U.S. patent application number 14/489837 was filed with the patent office on 2015-05-28 for neutralising antibody molecules having specificity for human il-17.
The applicant listed for this patent is UCB PHARMA S.A.. Invention is credited to Ralph Adams, Andrew George Popplewell, Stephen Edward Rapecki, Simon Peter Tickle.
Application Number | 20150147331 14/489837 |
Document ID | / |
Family ID | 33548589 |
Filed Date | 2015-05-28 |
United States Patent
Application |
20150147331 |
Kind Code |
A1 |
Adams; Ralph ; et
al. |
May 28, 2015 |
Neutralising Antibody Molecules having Specificity for Human
IL-17
Abstract
The invention relates to an antibody molecule having specificity
for antigenic determinants of IL-17, therapeutic uses of the
antibody molecule and methods for producing said antibody
molecule.
Inventors: |
Adams; Ralph; (Slough,
GB) ; Popplewell; Andrew George; (Slough, GB)
; Rapecki; Stephen Edward; (Slough, GB) ; Tickle;
Simon Peter; (Slough, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UCB PHARMA S.A. |
Brussels |
|
BE |
|
|
Family ID: |
33548589 |
Appl. No.: |
14/489837 |
Filed: |
September 18, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13709055 |
Dec 9, 2012 |
8865167 |
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14489837 |
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11791109 |
May 17, 2007 |
8231875 |
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PCT/GB2005/004392 |
Nov 16, 2005 |
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13709055 |
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Current U.S.
Class: |
424/139.1 |
Current CPC
Class: |
A61K 2039/505 20130101;
A61P 19/08 20180101; A61P 17/06 20180101; C07K 2317/20 20130101;
A61P 3/10 20180101; A61P 31/04 20180101; C07K 2317/92 20130101;
A61P 9/10 20180101; A61P 1/16 20180101; A61P 35/00 20180101; A61P
7/02 20180101; A61P 37/06 20180101; A61P 29/00 20180101; C07K
16/244 20130101; A61P 37/02 20180101; A61P 43/00 20180101; C07K
2317/24 20130101; A61P 3/00 20180101; A61P 31/12 20180101; A61P
41/00 20180101; C07K 2317/76 20130101; A61P 33/00 20180101; A61P
1/04 20180101; A61P 31/00 20180101; A61P 1/02 20180101; A61P 1/18
20180101; A61P 19/10 20180101; A61P 31/10 20180101; A61P 9/00
20180101; A61K 47/60 20170801; C07K 16/464 20130101; A61P 17/02
20180101; C07K 2317/55 20130101; C07K 2317/565 20130101; A61P 11/06
20180101; A61P 19/02 20180101; A61P 35/02 20180101; A61P 25/28
20180101 |
Class at
Publication: |
424/139.1 |
International
Class: |
C07K 16/24 20060101
C07K016/24 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 19, 2004 |
GB |
0425569.1 |
Claims
1. (canceled)
2. A method of treating an IL-17 mediated pathological condition in
a mammal comprising the step of administering to the mammal a
therapeutically effective amount of a pharmaceutical composition
comprising a neutralising antibody having specificity for human
IL-17, wherein a variable domain of a heavy chain comprises at
least one CDR having a sequence selected from SEQ ID NO:5 for
CDR-H1, SEQ ID NO:6 for CDR-H2, and SEQ ID NO:7 for CDR-H3 and
wherein a light chain comprises SEQ ID NO:13.
3. The method of claim 2, wherein the neutralising antibody
comprises a light chain and a heavy chain, wherein a variable
domain of the heavy chain comprises SEQ ID NO:5 for CDR-H1, SEQ ID
NO:6 for CDR-H2, and SEQ ID NO:7 for CDR-H3, and a variable domain
of the light chain comprises SEQ ID NO:8 for CDR-L1, SEQ ID NO:9
for CDR-L2, and SEQ ID NO:10 for CDR-L3 to human IL-17.
4. The method of claim 2, wherein the neutralising antibody
comprises a heavy chain and a light chain, wherein a variable
domain of the heavy chain comprises the sequence gH11 (SEQ ID
NO:11) and a variable domain of the light chain comprises the
sequence gL3 (SEQ ID NO: 13).
5. The method of claim 2, wherein the neutralising antibody
comprises a heavy chain comprising sequence given in SEQ ID NO:16
and a light chain comprising sequence given in SEQ ID NO:18.
6. The method of claim 2, wherein the neutralising antibody
comprises a light chain and a heavy chain wherein the heavy chain
comprises SEQ ID NO:11.
7. The method of claim 2, wherein the neutralising antibody binds
to the same epitope as an antibody in which a variable domain of a
heavy chain comprises sequence gH11 (SEQ ID NO:11) and a variable
domain of a light chain comprises the sequence gL3 (SEQ ID NO:
13).
8. The method of claim 2, wherein the neutralising antibody binds
to a same epitope as an antibody comprising a light chain and a
heavy chain, wherein a variable domain of the heavy chain comprises
SEQ ID NO:5 for CDR-H1, SEQ ID NO:6 for CDR-H2, and SEQ ID NO:7 for
CDR-H3 and wherein a variable domain of the light chain comprises
SEQ ID NO:8 for CDRL1, SEQ ID NO:9 for CDR-L2, and SEQ ID NO:10 for
CDR-L3 to human IL-17.
9. The method of claim 2, wherein the neutralising antibody
cross-blocks binding of an antibody in which a variable domain of a
heavy chain comprises sequence gH11 (SEQ ID NO: 11) and a variable
domain of a light chain comprises sequence gL3 (SEQ ID NO: 13) to
human IL-17.
10. The method of claim 2, wherein the neutralising antibody
cross-blocks binding of an antibody in which a variable domain of a
heavy chain comprises SEQ ID NO:5 for CDR-H1, SEQ ID NO:6 for
CDR-H2, and SEQ ID NO:7 for CDR-H3 and wherein a variable domain of
a light chain comprises SEQ ID NO:8 for CDRL1, SEQ ID NO:9 for
CDR-L2, and SEQ ID NO:10 for CDR-L3 to human IL-17.
11. The method of claim 2, wherein the neutralising antibody is
cross-blocked from binding human IL-17 by the antibody in which the
variable domain of the heavy chain comprises the sequence gH11 (SEQ
ID NO: 11) and the variable domain of the light chain comprises the
sequence gL3 (SEQ ID NO: 13) to human IL-17.
12. The method of claim 2, wherein the neutralising antibody
affinity for human IL-17 is between 100 pM and 400 pM.
13. The method of claim 2, wherein the neutralising antibody
affinity for human IL-17 is less than 500 pM.
14. The method of claim 2, wherein one or more effector or reporter
molecules are attached to the neutralising antibody.
15. The method of claim 2, wherein the pharmaceutical composition
further comprises one or more of a pharmaceutically acceptable
excipient, diluent or carrier.
16. The method of claim 2, wherein the IL-17 mediated pathological
condition is associated with an increased level of IL-17.
17. The method of claim 2, wherein the IL-17 mediated pathological
condition is an infection comprising viral, bacterial, fungal or
parasitic infection, endotoxic shock associated with infection,
arthritis, rheumatoid arthritis, asthma, pelvic inflammatory
disease, Alzheimer's disease, Crohn' s disease, inflammatory bowel
disease, ulcerative colitis, Peyronie's disease, coeliac disease,
gallbladder disease, Pilonidal disease, peritonitis, psoriasis,
vasculitis, surgical adhesions, stroke, Type I Diabetes, Lyme
arthritis, meningoencephalitis, immune mediated inflammatory
disorders of the central and peripheral nervous system such as
multiple sclerosis and Guillain-Barr syndrome, other autoimmune
disorders, pancreatitis, trauma, surgery, graft-versus-host
disease, transplant rejection, cancer comprising solid tumours
comprising melanomas, hepatoblastomas, sarcomas, squamous cell
carcinomas, transitional cell cancers, ovarian cancers and
hematologic malignancies and in particular acute myelogenous
leukaemia, chronic myelogenous leukemia, gastric cancer and colon
cancer, heart disease comprising ischaemic diseases, wherein the
diseases comprise myocardial infarction or atherosclerosis,
intravascular coagulation, bone resorption, osteoporosis,
periodontitis, hypochlorhydia, pain, rheumatoid arthritis, multiple
sclerosis, inflammatory disease, autoimmune disease or cancer.
18. A kit comprising the pharmaceutical composition of claim 2 and
instructions for use.
19. A method of treating an IL-17 mediated pathological condition
in a mammal comprising the step of administering to the mammal a
therapeutically effective amount of a pharmaceutical composition
comprising a neutralising antibody having specificity for human
IL-17, wherein the neutralising antibody binds to a same epitope as
an antibody in which a variable domain of a heavy chain comprises
sequence gH11 (SEQ ID NO:11) and a variable domain of a light chain
comprises sequence gL3 (SEQ ID NO: 13).
Description
[0001] The present invention relates to an antibody molecule having
specificity for antigenic determinants of IL-17. The present
invention also relates to the therapeutic uses of the antibody
molecule and methods for producing the antibody molecule.
[0002] Interleukin 17 (IL-17), also known as CTLA-8 or IL-17A, is a
pro-inflammatory cytokine which stimulates the secretion of a wide
range of other cytokines from various non-immune cells. IL-17 is
capable of inducing the secretion of IL-6, IL-8, PGE2, MCP-1 and
G-CSF by adherent cells like fibroblasts, keratinocytes, epithelial
and endothelial cells and is also able to induce ICAM-1 surface
expression, proliferation of T cells, and growth and
differentiation of CD34+ human progenitors into neutrophils when
cocultured in the presence of irradiated fibroblasts (Fossiez et
al., 1998, Int. Rev. Immunol. 16, 541-551). IL-17 is predominantly
produced by activated memory T cells and acts by binding to a
ubiquitously distributed cell surface receptor (IL-17R) (Yao et
al., 1997, Cytokine, 9, 794-800). A number of homologues of IL-17
have been identified which have both similar and distinct roles in
regulating inflammatory responses. For a review of IL-17
cytokine/receptor families see Dumont, 2003, Expert Opin. Ther.
Patents, 13, 287-303.
[0003] IL-17 may contribute to a number of diseases mediated by
abnormal immune responses, such as rheumatoid arthritis and air-way
inflammation, as well as organ transplant rejection and antitumour
immunity. Inhibitors of IL-17 activity are well known in the art
for example a murine IL-17R:human Fc fusion protein, a murine
soluble IL-17R and an anti-IL-17 monoclonal antibody have been used
to demonstrate the role of IL-17 in various models of rheumatoid
arthritis (Lubberts et al., J. Immunol. 2001, 167, 1004-1013;
Chabaud et al., Arthritis Res. 2001, 3, 168-177). In addition,
neutralising polyclonal antibodies have been used to reduce
peritoneal adhesion formation (Chung et al., 2002, J. Exp. Med.,
195, 1471-1478). To date no anti-human IL-17 antibodies have been
developed for use in therapy and hence there is a need for a high
affinity, anti-IL-17 antibody suitable for treating patients.
[0004] We have now identified a high affinity neutralising
anti-IL-17 antibody that is particularly efficacious in vivo, for
example in the in vivo inflammation models described herein.
[0005] The residues in antibody variable domains are conventionally
numbered according to a system devised by Kabat et al. This system
is set forth in Kabat et al., 1987, in Sequences of Proteins of
Immunological Interest, US Department of Health and Human Services,
NIH, USA (hereafter "Kabat et al. (supra)"). This numbering system
is used in the present specification except where otherwise
indicated.
[0006] The Kabat residue designations do not always correspond
directly with the linear numbering of the amino acid residues. The
actual linear amino acid sequence may contain fewer or additional
amino acids than in the strict Kabat numbering corresponding to a
shortening of, or insertion into, a structural component, whether
framework or complementarity determining region (CDR), of the basic
variable domain structure. The correct Kabat numbering of residues
may be determined for a given antibody by alignment of residues of
homology in the sequence of the antibody with a "standard" Kabat
numbered sequence.
