdB.
[0579] Residue R58 in CDR2 of the ID1B polypeptide sequence was
substituted with a histidine residue (making "ID24B").
Independently, residues R105 and R108 in CDR3 of the ID1B
polypeptide sequence were each substituted with a histidine residue
(the R105H substitution making "ID27B" and the R108H substitution
making "ID25B"). These ICVDs are summarised in Table 6. The impact
of these substitutions on intestinal stability and potency was
tested.
TABLE-US-00008 TABLE 6 ICVD Substitution(s) ID1B None ID24B R58H
(in CDR2) M34I ID25B R108H (in CDR3) M34I ID27B R105H (in CDR3)
M34I
[0580] DNA encoding ID1B, ID24B, ID25B and ID27B was cloned and
expressed in yeast.
[0581] 6.1 Potency--Vero Cell Cytotoxicity Standard Assay
[0582] Dose-response curves of each ICVD were generated using TcdB
from the 027 C. difficile ribotype in the Vero Cell Cytotoxicity
Standard Assay (FIG. 10A).
[0583] 6.2.1 Intestinal Stability--Standard Human Faecal
Supernatant Intestinal Tract Model
[0584] ID1B, ID24B, ID25B and ID27B were digested for 1 hour in
Faecal Pool 2 (FIG. 10B) according to the Standard Human Faecal
Supernatant Intestinal Tract Model. Percentage survival of ICVDs
was calculated using the Standard Toxin ELISA Assay.
[0585] 6.2.2 Intestinal Stability--The Standard Trypsin Intestinal
Tract Model
[0586] The ICVDs were assayed for trypsin stability, in the manner
described in Example 5 above (FIGS. 11A-C).
[0587] 6.3 Conclusion
[0588] The single CDR3 substitutions resulted in a minor reduction
in potency (FIG. 10A).
[0589] The density of the main band in the ID1B gel (FIG. 11A)
appears to reduce to a greater extent over the time periods tested
than that of the substituted ICVDs (FIGS. 11B-11C) and therefore
the substituted ICVDs appear to be more stable than unsubstituted
ID1B in this trypsin assay.
[0590] The faecal supernatant stability of all substituted ICVDs
was increased (FIG. 10B). The more central R105H CDR3 substitution
(FIG. 10B, ID27B) provided a greater faecal supernatant stability
increase than the more peripheral R108H CDR3 substitution (FIG.
10B, ID25B). This indicates that such substitutions may be more
stabilising when made in a central `window` of a CDR.
Example 7
Substitution of an Arginine Residue with a Histidine Residue in
CDR3 of One Arm of an Anti-TcdB Bivalent Construct
[0591] ID41B is an anti-TcdB bivalent construct consisting of
modified versions of wild type ICVDs Q31B1 and B10F1. An R108H
(CDR3) substitution was made in the B10F1 arm of ID41B (making
"ID43B"). The impact of this substitution on potency and intestinal
stability was tested. DNA encoding ID41B and ID43B was cloned and
expressed in yeast.
[0592] 7.1 Potency--Vero Cell Cytotoxicity Standard Assay
[0593] Dose-response curves of each construct were generated using
TcdB from the 017 C. difficile ribotype in the Vero Cell
Cytotoxicity Standard Assay (FIG. 12A).
[0594] 7.2 Intestinal stability--Standard Toxin ELISA Assay
[0595] Constructs were digested for 4 hours in Faecal Pools 2, 3
and 4 according to the Standard Human Faecal Supernatant Intestinal
Tract Model. Three repeat ELISAs were run for each faecal pool.
Percentage survival was calculated using the Standard Toxin ELISA
Assay (FIGS. 12B-12D).
[0596] 7.3 Conclusion
[0597] The R108H substitution (ID43B) had a very minor impact on
potency (FIG. 12A). In the majority of faecal supernatant assays
(six out of nine across all faecal pools), the R108H substitution
in ID43B resulted in increased stability (FIGS. 12B-12D).
Example 8
Substitution of an Arginine Residue with a Histidine Residue in
CDR3 of an Anti-TcdA Bivalent ICVD
[0598] ID17A is an anti-TcdA bivalent construct consisting of
modified versions of wild type ICVDs B4F10 and Q34A3 (B4F10 and
Q34A3 were isolated, cloned and purified from a llama immunised
with TcdA toxoids prepared by formalin inactivation of purified
TcdA).
[0599] An R109H (CDR3) substitution was made in the B4F10 arm of
ID17A (making "ID29A"). The impact of this substitution on potency
and intestinal stability was tested. DNA encoding ID17A and ID29A
was cloned and expressed in yeast.
[0600] 8.1 Potency--Vero Cell Cytotoxicity Standard Assay
[0601] Dose-response curves of each construct were generated using
TcdA in the Vero Cell Cytotoxicity Standard Assay (FIG. 13A).
[0602] 8.2 Intestinal stability--Standard Human Faecal Supernatant
Intestinal Tract Model
[0603] Constructs were digested for 1 hour in Faecal Pools 2, 3 and
4 according to the Standard Human Faecal Supernatant Intestinal
Tract Model. Percentage survival was calculated using the Standard
Toxin ELISA Assay (FIG. 13B).
[0604] 8.3 Conclusion
[0605] The R109H (CDR3) substitution in one arm of this anti-TcdA
bihead had a minor impact on potency (FIG. 13A). In all faecal
pools tested, this substitution resulted in highly increased
stability (FIG. 13B).
Example 9
Substitution of an Arginine Residue with a Histidine Residue in
CDR3 of an Anti-IL-6R ICVD 7F6
[0606] 7F6 is an anti-IL-6R ICVD. 7F6 was isolated, cloned and
purified from a llama immunised with soluble human recombinant
IL-6R.
[0607] Residue R102 in CDR3 of the 7F6 polypeptide sequence was
substituted with a histidine residue (making "ID-3V") and the
impact of this substitution on potency and intestinal stability was
tested. DNA encoding 7F6 and ID-3V was cloned and expressed in E.
coli.
[0608] 9.1 Potency--Standard qp130 ELISA Assay
[0609] Dose-response curves were generated using the standard gp130
ELISA assay and these were used to generate EC.sub.50 values (Table
7, graph not shown).
TABLE-US-00009 TABLE 7 Construct Substitution EC50 (nM) 7F6 None
(R102) 0.15 ID-3V R102H (in CDR3) 0.16
[0610] 9.2 Intestinal stability--Standard Mouse Small Intestinal
Supernatant Intestinal Tract Model
[0611] ICVDs were digested for 4 hours in mouse small intestinal
material according to the Standard Mouse Small Intestinal
Supernatant Intestinal Tract Model. Percentage stability of ICVDs
was calculated using the Standard gp130 ELISA assay. The results
are shown in Table 8.
TABLE-US-00010 TABLE 8 Construct Substitution % Stability 7F6 None
(R102) 1% ID-3V R102H (in CDR3) 12%
[0612] 9.3 Intestinal Stability--Standard Human Faecal Supernatant
Intestinal Tract Model
[0613] ICVDs were digested for 16 hours in human faecal supernatant
according to the Standard Human Faecal Supernatant Intestinal Tract
Model. Percentage stability of ICVDs was calculated using the
Standard gp130 ELISA assay. The results are shown in Table 9.
TABLE-US-00011 TABLE 9 Construct Substitution % Stability 7F6 None
(R102) 28% ID-3V R102H (in CDR3) 41%
[0614] 9.4 Conclusion
[0615] This R102H substitution in CDR3 of 7F6 further increased
intestinal stability of the ICVD according to both the Standard
Mouse Small Intestinal Supernatant Intestinal Tract Model (see
Tables 8 and 9), without significantly impacting potency (Table
7).