[0007] The CDRs of the heavy chain variable domain are located at
residues 31-35 (CDR-H1), residues 50-65 (CDR-H2) and residues
95-102 (CDR-H3) according to the Kabat numbering system. However,
according to Chothia (Chothia, C. and Lesk, A. M. J. Mol. Biol.,
196, 901-917 (1987)), the loop equivalent to CDR-H1 extends from
residue 26 to residue 32. Thus `CDR-H1`, as used herein, comprises
residues 26 to 35, as described by a combination of the Kabat
numbering system and Chothia's topological loop definition.
[0008] The CDRs of the light chain variable domain are located at
residues 24-34 (CDR-L1), residues 50-56 (CDR-L2) and residues 89-97
(CDR-L3) according to the Kabat numbering system.
[0009] As used herein, the term `neutralising antibody` describes
an antibody that is capable of neutralising the biological
signalling activity of IL-17, for example by blocking binding of
IL-17 to the IL-17R.
[0010] In a first aspect, the present invention provides a
neutralising antibody having specificity for human IL-17,
comprising a heavy chain, wherein the variable domain of the heavy
chain comprises at least one of a CDR having the sequence given in
SEQ ID NO:5 for CDR-H1, a CDR having the sequence given in SEQ ID
NO:6 for CDR-H2 and a CDR having the sequence given in SEQ ID NO:7
for CDR-H3.
[0011] Preferably, an antibody of the first aspect of the present
invention comprises a heavy chain wherein at least two of CDR-H1,
CDR-H2 and CDR-H3 of the variable domain of the heavy chain are
selected from the following: the sequence given in SEQ ID NO:5 for
CDR-H1, the sequence given in SEQ ID NO:6 for CDR-H2 and the
sequence given in SEQ ID NO:7 for CDR-H3. For example, the antibody
may comprise a heavy chain wherein CDR-H1 has the sequence given in
SEQ ID NO:5 and CDR-H2 has the sequence given in SEQ ID NO:6.
Alternatively, the antibody may comprise a heavy chain wherein
CDR-H1 has the sequence given in SEQ ID NO:5 and CDR-H3 has the
sequence given in SEQ ID NO:7, or the antibody may comprise a heavy
chain wherein CDR-H2 has the sequence given in SEQ ID NO:6 and
CDR-H3 has the sequence given in SEQ ID NO:7. For the avoidance of
doubt, it is understood that all permutations are included.
[0012] More preferably, the antibody of the first aspect of the
present invention comprises a heavy chain, wherein the variable
domain comprises the sequence given in SEQ ID NO:5 for CDR-H1, the
sequence given in SEQ ID NO:6 for CDR-H2 and the sequence given in
SEQ ID NO:7 for CDR-H3.
[0013] In one embodiment, the antibody of the first aspect of the
present invention comprises a heavy chain, wherein the variable
domain of the heavy chain comprises the sequence given in SEQ ID
NO:2.
[0014] In another embodiment, the antibody of the first aspect of
the present invention comprises a heavy chain, wherein the variable
domain of the heavy chain comprises a sequence having at least 60%
identity or similarity to the sequence given in SEQ ID NO:2. In one
embodiment, the antibody of the first aspect of the present
invention comprises a heavy chain, wherein the variable domain of
the heavy chain comprises a sequence having at least 90%, 95% or
98% identity or similarity to the sequence given in SEQ ID
NO:2.
[0015] "Identity", as used herein, indicates that at any particular
position in the aligned sequences, the amino acid residue is
identical between the sequences. "Similarity", as used herein,
indicates that, at any particular position in the aligned
sequences, the amino acid residue is of a similar type between the
sequences. For example, leucine may be substituted for isoleucine
or valine. Other amino acids which can often be substituted for one
another include but are not limited to: [0016] phenylalanine,
tyrosine and tryptophan (amino acids having aromatic side chains);
[0017] lysine, arginine and histidine (amino acids having basic
side chains); [0018] aspartate and glutamate (amino acids having
acidic side chains); [0019] asparagine and glutamine (amino acids
having amide side chains); and [0020] cysteine and methionine
(amino acids having sulphur-containing side chains). Degrees of
identity and similarity can be readily calculated (Computational
Molecular Biology, Lesk, A. M., ed., Oxford University Press, New
York, 1988; Biocomputing. Informatics and Genome Projects, Smith,
D. W., ed., Academic Press, New York, 1993; Computer Analysis of
Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds.,
Humana Press, New Jersey, 1994; Sequence Analysis in Molecular
Biology, von Heinje, G., Academic Press, 1987; and Sequence
Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton
Press, New York, 1991).
[0021] In a second aspect, the present invention provides a
neutralising antibody having specificity for human IL-17,
comprising a light chain, wherein the variable domain of the light
chain comprises at least one of a CDR having the sequence given in
SEQ ID NO:8 for CDR-L1, a CDR having the sequence given in SEQ ID
NO:9 for CDR-L2 and a CDR having the sequence given in SEQ ID NO:10
for CDR-L3.
[0022] Preferably, the antibody of the second aspect of the present
invention comprises a light chain, wherein at least two of CDR-L1,
CDR-L2 and CDR-L3 of the variable domain of the light chain are
selected from the following: the sequence given in SEQ ID NO:8 for
CDR-L1, the sequence given in SEQ ID NO:9 for CDR-L2 and the
sequence given in SEQ ID NO:10 for CDR-L3. For example, the
antibody may comprise a light chain wherein CDR-L1 has the sequence
given in SEQ ID NO:8 and CDR-L2 has the sequence given in SEQ ID
NO:9. Alternatively, the antibody may comprise a light chain
wherein CDR-L1 has the sequence given in SEQ ID NO:8 and CDR-L3 has
the sequence given in SEQ ID NO:10, or the antibody may comprise a
light chain wherein CDR-L2 has the sequence given in SEQ ID NO:9
and CDR-L3 has the sequence given in SEQ ID NO:10. For the
avoidance of doubt, it is understood that all permutations are
included.
[0023] More preferably, the antibody of the second aspect of the
present invention comprises a light chain, wherein the variable
domain comprises the sequence given in SEQ ID NO:8 for CDR-L1, the
sequence given in SEQ ID NO:9 for CDR-L2 and the sequence given in
SEQ ID NO:10 for CDR-L3.
[0024] In one embodiment, the antibody of the second aspect of the
present invention comprises a light chain, wherein the variable
domain of the light chain comprises the sequence given in SEQ ID
NO:4.
[0025] In another embodiment, the antibody of the second aspect of
the present invention comprises a light chain, wherein the variable
domain of the light chain comprises a sequence having at least 60%
identity or similarity to the sequence given in SEQ ID NO:4.
Preferably, the antibody of the second aspect of the present
invention comprises a light chain, wherein the variable domain of
the light chain comprises a sequence having at least 90%, 95% or
98% identity or similarity to the sequence given in SEQ ID
NO:4.
[0026] The antibody molecules of the first and second aspects of
the present invention preferably comprise a complementary light
chain or a complementary heavy chain, respectively.
[0027] Preferably, the antibody according to either of the first
and second aspects of the present invention comprises a heavy
chain, wherein the variable domain of the heavy chain comprises the
sequence given in SEQ ID NO:5 for CDR-H1, the sequence given in SEQ
ID NO:6 for CDR-H2 and the sequence given in SEQ ID NO:7 for CDR-H3
and a light chain wherein the variable domain of the light chain
comprises the sequence given in SEQ ID NO:8 for CDR-L1, the
sequence given in SEQ ID NO:9 for CDR-L2 and the sequence given in
SEQ ID NO:10 for CDR-L3.
[0028] In one embodiment of the first and second aspects of the
invention, the antibody comprises a heavy chain, wherein the
variable domain of the heavy chain comprises the sequence given in
SEQ ID NO:2 and a light chain, wherein the variable domain of the
light chain comprises the sequence given in SEQ ID NO:4.
[0029] Hence in one further embodiment of the first and second
aspects of the invention, the antibody comprises a heavy chain and
a light chain, wherein the variable domain of the heavy chain
comprises a sequence having at least 60% identity or similarity to
the sequence given in SEQ ID NO:2 and the variable domain of the
light chain comprises a sequence having at least 60% identity or
similarity to the sequence given in SEQ ID NO:4. Preferably, the
antibody comprises a heavy chain, wherein the variable domain of
the light chain comprises a sequence having at least 90%, 95% or
98% identity or similarity to the sequence given in SEQ ID NO:2 and
a light chain, wherein the variable domain of the light chain
comprises a sequence having at least 90%, 95% or 98% identity or
similarity to the sequence given in SEQ ID NO:4.
[0030] In a third aspect of the present invention, there is
provided an antibody according to either the first or the second
aspect of the invention, wherein said antibody is a monoclonal
antibody.
[0031] In a preferred embodiment of the third aspect of the
invention, the monoclonal antibody comprises a heavy chain, wherein
the variable domain of the heavy chain comprises the sequence given
in SEQ ID NO:2 and a light chain, wherein the variable domain of
the light chain comprises the sequence given in SEQ ID NO:4.
[0032] In an alternatively preferred embodiment of the third aspect
of the invention, the monoclonal antibody is a murine monoclonal
antibody, wherein the monoclonal antibody comprises a heavy chain
and a light chain, wherein the variable domain of the heavy chain
comprises the sequence given in SEQ ID NO:2, and wherein the
variable domain of the light chain comprises the sequence given in
SEQ ID NO:4. This murine monoclonal antibody is referred to herein
as `IL-17F4.100` or as the "donor" antibody. The complete
nucleotide and amino acid sequences of the variable domains of the
heavy and light chains of mouse monoclonal antibody IL-17F4.100 are
shown in FIGS. 1a and 1b and are given in SEQ ID NOS: 1 to 4. The
CDRs given in SEQ ID NOS: 5 to 10 are derived from murine
monoclonal antibody IL-17F4.100.
[0033] In a fourth aspect of the invention, there is provided a
CDR-grafted antibody molecule, wherein one or more of the CDRs have
been obtained from the murine monoclonal antibody IL-17F4.100 (SEQ
ID NOS:5 to 10). As used herein, the term `CDR-grafted antibody
molecule` refers to an antibody molecule wherein the heavy and/or
light chain contains one or more CDRs (including, if desired, one
or more modified CDRs) from a donor antibody (e.g. a murine
monoclonal antibody) grafted into a heavy and/or light chain
variable region framework of an acceptor antibody (e.g. a human
antibody). For a review, see Vaughan et al, Nature Biotechnology,
16, 535-539, 1998.
[0034] When the CDRs are grafted, any appropriate acceptor variable
region framework sequence may be used having regard to the
class/type of the donor antibody from which the CDRs are derived,
including mouse, primate and human framework regions. Preferably,
the CDR-grafted antibody of the fourth aspect of the present
invention has a variable domain comprising human acceptor framework
regions as well as one or more of the CDRs derived from the donor
antibody as referred to above. Thus, provided is a neutralising
CDR-grafted antibody wherein the variable domain comprises human
acceptor framework regions and non-human, preferably murine, donor
CDRs.
[0035] Examples of human frameworks which can be used in the
present invention are KOL, NEWM, REI, EU, TUR, TEI, LAY and POM
(Kabat et al., supra). For example, KOL and NEWM can be used for
the heavy chain, REI can be used for the light chain and EU, LAY
and POM can be used for both the heavy chain and the light chain.
Alternatively, human germline sequences may be used; these are
available at: http://vbase.mrc-cpe.cam.ac.uk/
[0036] In a CDR-grafted antibody of the present invention, the
acceptor heavy and light chains do not necessarily need to be
derived from the same antibody and may, if desired, comprise
composite chains having framework regions derived from different
chains.