Example 10
Substitution of an Arginine Residue with a Histidine Residue in
CDR3 of an Anti-IL-6R ICVD 5G9
[0616] 5G9 is an anti-IL-6R ICVD. 5G9 was isolated, cloned and
purified from a llama immunised with soluble human recombinant
IL-6R.
[0617] Residue R105 in CDR3 of the 5G9 polypeptide sequence was
substituted with a histidine residue (making "ID-54V") and the
impact of this substitution on potency and intestinal stability was
tested. DNA encoding 5G9 and ID-54V was cloned and expressed in E.
coli.
[0618] 10.1 Potency--Standard gp130 ELISA Assay
[0619] Dose-response curves were generated using the standard gp130
ELISA assay and these were used to generate EC50 values (Table 10,
graph not shown).
TABLE-US-00012 TABLE 10 Construct Substitution EC50 (nM) 5G9 None
(R105) 0.09 ID-54V R105H (in CDR3) 0.15
[0620] 10.2 Intestinal Stability--Standard Mouse Small Intestinal
Supernatant Intestinal Tract Model
[0621] ICVDs were digested for 4 hours in mouse small intestinal
material according to the Standard Mouse Small Intestinal
Supernatant Intestinal Tract Model. Percentage stability of ICVDs
was calculated using the Standard gp130 ELISA assay. The results
are shown in Table 11.
TABLE-US-00013 TABLE 11 Construct Substitution % Stability 5G9 None
(R105) 5% ID-54V R105H (in CDR3) 36%
[0622] 10.3 Intestinal stability--Standard Human Faecal Supernatant
Intestinal Tract Model
[0623] ICVDs were digested for 16 hours in human faecal supernatant
according to the Standard Human Faecal Supernatant Intestinal Tract
Model. Percentage stability of ICVDs was calculated using the
Standard gp130 ELISA assay. The results are shown in Table 12.
TABLE-US-00014 TABLE 12 Construct Substitution % Stability 5G9 None
(R105) 40% ID-54V R105H (in CDR3) 48%
[0624] 10.4 Conclusion
[0625] This R105H substitution in CDR3 of 5G9 further increased
intestinal stability of the ICVD according to both the Standard
Mouse Small Intestinal Supernatant Intestinal Tract Model (see
Tables 11 and 12), with only a minor impact on potency (Table
10).
[0626] Throughout the specification and the claims which follow,
unless the context requires otherwise, the word `comprise`, and
variations such as `comprises` and `comprising`, will be understood
to imply the inclusion of a stated integer, step, group of integers
or group of steps but not to the exclusion of any other integer,
step, group of integers or group of steps. All patents and patent
applications mentioned throughout the specification of the present
invention are herein incorporated in their entirety by reference.
The invention embraces all combinations of preferred and more
preferred groups and suitable and more suitable groups and
embodiments of groups recited above.