[0037] The preferred framework region for the heavy chain of the
CDR-grafted antibody of the present invention is derived from the
human sub-group VH3 sequence 1-3 3-33 together with JH4 (shown in
FIG. 2; SEQ ID NO:20 and 21). Accordingly, provided is a
neutralising CDR-grafted antibody comprising at least one non-human
donor CDR wherein the heavy chain framework region is derived from
the human subgroup sequence 1-3 3-33 together with JH4. The
sequence of human JH4 is as follows: (YFDY)WGQGTLVTVSS (SEQ ID
NO:21). The YFDY motif is part of CDR-H3 and is not part of
framework 4 (Ravetch, J V. et al., 1981, Cell, 27, 583-591). The
donor sequence is the IL-17F4.100 VH sequence (SEQ ID NO:2) shown
in FIG. 1a.
[0038] The preferred framework region for the light chain of the
CDR-grafted antibody of the present invention is derived from the
human germline sub-group VK1 sequence 2-1-(1) O12 together with JK1
shown in FIG. 2 (SEQ ID NO:22 and 23). Accordingly, provided is a
neutralising CDR-grafted antibody comprising at least one non-human
donor CDR wherein the light chain framework region is derived from
the human subgroup sequence VK1 2-1-(1) O12 together with JK1. The
JK1 sequence is as follows: (WT)FGQGTKVEIK (SEQ ID NO:23). The WT
motif is part of CDR-L3 and is not part of framework 4 (Hieter, P
A., et al., 1982, J. Biol. Chem., 257, 1516-1522). The donor
sequence is the IL-17F4.100 VL sequence (SEQ ID N0:4) shown in FIG.
1b.
[0039] Also, in a CDR-grafted antibody of the present invention,
the framework regions need not have exactly the same sequence as
those of the acceptor antibody. For instance, unusual residues may
be changed to more frequently-occurring residues for that acceptor
chain class or type. Alternatively, selected residues in the
acceptor framework regions may be changed so that they correspond
to the residue found at the same position in the donor antibody
(see Reichmann et al., 1998, Nature, 332, 323-324). Such changes
should be kept to the minimum necessary to recover the affinity of
the donor antibody. A protocol for selecting residues in the
acceptor framework regions which may need to be changed is set
forth in WO 91/09967.
[0040] Preferably, in a CDR-grafted antibody molecule of the
present invention, if the acceptor heavy chain has the human VH3
sequence 1-3 3-33 together with JH4, then the acceptor framework
regions of the heavy chain comprise, in addition to one or more
donor CDRs, a donor residue at at least one of positions 24 and 78,
preferably at both position 24 and 78 (according to Kabat et al.,
(supra)). Accordingly, provided is a CDR-grafted antibody, wherein
at least the residues at position 24 and 78 of the variable domain
of the heavy chain are donor residues.
[0041] Preferably, in a CDR-grafted antibody molecule according to
the present invention, if the acceptor light chain has the human
sub-group VK1 sequence 2-1-(1) O12 together with JK1, then the
acceptor framework regions of the light chain comprise a donor
residue at position 2 (according to Kabat et al., supra).
Accordingly, provided is a CDR-grafted antibody wherein at least
the residue at position 2 is a donor residue.
[0042] Donor residues are residues from the donor antibody, i.e.
the antibody from which the CDRs were originally derived, which in
the case of the present invention is the murine monoclonal antibody
IL-17F4.100.
[0043] In an alternative embodiment of the first or fourth aspects
of the present invention, the heavy chain preferably comprises the
sequence of gH11 (SEQ ID NO:11). The sequence of the variable
domain of this grafted heavy chain is shown in FIG. 3a (starting at
base 64).
[0044] In an alternative embodiment of the second or fourth aspects
of the present invention, the light chain preferably comprises the
sequence of gL3 (SEQ ID NO:13). The sequence of the variable domain
of this grafted light chain is shown in FIG. 3b (starting at base
64).
[0045] More preferably, an antibody molecule according to the
alternative embodiment of the first, second or fourth aspects of
the present invention comprises a heavy chain comprising the
sequence of gH11 (SEQ ID NO:11) and a light chain comprising the
sequence of gL3 (SEQ ID NO:13).
[0046] In one embodiment of the fourth aspect of the invention, the
antibody comprises a heavy chain and a light chain, wherein the
variable domain of the heavy chain comprises a sequence having at
least 60% identity or similarity to the sequence given in SEQ ID
NO:11 and the variable domain of the light chain comprises a
sequence having at least 60% identity or similarity to the sequence
given in SEQ ID NO:13. Preferably, the antibody comprises a heavy
chain, wherein the variable domain of the light chain comprises a
sequence having at least 90%, 95% or 98% identity or similarity to
the sequence given in SEQ ID NO:11 and a light chain, wherein the
variable domain of the light chain comprises a sequence having at
least 90%, 95% or 98% identity or similarity to the sequence given
in SEQ ID NO:13.
[0047] The antibody molecule of the present invention may comprise
a complete antibody molecule having full length heavy and light
chains or a fragment thereof, such as a Fab, modified Fab, Fab',
F(ab').sub.2, Fv or scFv fragment. Alternatively, it may comprise a
light chain or heavy chain monomer or dimer or a single chain
antibody, e.g. a single chain Fv in which the heavy and light chain
variable domains are joined by a peptide linker. Similarly, the
heavy and light chain variable regions may be combined with other
antibody domains as appropriate. The methods for creating and
manufacturing these antibody fragments are well known in the art
(see for example Verma et al., 1998, Journal of Immunological
Methods, 216, 165-181).
[0048] The constant region domains of the antibody molecule of the
present invention, if present, may be selected having regard to the
proposed function of the antibody molecule, and in particular the
effector functions which may be required. For example, the constant
region domains may be human IgA, IgD, IgE, IgG or IgM domains. In
particular, human IgG constant region domains may be used,
especially of the IgG1 and IgG3 isotypes when the antibody molecule
is intended for therapeutic uses and antibody effector functions
are required. Alternatively, IgG2 and IgG4 isotypes may be used
when the antibody molecule is intended for therapeutic purposes and
antibody effector functions are not required, e.g. for simply
blocking IL-17 activity.
[0049] Particular antibody fragments for use in the present
invention include Fab and Fab' fragments and those described in
International patent applications WO2005/003169, WO2005/003170 and
WO2005/003171 (Published 13.1.2005). In particular the modified
antibody Fab fragments described in International patent
application WO2005/003169 are preferred. These Fab fragments
comprise a heavy and light chain pair, V.sub.H/C.sub.H1 and
V.sub.L/C.sub.L covalently linked through interchain cysteines in
the heavy and light chain constant regions and are characterised in
that the heavy chain constant region terminates at the interchain
cysteine of C.sub.H1. The term `interchain cysteine` refers to a
cysteine in the heavy or light chain constant region that would be
disulphide linked to a cysteine in the corresponding heavy or light
chain constant region encoded in a naturally occurring germline
antibody gene. In particular the interchain cysteines are a
cysteine in the constant region of the light chain (C.sub.L) and a
cysteine in the first constant region of the heavy chain (C.sub.H1)
that are disulphide linked to each other in naturally occurring
antibodies. Examples of such cysteines may typically be found at
position 214 of the light chain and 233 of the heavy chain of human
IgG1, 127 of the heavy chain of human IgM, IgE, IgG2, IgG3, IgG4
and 128 of the heavy chain of human IgD and IgA2B, as defined by
Kabat et al., 1987, in Sequences of Proteins of Immunological
Interest, US Department of Health and Human Services, NIH, USA. In
murine IgG, interchain cysteines may be found at position 214 of
the light chain and 235 of the heavy chain. It will be appreciated
that the exact positions of these cysteines may vary from that of
naturally occurring antibodies if any modifications, such as
deletions, insertions and/or substitutions have been made to the
antibody Fab fragment. These antibody Fab fragments may be prepared
by any suitable method known in the art. For example, the antibody
Fab fragment may be obtained from any whole antibody, especially a
whole monoclonal antibody, using any suitable enzymatic cleavage
and/or digestion techniques, for example by treatment with pepsin
or papain and c-terminal proteases. Preferably these antibody Fab
fragments are prepared by the use of recombinant DNA techniques
involving the manipulation and re-expression of DNA encoding
antibody variable and constant regions. Standard molecular biology
techniques may be used to modify, add or delete further amino acids
or domains as desired. Any alterations to the variable or constant
regions are still encompassed by the terms `variable` and
`constant` regions as used herein. Preferably PCR is used to
introduce a stop codon immediately following the codon encoding the
interchain cysteine of C.sub.H1, such that translation of the
C.sub.H1 domain stops at the interchain cysteine. Methods for
designing suitable PCR primers are well known in the art and the
sequences of antibody C.sub.H1 domains are readily available (Kabat
et al., supra). Alternatively stop codons may be introduced using
site-directed mutagenesis techniques such as those described in
White (Ed.), PCR Protocols: Current Methods and Applications
(1993). In one example the constant regions in these fragments are
derived from IgG1 and the interchain cysteine of C.sub.L is at
position 214 of the light chain and the interchain cysteine of
C.sub.H1 is at position 233 of the heavy chain.
[0050] In a preferred embodiment of the first, second or fourth
aspects of the invention, the antibody provided by the present
invention is a neutralising antibody molecule, wherein its heavy
chain comprises or consists of the sequence given in SEQ ID NO:16
and the light chain comprises or consists of the sequence given in
SEQ ID:18. Most preferably, the antibody provided by the present
invention is a neutralising antibody molecule with an antibody
format as described in International patent application
WO2005/003169 wherein its heavy chain comprises or consists of the
sequence given in SEQ ID NO:16, and wherein its light chain
comprises or consists of the sequence given in SEQ ID NO:18.
[0051] In one embodiment of this aspect of the invention, the
antibody comprises a heavy chain and a light chain, wherein the
heavy chain comprises a sequence having at least 60% identity or
similarity to the sequence given in SEQ ID NO:16 and the light
chain comprises a sequence having at least 60% identity or
similarity to the sequence given in SEQ ID NO:18. Preferably, the
antibody comprises a heavy chain, wherein the heavy chain comprises
a sequence having at least 90%, 95% or 98% identity or similarity
to the sequence given in SEQ ID NO:16 and a light chain, wherein
the light chain comprises a sequence having at least 90%, 95% or
98% identity or similarity to the sequence given in SEQ ID
NO:18.
[0052] In a fifth aspect of the invention, there is provided a
specific region or epitope of human IL-17 wherein binding of
IL-17F4.100 or antibodies comprising the heavy chain sequence gH11
(SEQ ID NO:11) and the light chain sequence gL3 (SEQ ID NO:13)
completely neutralises the activity of the IL-17 protein.
[0053] This specific region or epitope of the human IL-17
polypeptide can be identified by any suitable epitope mapping
method known in the art in combination with any one of the
antibodies provided by the present invention. Examples of such
methods include screening peptides of varying lengths derived from
IL-17 for binding to the antibody of the present invention with the
smallest fragment that can specifically bind to the antibody
containing the sequence of the epitope recognised by the antibody.
The IL-17 peptides may be produced synthetically or by proteolytic
digestion of the IL-17 polypeptide. Peptides that bind the antibody
can be identified by, for example, mass spectrometric analysis. In
another example, NMR spectroscopy can be used to identify the
epitope bound by an antibody of the present invention. Once
identified, the epitopic fragment which binds an antibody of the
present invention can be used, if required, as an immunogen to
obtain additional neutralising antibodies which bind the same
epitope.
[0054] Antibodies which cross-block the binding of the antibodies
of the first to fourth aspects of the present invention to IL-17
may be similarly useful in neutralising IL-17 activity. In a sixth
aspect of the invention, therefore, there is provided a
neutralising antibody having specificity for human IL-17, which
cross-blocks the binding of any one of the antibodies provided in
the first to fourth aspects of the present invention to human IL-17
and/or is cross-blocked from binding IL-17 by any one of those
antibodies. In one embodiment the neutralising antibody of the
sixth aspect of the present invention binds to the same epitope as
an antibody provided by the first to fourth aspects of the present
invention. In further embodiments the neutralising antibody of the
sixth aspect of the present invention binds to an epitope which
borders and/or overlaps with the epitope bound by an antibody of
the first to fourth aspects of the invention. In a still further
embodiment the neutralising antibody of the sixth aspect of the
invention does not bind to the same epitope as an antibody of the
first to fourth aspects of the invention or an epitope that borders
and/or overlaps with said epitope.
[0055] Cross-blocking antibodies according to the sixth aspect of
the present invention can be identified using any suitable method
in the art, for example by using competition ELISA or BIAcore where
binding of the cross blocking antibody of the sixth aspect of the
invention to human IL-17 prevents the binding of an antibody
provided in the first to fourth aspects of the present invention or
vice versa.
[0056] In one embodiment there is provided a neutralising antibody
having specificity for human IL-17, which cross-blocks the binding
of IL17F4.100 or an antibody whose heavy chain comprises the
sequence gH11 (SEQ ID NO:11) and whose light chain comprises the
sequence gL3 (SEQ ID NO:13) to human IL-17. In one embodiment the
cross-blocking antibodies provided by the sixth aspect of the
invention inhibit the binding of IL17F4.100 or an antibody whose
heavy chain comprises the sequence gH11 (SEQ ID NO:11) and whose
light chain comprises the sequence gL3 (SEQ ID NO:13) by greater
than 80%, preferably by greater than 85%, more preferably by
greater than 90%, even more preferably by greater than 95%.
[0057] Alternatively or in addition, antibodies according to this
aspect of the invention may be cross-blocked from binding to human
IL-17 by any one of the antibodies of the first to fourth aspects
of the present invention. Also provided therefore is a neutralising
antibody molecule having specificity for human IL-17 which is
cross-blocked from binding human IL-17 by the antibody IL17F4.100
or an antibody whose heavy chain comprises the sequence gH11 (SEQ
ID NO:11) and whose light chain comprises the sequence gL3 (SEQ ID
NO:13). In one embodiment the cross-blocking antibodies provided by
the sixth aspect of the invention are inhibited from binding human
IL-17 by IL17F4.100 or an antibody whose heavy chain comprises the
sequence gH11 (SEQ ID NO:11) and whose light chain comprises the
sequence gL3 (SEQ ID NO:13) by greater than 80%, preferably by
greater than 85%, more preferably by greater than 90%, even more
preferably by greater than 95%.
[0058] The antibody molecule of any aspect of the present invention
preferably has a high binding affinity, preferably picomolar.
Preferably the antibody molecule of the present invention has a
binding affinity of between about 1 and 500 pM. In one embodiment
the antibody molecule of the present invention has a binding
affinity of between about 100 and about 400 pM. It will be
appreciated that the affinity of antibodies provided by the present
invention may be altered using any suitable method known in the
art. The present invention therefore also relates to variants of
the antibody molecules of the present invention, which have an
improved affinity for IL-17. Such variants can be obtained by a
number of affinity maturation protocols including mutating the CDRs
(Yang et al., J. Mol. Biol., 254, 392-403, 1995), chain shuffling
(Marks et al., Bio/Technology, 10, 779-783, 1992), use of mutator
strains of E. coli (Low et al., J. Mol. Biol., 250, 359-368, 1996),
DNA shuffling (Patten et al., Curr. Opin. Biotechnol., 8, 724-733,
1997), phage display (Thompson et al., J. Mol. Biol., 256, 77-88,
1996) and sexual PCR (Crameri et al., Nature, 391, 288-291, 1998).
Vaughan et al. (supra) discusses these methods of affinity
maturation.
[0059] If desired an antibody for use in the present invention
maybe conjugated to one or more effector molecule(s). It will be
appreciated that the effector molecule may comprise a single
effector molecule or two or more such molecules so linked as to
form a single moiety that can be attached to the antibodies of the
present invention. Where it is desired to obtain an antibody
fragment linked to an effector molecule, this may be prepared by
standard chemical or recombinant DNA procedures in which the
antibody fragment is linked either directly or via a coupling agent
to the effector molecule. Techniques for conjugating such effector
molecules to antibodies are well known in the art (see, Hellstrom
et al., Controlled Drug Delivery, 2nd Ed., Robinson et al., eds.,
1987, pp. 623-53; Thorpe et al., 1982, Immunol. Rev., 62:119-58 and
Dubowchik et al., 1999, Pharmacology and Therapeutics, 83, 67-123).
Particular chemical procedures include, for example, those
described in WO 93/06231, WO 92/22583, WO 89/00195, WO 89/01476 and
WO03031581. Alternatively, where the effector molecule is a protein
or polypeptide the linkage may be achieved using recombinant DNA
procedures, for example as described in WO 86/01533 and
EP0392745.
[0060] The term effector molecule as used herein includes, for
example, antineoplastic agents, drugs, toxins, biologically active
proteins, for example enzymes, other antibody or antibody
fragments, synthetic or naturally occurring polymers, nucleic acids
and fragments thereof e.g. DNA, RNA and fragments thereof,
radionuclides, particularly radioiodide, radioisotopes, chelated
metals, nanoparticles and reporter groups such as fluorescent
compounds or compounds which may be detected by NMR or ESR
spectroscopy.
[0061] Examples of effector molecules may include cytotoxins or
cytotoxic agents including any agent that is detrimental to (e.g.
kills) cells. Examples include combrestatins, dolastatins,
epothilones, staurosporin, maytansinoids, spongistatins, rhizoxin,
halichondrins, roridins, hemiasterlins, taxol, cytochalasin B,
gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,
tenoposide, vincristine, vinblastine, colchicin, doxorubicin,
daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,
actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,
tetracaine, lidocaine, propranolol, and puromycin and analogs or
homologs thereof.
[0062] Effector molecules also include, but are not limited to,
antimetabolites (e.g. methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g. mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines
(e.g. daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g. dactinomycin (formerly actinomycin), bleomycin,
mithramycin, anthramycin (AMC), calicheamicins or duocarmycins),
and anti-mitotic agents (e.g. vincristine and vinblastine).
[0063] Other effector molecules may include chelated radionuclides
such as .sup.111In and .sup.90Y, Lu.sup.177, Bismuth.sup.213,
Californium.sup.252, Iridium.sup.192 and
Tungsten.sup.188/Rhenium.sup.188; or drugs such as but not limited
to, alkylphosphocholines, topoisomerase I inhibitors, taxoids and
suramin.
[0064] Other effector molecules include proteins, peptides and
enzymes. Enzymes of interest include, but are not limited to,
proteolytic enzymes, hydrolases, lyases, isomerases, transferases.
Proteins, polypeptides and peptides of interest include, but are
not limited to, immunoglobulins, toxins such as abrin, ricin A,
pseudomonas exotoxin, or diphtheria toxin, a protein such as
insulin, tumour necrosis factor, .alpha.-interferon,
.beta.-interferon, nerve growth factor, platelet derived growth
factor or tissue plasminogen activator, a thrombotic agent or an
anti-angiogenic agent, e.g. angiostatin or endostatin, or, a
biological response modifier such as a lymphokine, interleukin-1
(IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte
macrophage colony stimulating factor (GM-CSF), granulocyte colony
stimulating factor (G-CSF), nerve growth factor (NGF) or other
growth factor and immunoglobulins.
[0065] Other effector molecules may include detectable substances
useful for example in diagnosis. Examples of detectable substances
include various enzymes, prosthetic groups, fluorescent materials,
luminescent materials, bioluminescent materials, radioactive
nuclides, positron emitting metals (for use in positron emission
tomography), and nonradioactive paramagnetic metal ions. See
generally U.S. Pat. No. 4,741,900 for metal ions which can be
conjugated to antibodies for use as diagnostics. Suitable enzymes
include horseradish peroxidase, alkaline phosphatase,
beta-galactosidase, or acetylcholinesterase; suitable prosthetic
groups include streptavidin, avidin and biotin; suitable
fluorescent materials include umbelliferone, fluorescein,
fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine
fluorescein, dansyl chloride and phycoerythrin; suitable
luminescent materials include luminol; suitable bioluminescent
materials include luciferase, luciferin, and aequorin; and suitable
radioactive nuclides include .sup.125I, .sup.131I, .sup.111In and
.sup.99Tc.
[0066] In another example the effector molecule may increase the
half-life of the antibody in vivo, and/or reduce immunogenicity of
the antibody and/or enhance the delivery of an antibody across an
epithelial barrier to the immune system. Examples of suitable
effector molecules of this type include polymers, albumin, albumin
binding proteins or albumin binding compounds such as those
described in PCT/GB2005/002084.
[0067] Where the effector molecule is a polymer it may, in general,
be a synthetic or a naturally occurring polymer, for example an
optionally substituted straight or branched chain polyalkylene,
polyalkenylene or polyoxyalkylene polymer or a branched or
unbranched polysaccharide, e.g. a homo- or
hetero-polysaccharide.
[0068] Particular optional substituents which may be present on the
above-mentioned synthetic polymers include one or more hydroxy,
methyl or methoxy groups.
[0069] Particular examples of synthetic polymers include optionally
substituted straight or branched chain poly(ethyleneglycol),
poly(propyleneglycol) poly(vinylalcohol) or derivatives thereof,
especially optionally substituted poly(ethyleneglycol) such as
methoxypoly(ethyleneglycol) or derivatives thereof.
[0070] Particular naturally occurring polymers include lactose,
amylose, dextran, glycogen or derivatives thereof.
[0071] "Derivatives" as used herein is intended to include reactive
derivatives, for example thiol-selective reactive groups such as
maleimides and the like. The reactive group may be linked directly
or through a linker segment to the polymer. It will be appreciated
that the residue of such a group will in some instances form part
of the product as the linking group between the antibody fragment
and the polymer.
[0072] The size of the polymer may be varied as desired, but will
generally be in an average molecular weight range from 500 Da to
50000 Da, preferably from 5000 to 40000 Da and more preferably from
20000 to 40000 Da. The polymer size may in particular be selected
on the basis of the intended use of the product for example ability
to localize to certain tissues such as tumors or extend circulating
half-life (for review see Chapman, 2002, Advanced Drug Delivery
Reviews, 54, 531-545). Thus, for example, where the product is
intended to leave the circulation and penetrate tissue, for example
for use in the treatment of a tumour, it may be advantageous to use
a small molecular weight polymer, for example with a molecular
weight of around 5000 Da. For applications where the product
remains in the circulation, it may be advantageous to use a higher
molecular weight polymer, for example having a molecular weight in
the range from 20000 Da to 40000 Da.
[0073] Particularly preferred polymers include a polyalkylene
polymer, such as a poly(ethyleneglycol) or, especially, a
methoxypoly(ethyleneglycol) or a derivative thereof, and especially
with a molecular weight in the range from about 15000 Da to about
40000 Da.
[0074] In one example antibodies for use in the present invention
are attached to poly(ethyleneglycol) (PEG) moieties. In one
particular example the antibody is an antibody fragment and the PEG
molecules may be attached through any available amino acid
side-chain or terminal amino acid functional group located in the
antibody fragment, for example any free amino, imino, thiol,
hydroxyl or carboxyl group. Such amino acids may occur naturally in
the antibody fragment or may be engineered into the fragment using
recombinant DNA methods (see for example U.S. Pat. No. 5,219,996;
U.S. Pat. No. 5,667,425; WO98/25971). In one example the antibody
molecule of the present invention is a modified Fab fragment
wherein the modification is the addition to the C-terminal end of
its heavy chain one or more amino acids to allow the attachment of
an effector molecule. Preferably, the additional amino acids form a
modified hinge region containing one or more cysteine residues to
which the effector molecule may be attached. Multiple sites can be
used to attach two or more PEG molecules.