Sequence CWU 1
1
351115PRTArtificial SequencePolypeptide sequence of anti-TNF-alpha
ICVD Q65B1 1Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp
Phe Ser Ser His 20 25 30 Trp Met Tyr Trp Val Arg Gln Ala Pro Gly
Lys Glu Leu Glu Trp Leu 35 40 45 Ser Glu Ile Asn Thr Asn Gly Leu
Ile Thr Lys Tyr Gly Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val
Ser Arg Asn Asn Ala Ala Asn Lys Met Tyr 65 70 75 80 Leu Glu Leu Thr
Arg Leu Glu Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Arg
Asn Gln Lys Gly Leu Asn Lys Gly Gln Gly Thr Gln Val Thr 100 105 110
Val Ser Ser 115 2115PRTArtificial SequencePolypeptide sequence of
anti-TNF-alpha ICVD ID8F-EV (ID32F) 2Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser
Cys Ala Ala Ser Gly Phe Asp Phe Ser Ser His 20 25 30 Trp Met Tyr
Trp Val Arg Gln Ala Pro Gly Lys Glu Leu Glu Trp Leu 35 40 45 Ser
Glu Ile Asn Thr Asn Gly Leu Ile Thr His Tyr Gly Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Val Ser Arg Asn Asn Ala Ala Asn Lys Met Tyr
65 70 75 80 Leu Glu Leu Thr Arg Leu Glu Pro Glu Asp Thr Ala Leu Tyr
Tyr Cys 85 90 95 Ala Arg Asn Gln Lys Gly Leu Asn Lys Gly Gln Gly
Thr Gln Val Thr 100 105 110 Val Ser Ser 115 3115PRTArtificial
SequencePolypeptide sequence of anti-TNF-alpha ICVD ID43F 3Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Ser His 20
25 30 Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Glu Leu Glu Trp
Leu 35 40 45 Ser Glu Ile Asn Thr Asn Gly Leu Ile Thr Ala Tyr Gly
Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asn Asn Ala
Ala Asn Lys Met Tyr 65 70 75 80 Leu Glu Leu Thr Arg Leu Glu Pro Glu
Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Arg Asn Gln Lys Gly Leu
Asn Lys Gly Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115
4115PRTArtificial SequencePolypeptide sequence of anti-TNF-alpha
ICVD ID44F 4Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp
Phe Ser Ser His 20 25 30 Trp Met Tyr Trp Val Arg Gln Ala Pro Gly
Lys Glu Leu Glu Trp Leu 35 40 45 Ser Glu Ile Asn Thr Asn Gly Leu
Ile Thr Gln Tyr Gly Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val
Ser Arg Asn Asn Ala Ala Asn Lys Met Tyr 65 70 75 80 Leu Glu Leu Thr
Arg Leu Glu Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Arg
Asn Gln Lys Gly Leu Asn Lys Gly Gln Gly Thr Gln Val Thr 100 105 110
Val Ser Ser 115 5115PRTArtificial SequencePolypeptide sequence of
anti-TNF-alpha ICVD ID34F 5Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala
Ser Gly Phe Asp Phe Ser Ser His 20 25 30 Trp Met Tyr Trp Val Arg
Gln Ala Pro Gly Lys Glu Leu Glu Trp Leu 35 40 45 Ser Glu Ile Asn
Thr Asn Gly Leu Ile Thr His Tyr Gly Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Val Ser Arg Asn Asn Ala Ala Asn Lys Met Tyr 65 70 75 80
Leu Glu Leu Thr Arg Leu Glu Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85
90 95 Ala Arg Asn Gln His Gly Leu Asn Lys Gly Gln Gly Thr Gln Val
Thr 100 105 110 Val Ser Ser 115 6122PRTArtificial
SequencePolypeptide sequence of anti-TcdB ICVD B10F1 6Gln Val Gln
Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr 20 25
30 Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45 Ala Ala Ile Asn Gly Ser Gly Gly Asn Arg Ile Ser Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Thr Val Tyr 65 70 75 80 Leu Gln Leu Asn Ser Leu Lys Pro Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ser Leu Thr Tyr Tyr Gly
Arg Ser Ala Arg Tyr Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val
Thr Val Ser Ser 115 120 7124PRTArtificial SequencePolypeptide
sequence of anti-TcdB ICVD Q31B1 7Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Ala Gly Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Arg Thr Leu Ser Ser Tyr 20 25 30 Thr Met Gly Trp
Phe Arg Gln Ala Pro Glu Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly
Ser Ser Arg Asp Gly Arg Thr Asn Tyr Tyr Ala Asn Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Ala His Thr Thr Ser Gly Val Pro Val Arg Glu
Arg Ser Tyr Ala 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val
Ser Ser 115 120 8122PRTArtificial SequencePolypeptide sequence of
anti-TcdB ICVD ID1B 8Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Ala Thr Phe Ser Ser Tyr 20 25 30 Tyr Met Gly Trp Phe Arg Gln
Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Asn Gly
Ser Gly Gly Asn Arg Ile Ser Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu
Gln Leu Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Ala Ser Leu Thr Tyr Tyr Gly Arg Ser Ala Arg Tyr Asp Tyr Trp
100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
9124PRTArtificial SequencePolypeptide sequence of anti-TcdB ICVD
ID2B 9Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Leu
Ser Ser Tyr 20 25 30 Thr Met Gly Trp Phe Arg Gln Ala Pro Glu Lys
Glu Arg Glu Phe Val 35 40 45 Ala Gly Ser Ser Arg Asp Gly Arg Thr
Asn Tyr Tyr Ala Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His
Thr Thr Ser Gly Val Pro Val Arg Glu Arg Ser Tyr Ala 100 105 110 Tyr
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
10124PRTArtificial SequencePolypeptide sequence of anti-TcdB ICVD
ID20B 10Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Leu
Ser Ser Tyr 20 25 30 Thr Ile Gly Trp Phe Arg Gln Ala Pro Glu Lys
Glu Arg Glu Phe Val 35 40 45 Ala Gly Ser Ser His Asp Gly His Thr
Asn Tyr Tyr Ala Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His
Thr Thr Ser Gly Val Pro Val Arg Glu Arg Ser Tyr Ala 100 105 110 Tyr
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
11124PRTArtificial SequencePolypeptide sequence of anti-TcdB ICVD
ID21B 11Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Leu
Ser Ser Tyr 20 25 30 Thr Ile Gly Trp Phe Arg Gln Ala Pro Glu Lys
Glu Arg Glu Phe Val 35 40 45 Ala Gly Ser Ser Arg Asp Gly Arg Thr
Asn Tyr Tyr Ala Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His
Thr Thr Ser Gly Val Pro Val His Glu Arg Ser Tyr Ala 100 105 110 Tyr
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
12124PRTArtificial SequencePolypeptide sequence of anti-TcdB ICVD
ID22B 12Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Leu
Ser Ser Tyr 20 25 30 Thr Ile Gly Trp Phe Arg Gln Ala Pro Glu Lys
Glu Arg Glu Phe Val 35 40 45 Ala Gly Ser Ser Arg Asp Gly Arg Thr
Asn Tyr Tyr Ala Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His
Thr Thr Ser Gly Val Pro Val Arg Glu His Ser Tyr Ala 100 105 110 Tyr
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
13122PRTArtificial SequencePolypeptide sequence of anti-TcdB ICVD
ID24B 13Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Phe
Ser Ser Tyr 20 25 30 Tyr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys
Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Asn Gly Ser Gly Gly Asn
His Ile Ser Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Leu Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ser
Leu Thr Tyr Tyr Gly Arg Ser Ala Arg Tyr Asp Tyr Trp 100 105 110 Gly
Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 14122PRTArtificial
SequencePolypeptide sequence of anti-TcdB ICVD ID25B 14Asp Val Gln
Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Phe Ser Ser Tyr 20 25
30 Tyr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45 Ala Ala Ile Asn Gly Ser Gly Gly Asn Arg Ile Ser Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Thr Val Tyr 65 70 75 80 Leu Gln Leu Asn Ser Leu Lys Pro Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ser Leu Thr Tyr Tyr Gly
Arg Ser Ala His Tyr Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val
Thr Val Ser Ser 115 120 15122PRTArtificial SequencePolypeptide