[0075] Preferably PEG molecules are covalently linked through a
thiol group of at least one cysteine residue located in the
antibody fragment. Each polymer molecule attached to the modified
antibody fragment may be covalently linked to the sulphur atom of a
cysteine residue located in the fragment. The covalent linkage will
generally be a disulphide bond or, in particular, a sulphur-carbon
bond. Where a thiol group is used as the point of attachment
appropriately activated effector molecules, for example thiol
selective derivatives such as maleimides and cysteine derivatives
may be used. An activated polymer may be used as the starting
material in the preparation of polymer-modified antibody fragments
as described above. The activated polymer may be any polymer
containing a thiol reactive group such as an .alpha.-halocarboxylic
acid or ester, e.g. iodoacetamide, an imide, e.g. maleimide, a
vinyl sulphone or a disulphide. Such starting materials may be
obtained commercially (for example from Nektar, formerly Shearwater
Polymers Inc., Huntsville, Ala., USA) or may be prepared from
commercially available starting materials using conventional
chemical procedures. Particular PEG molecules include 20K
methoxy-PEG-amine (obtainable from Nektar, formerly Shearwater;
Rapp Polymere; and SunBio) and M-PEG-SPA (obtainable from Nektar,
formerly Shearwater).
[0076] In one embodiment, the antibody is a modified Fab fragment
which is PEGylated, i.e. has PEG (poly(ethyleneglycol)) covalently
attached thereto, e.g. according to the method disclosed in EP
0948544 [see also "Poly(ethyleneglycol) Chemistry, Biotechnical and
Biomedical Applications", 1992, J. Milton Harris (ed), Plenum
Press, New York, "Poly(ethyleneglycol) Chemistry and Biological
Applications", 1997, J. Milton Harris and S. Zalipsky (eds),
American Chemical Society, Washington D.C. and "Bioconjugation
Protein Coupling Techniques for the Biomedical Sciences", 1998, M.
Aslam and A. Dent, Grove Publishers, New York; Chapman, A. 2002,
Advanced Drug Delivery Reviews 2002, 54:531-545]. In one example
PEG is attached to a cysteine in the hinge region. In one example,
a PEG modified Fab fragment has a maleimide group covalently linked
to a single thiol group in a modified hinge region. A lysine
residue may be covalently linked to the maleimide group and to each
of the amine groups on the lysine residue may be attached a
methoxypoly(ethyleneglycol) polymer having a molecular weight of
approximately 20,000 Da. The total molecular weight of the PEG
attached to the Fab fragment may therefore be approximately 40,000
Da.
[0077] In one embodiment, the present invention provides a
neutralising antibody molecule having specificity for human IL-17,
which is a modified Fab fragment having a heavy chain comprising
the sequence given in SEQ ID NO:11 and a light chain comprising the
sequence given in SEQ ID NO:13 and having at the C-terminal end of
its heavy chain a modified hinge region containing at least one
cysteine residue to which an effector molecule is attached.
Preferably the effector molecule is PEG and is attached using the
methods described in (WO98/25971 and WO2004072116) whereby a
lysyl-maleimide group is attached to the cysteine residue at the
C-terminal end of the heavy chain, and each amino group of the
lysyl residue has covalently linked to it a
methoxypoly(ethyleneglycol) residue having a molecular weight of
about 20,000 Da. The total molecular weight of the PEG attached to
the antibody is therefore approximately 40,000 Da.
[0078] In another example effector molecules may be attached to
antibody fragments using the methods described in International
patent applications WO2005/003169, WO2005/003170 and
WO2005/003171.
[0079] In another preferred embodiment an antibody fragment for use
in the present invention is a PEGylated (i.e. has PEG
(poly(ethyleneglycol)) covalently attached thereto) Fab fragment as
described in International Application Number WO2005/003169. This
PEGylated Fab fragment is a Fab fragment in which the heavy chain
terminates at the interchain cysteine of C.sub.H1 and the PEG
attached to the fragment, preferably PEG-maleimide, is covalently
linked to the interchain cysteine of C.sub.L and the interchain
cysteine of C.sub.H1. Preferably the interchain cysteine of C.sub.L
is at position 214 of the light chain and the interchain cysteine
of C.sub.H1 is at position 233 of the heavy chain. As discussed
above the total amount of PEG attached to the fragment may be
varied as desired. In one example each polymer attached to the Fab
preferably has a molecular weight of approximately 20,000 Da. For
example, the molecular weight may be 15,000-25,000 Da, or
preferably 18,000-22,000 Da, and even more preferably 20,000 Da.
The total molecular weight of the PEG attached to the antibody is
therefore approximately 30,000 to 50,000 Da, preferably 40,000
Da.
[0080] PEG is attached to these fragments by first reducing the
interchain disulphide bond between the interchain cysteines of
C.sub.L and C.sub.H1 and subsequently attaching the PEG to the free
thiols. Once PEG is attached to the interchain cysteines there is
no interchain disulphide linkage between the heavy and light chain.
Suitable reducing agents for reducing the interchain disulphide
bond are widely known in the art for example those described in
Singh et al., 1995, Methods in Enzymology, 251, 167-73. Particular
examples include thiol based reducing agents such as reduced
glutathione (GSH), .beta.-mercaptoethanol (.beta.-ME),
.beta.-mercaptoethylamine (.beta.-MA) and dithiothreitol (DTT).
Other methods include using electrolytic methods, such as the
method described in Leach et al., 1965, Div. Protein. Chem, 4,
23-27 and using photoreduction methods, such as the method
described in Ellison et al., 2000, Biotechniques, 28 (2), 324-326.
Preferably however, the reducing agent is a non-thiol based
reducing agent, preferably one of the trialkylphosphine reducing
agents (Ruegg U T and Rudinger, J., 1977, Methods in Enzymology,
47, 111-126; Burns J et al., 1991, J. Org. Chem, 56, 2648-2650;
Getz et al., 1999, Analytical Biochemistry, 273, 73-80; Han and
Han, 1994, Analytical Biochemistry, 220, 5-10; Seitz et al., 1999,
Euro. J. Nuclear Medicine, 26, 1265-1273), particular examples of
which include tris(2-carboxyethyl)phosphine (TCEP), tris butyl
phosphine (TBP), tris-(2-cyanoethyl)phosphine,
tris-(3-hydroxypropyl)phosphine (THP) and
tris-(2-hydroxyethyl)phosphine. Most preferred are the reducing
agents TCEP and THP. It will be clear to a person skilled in the
art that the concentration of reducing agent can be determined
empirically, for example, by varying the concentration of reducing
agent and measuring the number of free thiols produced. Typically
the reducing agent is used in excess over the antibody fragment for
example between 2 and 1000 fold molar excess. Preferably the
reducing agent is in 2, 3, 4, 5, 10, 100 or 1000 fold excess. In
one embodiment the reductant is used at between 2 and 5 mM.
[0081] The reduction and PEGylation reactions may generally be
performed in a solvent, for example an aqueous buffer solution such
as acetate or phosphate, at around neutral pH, for example around
pH 4.5 to around pH 8.5, typically pH 4.5 to 8, suitably pH6 to 7.
The reactions may generally be performed at any suitable
temperature, for example between about 5.degree. C. and about
70.degree. C., for example at room temperature. The solvent may
optionally contain a chelating agent such as EDTA, EGTA, CDTA or
DTPA. Preferably the solvent contains EDTA at between 1 and 5 mM,
preferably 2 mM. Alternatively or in addition the solvent may be a
chelating buffer such as citric acid, oxalic acid, folic acid,
bicine, tricine, tris or ADA. The PEG will generally be employed in
excess concentration relative to the concentration of the antibody
fragment. Typically the PEG is in between 2 and 100 fold molar
excess, preferably 5, 10 or 50 fold excess.
[0082] Where necessary, the desired product containing the desired
number of PEG molecules may be separated from any starting
materials or other product generated during the production process
by conventional means, for example by chromatography techniques
such as ion exchange, size exclusion, protein A, G or L affinity
chromatography or hydrophobic interaction chromatography.
[0083] Hence in one preferred embodiment, the present invention
provides a neutralising antibody molecule having specificity for
human IL-17, which is a Fab fragment as described in International
Application Number WO2005/003169, having a heavy chain comprising
the sequence given in SEQ ID NO:16 and a light chain comprising the
sequence given in SEQ ID NO:18 to which one or more effector
molecules is attached, preferably two or more.
[0084] Most preferably, the antibody of the present invention is a
PEGylated (i.e. has PEG (poly(ethyleneglycol)) covalently attached
thereto) Fab fragment as described in International Application
Number WO2005/003169. The present invention therefore provides a
PEGylated Fab fragment, CDP435, which is a neutralising antibody
molecule having specificity for human IL-17, having a heavy chain
comprising the sequence given in SEQ ID NO:16 and a light chain
comprising the sequence given in SEQ ID NO:18 to which PEG,
preferably PEG-maleimide, is covalently linked to the interchain
cysteine of C.sub.L and the interchain cysteine of C.sub.H1.
Preferably the interchain cysteine of C.sub.L is at position 214 of
the light chain and the interchain cysteine of C.sub.H1 is at
position 233 of the heavy chain (Kabat et al. (supra)). In the
antibody fragment of CDP435 these cysteines can be found by
sequential numbering at positions 222 and 218 of the heavy and
light chain respectively. Preferably each PEG attached to the Fab
has a molecular weight of approximately 20,000 Da and the total
molecular weight of the PEG attached to the Fab is therefore
approximately 40,000 Da. A diagrammatic representation of the
structure of the PEGylated Fab fragment, CDP435 is shown in FIG.
15. n is typically between about 400 and about 520. In one example
n is between 415 and 505. In one example n is about 460.
[0085] The present invention also provides an isolated DNA sequence
encoding the heavy and/or light chain(s) of an antibody molecule of
the present invention. Preferably, the DNA sequence encodes the
heavy or the light chain of an antibody molecule of the present
invention. The DNA sequence of the present invention may comprise
synthetic DNA, for instance produced by chemical processing, cDNA,
genomic DNA or any combination thereof.
[0086] DNA sequences which encode an antibody molecule of the
present invention can be obtained by methods well known to those
skilled in the art. For example, DNA sequences coding for part or
all of the antibody heavy and light chains may be synthesised as
desired from the determined DNA sequences or on the basis of the
corresponding amino acid sequences.
[0087] DNA coding for acceptor framework sequences is widely
available to those skilled in the art and can be readily
synthesised on the basis of their known amino acid sequences.
[0088] Standard techniques of molecular biology may be used to
prepare DNA sequences coding for the antibody molecule of the
present invention. Desired DNA sequences may be synthesised
completely or in part using oligonucleotide synthesis techniques.
Site-directed mutagenesis and polymerase chain reaction (PCR)
techniques may be used as appropriate.
[0089] Examples of suitable sequences are provided in SEQ ID NO:1;
SEQ ID NO:3; SEQ ID NO:12; SEQ ID NO:14; SEQ ID NO:15 and SEQ ID
NO:17.
[0090] The present invention also relates to a cloning or
expression vector comprising one or more DNA sequences of the
present invention. Accordingly, provided is a cloning or expression
vector comprising one or more DNA sequences encoding an antibody of
the present invention. Preferably, the cloning or expression vector
comprises two DNA sequences, encoding the light chain and the heavy
chain of the antibody molecule of the present invention,
respectively. Preferably, a vector according to the present
invention comprises the sequence given in SEQ ID NO:19. Bases 1-63
and 722-784 encode the E. coli OmpA leader sequence which is most
preferably cleaved to give a neutralising antibody molecule of the
present invention. Bases 718 to 721 between the light and heavy
chain sequences represent an intergenic sequence for use in
antibody expression in E. coli (WO03/048208).
[0091] General methods by which the vectors may be constructed,
transfection methods and culture methods are well known to those
skilled in the art. In this respect, reference is made to "Current
Protocols in Molecular Biology", 1999, F. M. Ausubel (ed), Wiley
Interscience, New York and the Maniatis Manual produced by Cold
Spring Harbor Publishing.
[0092] Also provided is a host cell comprising one or more cloning
or expression vectors comprising one or more DNA sequences encoding
an antibody of the present invention. Any suitable host cell/vector
system may be used for expression of the DNA sequences encoding the
antibody molecule of the present invention. Bacterial, for example
E. coli, and other microbial systems may be used or eukaryotic, for
example mammalian, host cell expression systems may also be used.
Suitable mammalian host cells include CHO, myeloma or hybridoma
cells.
[0093] The present invention also provides a process for the
production of an antibody molecule according to the present
invention comprising culturing a host cell containing a vector of
the present invention under conditions suitable for leading to
expression of protein from DNA encoding the antibody molecule of
the present invention, and isolating the antibody molecule.
[0094] The antibody molecule may comprise only a heavy or light
chain polypeptide, in which case only a heavy chain or light chain
polypeptide coding sequence needs to be used to transfect the host
cells. For production of products comprising both heavy and light
chains, the cell line may be transfected with two vectors, a first
vector encoding a light chain polypeptide and a second vector
encoding a heavy chain polypeptide. Alternatively, a single vector
may be used, the vector including sequences encoding light chain
and heavy chain polypeptides.
[0095] As the antibodies of the present invention are useful in the
treatment and/or prophylaxis of a pathological condition, the
present invention also provides a pharmaceutical or diagnostic
composition comprising an antibody molecule of the present
invention in combination with one or more of a pharmaceutically
acceptable excipient, diluent or carrier. Accordingly, provided is
the use of an antibody of the invention for the manufacture of a
medicament. The composition will usually be supplied as part of a
sterile, pharmaceutical composition that will normally include a
pharmaceutically acceptable carrier. A pharmaceutical composition
of the present invention may additionally comprise a
pharmaceutically-acceptable adjuvant.
[0096] The present invention also provides a process for
preparation of a pharmaceutical or diagnostic composition
comprising adding and mixing the antibody molecule of the present
invention together with one or more of a pharmaceutically
acceptable excipient, diluent or carrier.
[0097] The antibody molecule may be the sole active ingredient in
the pharmaceutical or diagnostic composition or may be accompanied
by other active ingredients including other antibody ingredients,
for example anti-TNF, anti-IL-1.beta., anti-T cell, anti-IFN.gamma.
or anti-LPS antibodies, or non-antibody ingredients such as
xanthines.
[0098] The pharmaceutical compositions preferably comprise a
therapeutically effective amount of the antibody of the invention.
The term "therapeutically effective amount" as used herein refers
to an amount of a therapeutic agent needed to treat, ameliorate or
prevent a targeted disease or condition, or to exhibit a detectable
therapeutic or preventative effect. For any antibody, the
therapeutically effective amount can be estimated initially either
in cell culture assays or in animal models, usually in rodents,
rabbits, dogs, pigs or primates. The animal model may also be used
to determine the appropriate concentration range and route of
administration. Such information can then be used to determine
useful doses and routes for administration in humans.
[0099] The precise therapeutically effective amount for a human
subject will depend upon the severity of the disease state, the
general health of the subject, the age, weight and gender of the
subject, diet, time and frequency of administration, drug
combination(s), reaction sensitivities and tolerance/response to
therapy. This amount can be determined by routine experimentation
and is within the judgment of the clinician. Generally, a
therapeutically effective amount will be from 0.01 mg/kg to 50
mg/kg, preferably 0.1 mg/kg to 20 mg/kg. Pharmaceutical
compositions may be conveniently presented in unit dose forms
containing a predetermined amount of an active agent of the
invention per dose.
[0100] Compositions may be administered individually to a patient
or may be administered in combination (e.g. simultaneously,
sequentially or separately) with other agents, drugs or
hormones.
[0101] The dose at which the antibody molecule of the present
invention is administered depends on the nature of the condition to
be treated, the extent of the inflammation present and on whether
the antibody molecule is being used prophylactically or to treat an
existing condition.
[0102] The frequency of dose will depend on the half-life of the
antibody molecule and the duration of its effect. If the antibody
molecule has a short half-life (e.g. 2 to 10 hours) it may be
necessary to give one or more doses per day. Alternatively, if the
antibody molecule has a long half life (e.g. 2 to 15 days) it may
only be necessary to give a dosage once per day, once per week or
even once every 1 or 2 months.
[0103] The pharmaceutically acceptable carrier should not itself
induce the production of antibodies harmful to the individual
receiving the composition and should not be toxic. Suitable
carriers may be large, slowly metabolised macromolecules such as
proteins, polypeptides, liposomes, polysaccharides, polylactic
acids, polyglycolic acids, polymeric amino acids, amino acid
copolymers and inactive virus particles.
[0104] Pharmaceutically acceptable salts can be used, for example
mineral acid salts, such as hydrochlorides, hydrobromides,
phosphates and sulphates, or salts of organic acids, such as
acetates, propionates, malonates and benzoates.
[0105] Pharmaceutically acceptable carriers in therapeutic
compositions may additionally contain liquids such as water,
saline, glycerol and ethanol. Additionally, auxiliary substances,
such as wetting or emulsifying agents or pH buffering substances,
may be present in such compositions. Such carriers enable the
pharmaceutical compositions to be formulated as tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries and suspensions,
for ingestion by the patient.
[0106] Preferred forms for administration include forms suitable
for parenteral administration, e.g. by injection or infusion, for
example by bolus injection or continuous infusion. Where the
product is for injection or infusion, it may take the form of a
suspension, solution or emulsion in an oily or aqueous vehicle and
it may contain formulatory agents, such as suspending,
preservative, stabilising and/or dispersing agents. Alternatively,
the antibody molecule may be in dry form, for reconstitution before
use with an appropriate sterile liquid.
[0107] Once formulated, the compositions of the invention can be
administered directly to the subject. The subjects to be treated
can be animals. However, it is preferred that the compositions are
adapted for administration to human subjects.
[0108] The pharmaceutical compositions of this invention may be
administered by any number of routes including, but not limited to,
oral, intravenous, intramuscular, intra-arterial, intramedullary,
intrathecal, intraventricular, transdermal, transcutaneous (for
example, see WO 98/20734), subcutaneous, intraperitoneal,
intranasal, enteral, topical, sublingual, intravaginal or rectal
routes. Hyposprays may also be used to administer the
pharmaceutical compositions of the invention. Typically, the
therapeutic compositions may be prepared as injectables, either as
liquid solutions or suspensions. Solid forms suitable for solution
in, or suspension in, liquid vehicles prior to injection may also
be prepared.
[0109] Direct delivery of the compositions will generally be
accomplished by injection, subcutaneously, intraperitoneally,
intravenously or intramuscularly, or delivered to the interstitial
space of a tissue. The compositions can also be administered into a
lesion. Dosage treatment may be a single dose schedule or a
multiple dose schedule.
[0110] It will be appreciated that the active ingredient in the
composition will be an antibody molecule. As such, it will be
susceptible to degradation in the gastrointestinal tract. Thus, if
the composition is to be administered by a route using the
gastrointestinal tract, the composition will need to contain agents
which protect the antibody from degradation but which release the
antibody once it has been absorbed from the gastrointestinal
tract.
[0111] A thorough discussion of pharmaceutically acceptable
carriers is available in Remington's Pharmaceutical Sciences (Mack
Publishing Company, N.J. 1991).
[0112] It is also envisaged that the antibody of the present
invention will be administered by use of gene therapy. In order to
achieve this, DNA sequences encoding the heavy and light chains of
the antibody molecule under the control of appropriate DNA
components are introduced into a patient such that the antibody
chains are expressed from the DNA sequences and assembled in
situ.
[0113] The present invention also provides an antibody molecule for
use in the control of inflammatory diseases. Preferably, the
antibody molecule can be used to reduce the inflammatory process or
to prevent the inflammatory process.
[0114] The present invention also provides the antibody molecule of
the present invention for use in the treatment or prophylaxis of a
pathological disorder that is mediated by IL-17 or associated with
an increased level of IL-17. Preferably, the pathological condition
is selected from the group consisting of infections (viral,
bacterial, fungal and parasitic), endotoxic shock associated with
infection, arthritis, rheumatoid arthritis, asthma, pelvic
inflammatory disease, Alzheimer's Disease, Crohn's disease,
Peyronie's Disease, coeliac disease, gallbladder disease, Pilonidal
disease, peritonitis, psoriasis, vasculitis, surgical adhesions,
stroke, Type I Diabetes, lyme arthritis, meningoencephalitis,
immune mediated inflammatory disorders of the central and
peripheral nervous system such as multiple sclerosis and
Guillain-Barr syndrome, other autoimmune disorders, pancreatitis,
trauma (surgery), graft-versus-host disease, transplant rejection,
cancer (both solid tumours such as melanomas, hepatoblastomas,
sarcomas, squamous cell carcinomas, transitional cell cancers,
ovarian cancers and hematologic malignancies and in particular
acute myelogenous leukaemia, chronic myelogenous leukemia, gastric
cancer and colon cancer), heart disease including ischaemic
diseases such as myocardial infarction as well as atherosclerosis,
intravascular coagulation, bone resorption, osteoporosis,
periodontitis and hypochlorhydia.
[0115] The present invention also provides an antibody molecule
according to the present invention for use in the treatment or
prophylaxis of pain.
[0116] The present invention further provides the use of an
antibody molecule according to the present invention in the
manufacture of a medicament for the treatment or prophylaxis of a
pathological disorder that is mediated by IL-17 or associated with
an increased level of IL-17. Preferably the pathological disorder
is rheumatoid arthritis or multiple sclerosis.
[0117] The present invention further provides the use of an
antibody molecule according to the present invention in the
manufacture of a medicament for the treatment or prophylaxis of
pain.
[0118] An antibody molecule of the present invention may be
utilised in any therapy where it is desired to reduce the effects
of IL-17 in the human or animal body. IL-17 may be circulating in
the body or may be present in an undesirably high level localised
at a particular site in the body, for example a site of
inflammation.
[0119] The antibody molecule of the present invention is preferably
used for the control of inflammatory disease.
[0120] The present invention also provides a method of treating
human or animal subjects suffering from or at risk of a disorder
mediated by IL-17, the method comprising administering to the
subject an effective amount of the antibody molecule of the present
invention.
[0121] The antibody molecule of the present invention may also be
used in diagnosis, for example in the in vivo diagnosis and imaging
of disease states involving IL-17.
[0122] The present invention is further described by way of
illustration only in the following examples, which refer to the
accompanying Figures, in which:
[0123] FIG. 1a) shows the nucleotide and amino acid sequence (SEQ
ID NOS:1 and 2, respectively) of the variable domains of the heavy
chain, and FIG. 1b) shows the nucleotide and amino acid sequence
(SEQ ID NOS:3 and 4, respectively) of the variable domains of the
light chain of murine monoclonal antibody IL-17F4.100. In both
figures positions 1-57 (nucleotide sequence numbering) are the
natural mouse leader sequences associated with these variable
regions.
[0124] FIG. 2 shows the graft design for the IL-17F4.100 heavy
(FIG. 2a; SEQ ID NO:11) and light chain (FIG. 2b; SEQ ID NO:13)
sequences. The symbol (|) highlights differences between
donor:acceptor:grafted framework sequences. CDR's are single
underlined. These are as defined by Kabat, except for CDR-H1 which
encompasses both Kabat and Chothia definitions. Double-underlined
sequences are donor residues retained in the grafts. Starred (*)
residues are common in human sub-group VH3 germline sequences, but
not present in this particular germline.
[0125] FIG. 3 shows the nucleotide and amino acid sequences of the
designed genes gH11 (FIG. 3a) and gL3 (FIG. 3b). In both chains the
E. coli OmpA leader sequence is shown (bases 1-63 of the nucleotide
sequence).