sequence of anti-TcdB ICVD ID27B 15Asp Val Gln Leu Gln Glu Ser Gly
Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Ala Thr Phe Ser Ser Tyr 20 25 30 Tyr Ile Gly Trp
Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala
Ile Asn Gly Ser Gly Gly Asn Arg Ile Ser Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65
70 75 80 Leu Gln Leu Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Ala Ser Leu Thr Tyr Tyr Gly His Ser Ala Arg
Tyr Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 16266PRTArtificial SequencePolypeptide sequence of
anti-TcdB construct ID41B 16Asp Val Gln Leu Gln Glu Ser Gly Gly Gly
Leu Val Gln Ala Gly Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Ala Thr Leu Ser Ser Tyr 20 25 30 Thr Met Gly Trp Phe Arg
Gln Ala Pro Glu Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ser Ser
Arg Asp Gly Arg Thr Asn Tyr Tyr Ala Asn Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Ala His Thr Thr Ser Gly Val Pro Val His Glu Arg Ser Tyr
Ala 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly
Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser 130 135 140 Asp Val Gln Leu Gln Glu Ser Gly Gly
Gly Leu Val Gln Ala Gly Gly 145 150 155 160 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Ala Thr Phe Ser Ser Tyr 165 170 175 Tyr Met Gly Trp
Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 180 185 190 Ala Ala
Ile Asn Gly Ser Gly Gly Asn Arg Ile Ser Ala Asp Ser Val 195 200 205
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 210
215 220 Leu Gln Leu Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
Cys 225 230 235 240 Ala Ala Ser Leu Thr Tyr Tyr Gly His Ser Ala Arg
Tyr Asp Tyr Trp 245 250 255 Gly Gln Gly Thr Gln Val Thr Val Ser Ser
260 265 17266PRTArtificial SequencePolypeptide sequence of
anti-TcdB construct ID43B 17Asp Val Gln Leu Gln Glu Ser Gly Gly Gly
Leu Val Gln Ala Gly Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Ala Thr Leu Ser Ser Tyr 20 25 30 Thr Met Gly Trp Phe Arg
Gln Ala Pro Glu Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ser Ser
Arg Asp
Gly Arg Thr Asn Tyr Tyr Ala Asn Ser Val 50 55 60 Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln
Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Ala His Thr Thr Ser Gly Val Pro Val His Glu Arg Ser Tyr Ala 100
105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly
Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser 130 135 140 Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
Val Gln Ala Gly Gly 145 150 155 160 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Ala Thr Phe Ser Ser Tyr 165 170 175 Tyr Met Gly Trp Phe Arg
Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 180 185 190 Ala Ala Ile Asn
Gly Ser Gly Gly Asn Arg Ile Ser Ala Asp Ser Val 195 200 205 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 210 215 220
Leu Gln Leu Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 225
230 235 240 Ala Ala Ser Leu Thr Tyr Tyr Gly His Ser Ala His Tyr Asp
Tyr Trp 245 250 255 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 260 265
18124PRTArtificial SequencePolypeptide sequence of anti-TcdB ICVD
ID45B 18Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Leu
Ser Ser Tyr 20 25 30 Thr Met Gly Trp Phe Arg Gln Ala Pro Glu Lys
Glu Arg Glu Phe Val 35 40 45 Ala Gly Ser Ser Arg Asp Gly Arg Thr
Asn Tyr Tyr Ala Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His
Thr Thr Ser Gly Val Pro Val Arg Glu Arg Ser Tyr Ala 100 105 110 Tyr
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
19124PRTArtificial SequencePolypeptide sequence of anti-TcdB ICVD
ID46B 19Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Leu
Ser Ser Tyr 20 25 30 Thr Met Gly Trp Phe Arg Gln Ala Pro Glu Lys
Glu Arg Glu Phe Val 35 40 45 Ala Gly Ser Ser Arg Asp Gly Arg Thr
Asn Tyr Tyr Ala Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His
Thr Thr Ser Gly Val Pro Val His Glu Arg Ser Tyr Ala 100 105 110 Tyr
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
20124PRTArtificial SequencePolypeptide sequence of anti-TcdB ICVD
ID47B 20Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Leu
Ser Ser Tyr 20 25 30 Thr Met Gly Trp Phe Arg Gln Ala Pro Glu Lys
Glu Arg Glu Phe Val 35 40 45 Ala Gly Ser Ser Arg Asp Gly Arg Thr
Asn Tyr Tyr Ala Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His
Thr Thr Ser Gly Val Pro Val Ala Glu Arg Ser Tyr Ala 100 105 110 Tyr
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
21124PRTArtificial SequencePolypeptide sequence of anti-TcdB ICVD
ID48B 21Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Leu
Ser Ser Tyr 20 25 30 Thr Met Gly Trp Phe Arg Gln Ala Pro Glu Lys
Glu Arg Glu Phe Val 35 40 45 Ala Gly Ser Ser Arg Asp Gly Arg Thr
Asn Tyr Tyr Ala Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His
Thr Thr Ser Gly Val Pro Val Gln Glu Arg Ser Tyr Ala 100 105 110 Tyr
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
22124PRTArtificial SequencePolypeptide sequence of anti-TcdB ICVD
ID49B 22Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Leu
Ser Ser Tyr 20 25 30 Thr Met Gly Trp Phe Arg Gln Ala Pro Glu Lys
Glu Arg Glu Phe Val 35 40 45 Ala Gly Ser Ser Arg Asp Gly Arg Thr
Asn Tyr Tyr Ala Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His
Thr Thr Ser Gly Val Pro Val Phe Glu Arg Ser Tyr Ala 100 105 110 Tyr
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
23124PRTArtificial SequencePolypeptide sequence of anti-TcdB ICVD
ID50B 23Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Leu
Ser Ser Tyr 20 25 30 Thr Met Gly Trp Phe Arg Gln Ala Pro Glu Lys
Glu Arg Glu Phe Val 35 40 45 Ala Gly Ser Ser Arg Asp Gly Arg Thr
Asn Tyr Tyr Ala Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His
Thr Thr Ser Gly Val Pro Val Trp Glu Arg Ser Tyr Ala 100 105 110 Tyr
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
24257PRTArtificial SequencePolypeptide sequence of anti-TcdA
construct ID17A 24Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val
Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Ala Thr Ser Asp Val Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Val
Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Thr Ile Asn Arg Ser
Gly Ser Asp Ser Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln
Met Asn Ser Leu Lys Pro Glu Glu Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Ala Ser Arg Ser Asp Cys Ile Gly Tyr Gly Cys Arg Arg Val Ser 100
105 110 Gln Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly
Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly 130 135 140 Gly Ser Asp Val Gln Leu Gln Glu Ser Gly Gly
Gly Leu Val Gln Ala 145 150 155 160 Gly Gly Ser Leu Arg Leu Ser Cys
Val Ile Ser Gly Met Asp Phe Ser 165 170 175 His Lys Pro Ala Gly Trp
Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 180 185 190 Phe Val Ala Ser
Ile Thr Thr Arg Ala Ser Thr His Tyr Ala Asp Ser 195 200 205 Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val 210 215 220
Tyr Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr 225
230 235 240 Cys Asn Ser Glu Tyr Tyr Trp Gly Gln Gly Thr Gln Val Thr
Val Ser 245 250 255 Ser 25257PRTArtificial SequencePolypeptide
sequence of anti-TcdA construct ID29A 25Asp Val Gln Leu Gln Glu Ser
Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Ala Thr Ser Asp Val Tyr 20 25 30 Ala Met Gly
Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala
Thr Ile Asn Arg Ser Gly Ser Asp Ser Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Glu Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Ala Ser Arg Ser Asp Cys Ile Gly Tyr Gly Cys