[0126] FIG. 4. Shows the amino acid sequence of the antibody Fab
fragment of CDP435 (a) light chain and (b) heavy chain.
[0127] FIG. 5. Shows the amino acid and nucleotide sequence of the
antibody Fab fragment of CDP435. Bases 1-63 and 722-784 represent
the E. coli OmpA, leader sequence.
[0128] FIG. 6. Plasmid map of pTTOD (CDP435)
[0129] FIG. 7. A comparison of the effect of CDP435 and IL17F4.100
on human IL-17 induced IL-6 production from Hela cells.
[0130] FIG. 8. The effect of CDP435 on human IL-17 induced IL-6
production from Hela cells.
[0131] FIG. 9. The effect of CDP435 on monkey IL-17 induced IL-6
production from Hela cells.
[0132] FIG. 10. The effect of CDP435 on human IL-17F induced IL-6
production from Hela cells.
[0133] FIG. 11. The effect of CDP435 on mouse IL-17 induced IL-6
production from 3T3-NIH cells.
[0134] FIG. 12. Pharmacokinetics of .sup.125I labelled CDP435
administered subcutaneously in rats
[0135] FIG. 13. In vivo neutralisation of hIL-17 induced neutrophil
accumulation in mice by local administration of CDP435.
[0136] FIG. 14. In vivo neutralisation of hIL-17 induced neutrophil
accumulation in mice by subcutaneous administration of CDP435.
[0137] FIG. 15. A diagrammatic representation of the structure of
CDP435. n is between 400 and 520.
DNA MANIPULATIONS AND GENERAL METHODS
[0138] E. coli strain INV.alpha.F' (Invitrogen) was used for
transformation and routine culture growth. DNA restriction and
modification enzymes were obtained from Roche Diagnostics Ltd. and
New England Biolabs. Plasmid preparations were performed using Maxi
Plasmid purification kits (QIAGEN, catalogue No. 12165). DNA
sequencing reactions were performed using the ABI Prism Big Dye
terminator sequencing kit (catalogue No. 4304149) and run on an ABI
3100 automated sequencer (Applied Biosystems). Data was analysed
using the program AutoAssembler (Applied Biosystems).
Oligonucleotides were obtained from OSWEL. The concentration of Fab
was determined using Fab assembly ELISA.
In Vitro Neutralisation Assay: Primary Fibroblasts
[0139] Human dermal fibroblasts were grown to 80% confluence in 96
well plates. Antibodies were titrated in half log dilutions from 1
.mu.g/ml and human IL-17 was added to give 25 .mu.g/ml final
concentration. The plates containing antibody and human IL-17 were
incubated at room temperature for 30 min. Culture medium was
removed from fibroblast cultures and 100 .mu.l antibody/IL-17 mix
added to the appropriate wells and cultured overnight at 37.degree.
C. The amount of IL-8 produced in response to IL-17 was then
estimated using the R&D Systems Human IL-8 Duoset Kit
DY208.
EXAMPLE 1
Isolation of IL-17F4.100
[0140] Antibody IL-17F4.100 was obtained using conventional
hybridoma techniques. Female BALB/C mice were immunised with
recombinant human IL-17 (purchased from R & D systems). Mice
received three intra peritoneal immunisations at two weekly
intervals of 10 .mu.g IL-17 in 100 .mu.l Freund's adjuvant. Three
days prior to performing the fusion the mouse was boosted with 1
.mu.g human IL-17 in 100 .mu.l PBS intravenously. The fusion was
performed using the method of Galfre et al., 1977, Nature, 266,
550-552 with the mouse myeloma cell line SP2/0 used as the fusion
partner. The fusion was screened for antibodies that bound to human
IL-17 by ELISA and a number of antibody producing hybridomas were
selected from this primary screen one of which was named
IL-17F4.100. The hybridoma cells producing IL-17F4.100, were cloned
by limiting dilution. The antibody was isotyped and found to be an
IgG.gamma.2b with a kappa light chain.
EXAMPLE 2
Gene Cloning and Expression of the Variable Regions from Murine
Monoclonal Antibody IL-17F4.100
PCR Cloning of VH and VL Regions
[0141] Genes for the heavy chain variable domain (VH) and light
chain variable domain (VL) of IL-17F4.100 were isolated and
sequenced following cloning via reverse transcription PCR.
[0142] The V-region sequences are shown in FIG. 1 (starting at base
58) and in SEQ ID NOS:1 to 4.
[0143] The murine V-region genes were sub-cloned into expression
vectors containing the human antibody constant region genes (human
kappa light chain and gamma-4 heavy chain) and a mouse/human
chimeric expressed transiently in CHO cells. Transfections of CHO
cells were performed using the lipofectamine procedure according to
manufacturer's instructions (InVitrogen, catalogue No. 18324).
EXAMPLE 3
CDR-Grafting of IL-17F4.100
[0144] A series of humanised VL and VH regions were designed in
which the CDR hypervariable regions plus a varying number of
framework residues from IL-17F4.100 were grafted onto human
V-region acceptor frameworks.
[0145] Three grafted VL regions (gL1-3) were designed and genes
were built by oligonucleotide assembly and PCR mutagenesis. A total
of 16 grafted VH regions were also constructed (gH1-16). These
humanised sequences were sub-cloned into vectors containing human
antibody constant region genes, were expressed transiently in CHO
cells and their activity in IL-17 binding and neutralisation assays
was compared to the chimeric antibody comprising the IL-17F4.100
variable regions and human constant regions.
[0146] The graft most potent at neutralising IL17 was gH11 gL3
which contains 1 mouse framework residue in the L chain (Val-2) and
2 mouse framework residues in the H chain (Val-24, Val-78).
[0147] FIG. 2 shows an alignment between the donor mouse sequence
and the acceptor human frameworks. The heavy chain acceptor
framework is the human germline sequence VH3 1-3 3.33, with
framework 4 coming from this portion of the human JH-region
germline JH4. The light chain acceptor framework is the human
germline sequence VK1 2-1-(1) O12, with framework 4 coming from
this portion of the human 3K-region germline JK1. The graft
sequences for gH11 and gL3 are given in FIGS. 3a (bases 64-420) and
3b (bases 64-399) respectively (SEQ ID NOS:11-14).
EXAMPLE 4
Production and Characterisation of CDP435
[0148] CDP435 is a PEGylated antibody fragment according to the
present invention in which the antibody component is an antibody
Fab fragment contructed from the grafts produced in Example 3. The
antibody Fab fragment component of CDP435 was constructed using the
genes encoding the selected humanised variable domain graft
(gH11gL3) which were sub-cloned into Celltech's E. coli expression
vector pTTOD, which contains DNA encoding the human C.gamma.1 heavy
chain CH1 domain and the human C kappa light chain domain (as
previously described in WO03/048208). In contrast to WO03/048208
the human heavy chain was truncated in the constant region such
that the interchain disulphide cysteine (cys-233 by Kabat numbering
system, cys-222 by sequential numbering) is the C-terminal residue.
The protein sequence of this CDR-grafted Fab is shown in FIGS. 4a
and 4b (Seq ID NOS: 15-18). A map of the pTTOD(CDP435) dicistronic
expression vector is shown in FIG. 6 which comprises the construct
provided in FIG. 5 and SEQ ID NO:19. The construct contains an
intergenic sequence, IGS-2, between the light and heavy chain genes
(See WO03/048208) and the OmpA leader sequence at the start of both
the light and heavy chain genes.
[0149] The pTTOD(CDP435) vector was transformed into the host
strain E. coli K12 W3110 and the antibody Fab fragment component of
CDP435 produced in E. coli by high cell density cultivation using
standard methods. Antibodies were purified using cation exchange
followed by anion exchange chromatography using standard methods
(Humphreys et al., 2002, Protein Expression and Purification, 26,
309-320).
Production of CDP435
[0150] Two 20 kDa PEG molecules were attached to the purified
antibody Fab fragment component of CDP435 using the following
method (See also the method provided in International patent
application WO2005/003169). The purified antibody Fab fragment
produced as described above was reduced to produce 2 thiols per Fab
(both interchain cysteines) with 10 mM
tris-(2-carboxyethyl)-phosphine (TCEP) for 1 hour at ambient
temperature. The reductant was removed by diafiltration into 0.1M
phosphate+2 mM EDTA, pH 6.0. The reduced antibody fragment of
CDP435 was DiPEGylated on the interchain cysteines with a 3-fold
molar excess of 20 kDa PEG-maleimide over Fab, overnight at ambient
temperature in order to attach a total of 40 kDa PEG (i.e.
2.times.20 kDa PEG) to produce CDP435. A diagrammatic
representation of CDP435 is shown in FIG. 15.
[0151] After PEGylation the reaction was conditioned for
purification of CDP435 by reducing the pH to 4.5 (addition of
acetic acid) and reducing the conductivity to 3 mS/cm (addition of
water). CDP435 was purified by SP Sepharose HP chromatography in 50
mM acetate pH 4.5. Purified material was concentrated and
diafiltered into 50 mM acetate, 125 mM NaCl, pH 5.5, and 0.22 .mu.m
sterile filtered.
BIAcore Assay
[0152] The assay format used CDP435 captured by anti-human IgG
F(ab).sub.2 with a titration of recombinant human IL-17 in the
solution phase. BIA (Biamolecular Interaction Analysis) was
performed using a BIAcore 3000 (BIAcore AB). Affinipure
F(ab').sub.2 Fragment goat anti-human IgG, F(ab).sub.2 fragment
specific (Jackson ImmunoResearch) was immobilised on a CM5 Sensor
Chip via amine coupling chemistry to a capture level of
.apprxeq.9000 response units (RUs). HBS-EP buffer (10 mM HEPES pH
7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, BIAcore AB) was
used as the running buffer with a flow rate of 10 .mu.l/min. An
injection of CDP435 was made at 10 .mu.l/min in order to obtain
around 200Ru of Fab captured by the immobilised anti-human
IgG-F(ab).sub.2 to the surface. Human IL-17 was titrated over the
captured antibody Fab fragment at various concentrations at a flow
rate of 30 .mu.l/min. The surface was regenerated by a 2.times.10
.mu.l injection of 40 mM HCl, followed by a 5 .mu.l injection of 5
mM NaOH at a flow rate of 10 .mu.l/min.
[0153] Background subtraction binding curves were analysed using
the BIAevaluation software (version 3.2) following standard
procedures. Kinetic parameters were determined from the fitting
algorithm. The affinity was measured at human IL-17 concentrations
at or below 12.5 nM. The affinity value determined for CDP435 was
in the range 133-365 pM with a mean.+-.SD of 223.8.+-.94.5 pM
(Table 1).
TABLE-US-00001 TABLE 1 Affinity by BIAcore Replicate k.sub.a
(M.sup.-1s.sup.-1) k.sub.d (s.sup.-1) K.sub.d (M) K.sub.d pM 1
1.71E+06 3.23E-04 1.891E-10 189 2 1.35E+06 1.79E-04 1.33E-10 133 3
1.83E+06 4.99E-04 2.72E-10 272 4 2.57E+06 4.11E-04 1.60E-10 160 5
1.62E+06 5.92E-04 3.65E-10 365
FIG. 7 demonstrates that the neutralisation activity of the
antibody Fab fragment of CDP435 is equivalent to that of the murine
parental antibody IL-17F4.100 in the Hela cell human IL-17
neutralisation assay (methods as described in Example 5).