His Arg Val Ser 100 105 110 Gln Asp Tyr Trp Gly Gln Gly Thr Gln Val
Thr Val Ser Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly 130 135 140 Gly Ser Asp Val Gln Leu
Gln Glu Ser Gly Gly Gly Leu Val Gln Ala 145 150 155 160 Gly Gly Ser
Leu Arg Leu Ser Cys Val Ile Ser Gly Met Asp Phe Ser 165 170 175 His
Lys Pro Ala Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 180 185
190 Phe Val Ala Ser Ile Thr Thr Arg Ala Ser Thr His Tyr Ala Asp Ser
195 200 205 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Thr Val 210 215 220 Tyr Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Tyr 225 230 235 240 Cys Asn Ser Glu Tyr Tyr Trp Gly Gln
Gly Thr Gln Val Thr Val Ser 245 250 255 Ser 2615PRTArtificial
SequenceExample CDR A 26Ala Arg Asn Glu Cys Asp Gln Gly His Ile Leu
Lys Met Phe Pro 1 5 10 15 275PRTArtificial SequenceFirst third of
Example CDR A 27Ala Arg Asn Glu Cys 1 5 285PRTArtificial
SequenceSecond third of Example CDR A 28Asp Gln Gly His Ile 1 5
295PRTArtificial SequenceThird third of Example CDR A 29Leu Lys Met
Phe Pro 1 5 308PRTArtificial SequenceExample CDR B 30Ala Arg Asn
Glu Cys Asp Gln Gly 1 5 314PRTArtificial SequenceSecond third of
Example CDR B 31Asn Glu Cys Asp 1 32120PRTArtificial
SequencePolypeptide sequence of anti-IL6R ICVD 7F6 32Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser
Thr Arg Leu Thr Cys Leu Ala Ser Gly Ser Ile Ser Ser Ile Asn 20 25
30 Val Ile Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45 Ala Met Ile Gly Arg Gly Glu Gly Ala Asn Tyr Gly Asp Phe
Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Tyr Cys Tyr 85 90 95 Ala Asp Tyr Glu Asp Arg Asp Ser
Pro Phe Asn Gly Ser Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val
Ser Ser 115 120 33120PRTArtificial SequencePolypeptide sequence of
anti-IL6R ICVD ID-3V 33Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Ala Gly Gly 1 5 10 15 Ser Thr Arg Leu Thr Cys Leu Ala Ser
Gly Ser Ile Ser Ser Ile Asn 20 25 30 Val Ile Gly Trp Tyr Arg Gln
Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Met Ile Gly Arg
Gly Glu Gly Ala Asn Tyr Gly Asp Phe Ala Lys 50 55 60 Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln
Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr 85 90
95 Ala Asp Tyr Glu Asp His Asp Ser Pro Phe Asn Gly Ser Trp Gly Gln
100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120
34123PRTArtificial SequencePolypeptide sequence of anti-IL6R ICVD
5G9 34Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
Gly 1 5 10 15 Ser Thr Arg Leu Thr Cys Lys Ala Ser Gly Ser Ile Phe
Asn Ile Asn 20 25 30 Ser Ile Asn Val Met Ala Trp Tyr Arg Gln Ala
Pro Gly Lys Gln Arg 35 40 45 Glu Leu Val Ala Ile Ile Gly Lys Gly
Gly Gly Thr Asn Tyr Ala Asp 50 55 60 Phe Val Lys Gly Arg Phe Thr
Ile Ser Arg Asp Ala Ala Lys Asn Thr 65 70 75 80 Val Asn Leu Gln Met
Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Tyr
Ala Asp Tyr Glu Asp Arg Asp Ser Pro Phe Asn Ala Ser 100 105 110 Trp
Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 35123PRTArtificial
SequencePolypeptide sequence of anti-IL6R ICVD ID-54V 35Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser
Thr Arg Leu Thr Cys Lys Ala Ser Gly Ser Ile Phe Asn Ile Asn 20 25
30 Ser Ile Asn Val Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg
35 40 45 Glu Leu Val Ala Ile Ile Gly Lys Gly Gly Gly Thr Asn Tyr
Ala Asp 50 55 60 Phe Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Ala
Ala Lys Asn Thr 65 70 75 80 Val Asn Leu Gln Met Asn Ser Leu Lys Pro
Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Tyr Ala Asp Tyr Glu Asp
His Asp Ser Pro Phe Asn Ala Ser 100 105 110 Trp Gly Gln Gly Thr Gln
Val Thr Val Ser Ser 115 120
* * * * *