EXAMPLE 5
In Vitro Neutralisation Assays Using CDP435
Hela Cells
[0154] The potency of CDP435 against human recombinant IL-17,
monkey recombinant IL-17 and human recombinant IL-17F in Hela cells
was tested. Hela cells were obtained from the cell bank at ATCC
(ATCC CCL-2). Cells were grown in Dulbecco's modified Eagle's
medium (DMEM) supplemented with 10% foetal calf serum, penicillin,
gentamycin and glutamine. 1.times.10.sup.4 cells were plated out
into 96 well flat bottomed tissue culture plates. Cells were
incubated overnight and washed once in assay buffer. Either human
IL-17 (25 ng ml.sup.-1), monkey IL-17 (25 ng ml.sup.-1) or human
IL-17F (100 ng ml.sup.-1) was incubated in the presence of a fixed
concentration of human TNF-.alpha. this mixture was preincubated
with CDP435. Cytokine plus antibody was then added to the Hela
cells which were incubated overnight. The production of IL-6 in the
cell culture supernatant was proportionate to the amount of
IL-17/IL-17F added to the cells. Human IL-6 levels were measured by
ELISA and quantified by comparison with known standard
concentrations of human IL-6.
[0155] The data (FIGS. 8, 9 and 10) indicates that CDP435 potently
neutralised both human recombinant M-17 and monkey recombinant
IL-17 but did not inhibit the activity of human recombinant IL-17F.
The data from these experiments indicated that CDP435 gave an
IC.sub.50 of 158 ng ml.sup.-1.+-.48 against human recombinant IL-17
(25 ng ml.sup.-1) and 147 ng ml.sup.-1.+-.45 against monkey
recombinant IL-17 (25 ng ml.sup.-1).
Mouse IL-17 Neutralisation Assay (3T3-NIH Cells)
[0156] The neutralisation potency of CDP435 against mouse
recombinant IL-17 was determined. 3T3-NIH cells were obtained from
the cell bank at ATCC (ATCC CRL-1658). Cells were grown in DMEM
supplemented with 10% calf serum, penicillin, gentamycin and
glutamine. The assay buffer used was identical to this buffer with
foetal calf serum replacing calf serum. 1.times.10.sup.4 cells were
plated out into 96 well flat bottomed tissue culture plates. Cells
were incubated overnight and washed once in assay buffer. Murine
IL-17 in the presence of a fixed concentration of human TNF-.alpha.
was preincubated with CDP435. Cytokine plus CDP435 was then added
to the 3T3-NIH cells which were incubated overnight. The production
of IL-6 in the cell culture supernatant was proportionate to the
amount of mouse IL-17 added to the cells. Mouse IL-6 levels were
measured by ELISA and quantified by comparison with known standard
concentrations of murine IL-6.
[0157] The data indicates that CDP435 did not inhibit the activity
of mouse recombinant IL-17 (FIG. 11).
EXAMPLE 6
Rat Pharmacokinetic Study with CDP435
[0158] Rats were injected s.c. with .sup.125I labelled CDP435. At
various times the animals were bled and the blood counted for
radioactivity. The pharmacokinetic trace is shown in FIG. 12.
AUC.sub.0-.infin.=2651% dose*h, t1/2.beta.=52 h, C.sub.max=22.7%
dose. The results showed that CDP435 had good pharmacokinetics with
a half life of 52 hours.
[0159] CDP435 was labelled with .sup.125I at a specific activity of
0.07 .mu.Ci/.mu.g and 77.6 .mu.s antibody administered s.c. in a
volume of 100 .mu.l.
In Vivo Neutralisation Assay
[0160] To determine the neutralisation efficacy of CDP435 in vivo,
CDP485 was tested in two in vivo models of inflammation.
Intraperitoneal CDP435/Intraperitoneal hIL-17 in Mice
[0161] Male Balb/c mice (18-25 g) were injected intraperitoneally
(i.p.) with CDP435 or control Fab' A33-PEG and then injected i.p. 5
minutes later with hIL-17. After 180 minutes, mice were killed by
cervical dislocation and peritoneal lavage performed (3 ml HBSS
(Hanks' Balanced Salts)+0.25% BSA, 12 mM HEPES) and neutrophil
accumulation quantified by FACS (Neutrophils were identified as
those cells expressing CD45 and high levels of GR1 by staining with
anti-CD45 CyChrome and anti-GR1 Phycoerythrin antibodies).
Neutrophil accumulation in response to 300 ng hIL-17 was
significantly reduced with CDP435 at doses of 0.01 and 0.1 mg/kg
(FIG. 13).
[0162] In a separate experiment animals were dosed s.c. with 20
mg/kg CDP435 and challenged i.p. with 300 ng hIL-17 24 hours later.
After a further 3 hours, peritoneal lavage showed that the CDP435
treatment had blocked neutrophil accumulation (FIG. 14). Thus
CDP435 is effective against hIL-17 when given locally with the
antigen or administered s.c. at a distant site.
[0163] It will of course be understood that the present invention
has been described by way of example only, is in no way meant to be
limiting, and that modifications of detail can be made within the
scope of the claims hereinafter. Preferred features of each
embodiment of the invention are as for each of the other
embodiments mutatis mutandis. All publications, including but not
limited to patents and patent applications, cited in this
specification are herein incorporated by reference as if each
individual publication were specifically and individually indicated
to be incorporated by reference herein as though fully set forth.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 23 <210> SEQ ID NO 1 <211> LENGTH: 357 <212>
TYPE: DNA <213> ORGANISM: Mus musculus <400> SEQUENCE:
1 caggtgcagc tgaaggagtc aggacctggc ctggtggcgc cctcacagag cctgtccatc
60 acatgcaccg tctcagggtt ctcattaact acctatggtg tacactggat
tcgccagcct 120 ccaggaaagg gtctggagtg gctggtagtg atttggagtg
atggatacac aacctataat 180 tcagctctca aatccagact gagcatcacc
aaggacaact ccaagagcca agttttctta 240 aaaatgaaca gtctccaaac
tgatgacaca gccatgtact actgtgccag aaatgatggt 300 gactacttct
attctatgga ctactggggt caaggaacct cagtcaccgt ctcctca 357 <210>
SEQ ID NO 2 <211> LENGTH: 119 <212> TYPE: PRT
<213> ORGANISM: Mus musculus <400> SEQUENCE: 2 Gln Val
Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Thr Tyr 20
25 30 Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp
Leu 35 40 45 Val Val Ile Trp Ser Asp Gly Tyr Thr Thr Tyr Asn Ser
Ala Leu Lys 50 55 60 Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys
Ser Gln Val Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Gln Thr Asp Asp
Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Arg Asn Asp Gly Asp Tyr Phe
Tyr Ser Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val
Ser Ser 115 <210> SEQ ID NO 3 <211> LENGTH: 339
<212> TYPE: DNA <213> ORGANISM: Mus musculus
<400> SEQUENCE: 3 gatgttgtga tgacccaaac tccactctcc ctgcctgtca
gtcttggaga tcaagcctcc 60 ttctcttgca gatctagtca gagccttgta
cacagtaatg gaaacaccta tttacattgg 120 tacctgcaga agccaggcca
gtctccaaag ctcctgatct acaaagtttc caaccgattt 180 tctggggtcc
cagacaggtt cagtggcagt gggtcaggga cagatttcac actcaagatc 240
agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac acatgttccg
300 acgttcggtg gaggcaccaa gctggaaatc aaacgtacg 339 <210> SEQ
ID NO 4 <211> LENGTH: 112 <212> TYPE: PRT <213>
ORGANISM: Mus musculus <400> SEQUENCE: 4 Asp Val Val Met Thr
Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala
Ser Phe Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn
Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40
45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe
Cys Ser Gln Ser 85 90 95 Thr His Val Pro Thr Phe Gly Gly Gly Thr
Lys Leu Glu Ile Lys Arg 100 105 110 <210> SEQ ID NO 5
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Mus musculus <400> SEQUENCE: 5 Gly Phe Ser Leu Thr Thr Tyr
Gly Val His 1 5 10 <210> SEQ ID NO 6 <211> LENGTH: 16
<212> TYPE: PRT <213> ORGANISM: Mus musculus
<400> SEQUENCE: 6 Val Ile Trp Ser Asp Gly Tyr Thr Thr Tyr Asn
Ser Ala Leu Lys Ser 1 5 10 15 <210> SEQ ID NO 7 <211>
LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Mus musculus
<400> SEQUENCE: 7 Asn Asp Gly Asp Tyr Phe Tyr Ser Met Asp Tyr
1 5 10 <210> SEQ ID NO 8 <211> LENGTH: 16 <212>
TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE:
8 Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His 1
5 10 15 <210> SEQ ID NO 9 <211> LENGTH: 7 <212>
TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE:
9 Lys Val Ser Asn Arg Phe Ser 1 5 <210> SEQ ID NO 10
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Mus musculus <400> SEQUENCE: 10 Ser Gln Ser Thr His Val Pro
Thr 1 5 <210> SEQ ID NO 11 <211> LENGTH: 119
<212> TYPE: PRT <213> ORGANISM: Artificial <220>
FEATURE: <223> OTHER INFORMATION: gH11 <400> SEQUENCE:
11 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Thr
Thr Tyr 20 25 30 Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Ser Asp Gly Tyr Thr Thr
Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asn Asp Gly
Asp Tyr Phe Tyr Ser Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu
Val Thr Val Ser Ser 115 <210> SEQ ID NO 12 <211>
LENGTH: 357 <212> TYPE: DNA <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: gH11
<400> SEQUENCE: 12 gaggttcagc tggtcgagtc tggaggcggg
gttgtccagc ctggagggag cctgcgtctc 60 tcttgtgcag ttagcggctt
ctcattaact acctatggtg tacactgggt gcggcaggca 120 cctgggaagg
gcctggagtg ggtggccgtg atttggagtg atggatacac aacctataat 180
tcagctctca aatcccgttt caccatttcc cgcgacaatt ctaagaacac cgtttacctc
240 cagatgaact ctctccgcgc agaggacaca gcagtctatt actgtgcacg
gaatgatggt 300 gactacttct attctatgga ctactgggga caggggaccc
ttgtgacagt ctcgagt 357 <210> SEQ ID NO 13 <211> LENGTH:
112 <212> TYPE: PRT <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: gL3 <400>
SEQUENCE: 13 Asp Val Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln
Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr
Gln Gln Lys Pro Gly Lys Ala 35 40 45 Pro Lys Leu Leu Ile Tyr Lys
Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Ser Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr
His Val Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
110 <210> SEQ ID NO 14 <211> LENGTH: 336 <212>
TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: gL3 <400> SEQUENCE: 14
gatgtgcaga tgacccagag tccaagcagt ctctccgcca gcgtaggcga tcgtgtgact
60 attacctgta gatctagtca gagccttgta cacagtaatg gaaacaccta
tttacattgg 120 tatcagcaaa aaccgggcaa agccccgaag ctgctcatct
ataaagtttc caaccgattt 180 tctggtgtgc catctcgttt cagtggcagt
ggcagcggta ccgactttac cctcacaatt 240 tcgtctctcc agccggaaga
tttcgccact tactattgtt ctcaaagtac acatgttccg 300 acgttcggtc
agggcactaa agtagaaatc aaacgt 336 <210> SEQ ID NO 15
<211> LENGTH: 672 <212> TYPE: DNA <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
CDP435 Heavy chain <400> SEQUENCE: 15 gaggttcagc tggtcgagtc
tggaggcggg gttgtccagc ctggagggag cctgcgtctc 60 tcttgtgcag
ttagcggctt ctcattaact acctatggtg tacactgggt gcggcaggca 120
cctgggaagg gcctggagtg ggtggccgtg atttggagtg atggatacac aacctataat
180 tcagctctca aatcccgttt caccatttcc cgcgacaatt ctaagaacac
cgtttacctc 240 cagatgaact ctctccgcgc agaggacaca gcagtctatt
actgtgcacg gaatgatggt 300 gactacttct attctatgga ctactgggga
caggggaccc ttgtgacagt ctcgagtgct 360 tctacaaagg gcccatcggt
cttccccctg gcaccctcct ccaagagcac ctctgggggc 420 acagcggccc
tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 480
aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga
540 ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac
ccagacctac 600 atctgcaacg tgaatcacaa gcccagcaac accaaggtcg
acaagaaagt tgagcccaaa 660 tcttgttaat ga 672 <210> SEQ ID NO
16 <211> LENGTH: 222 <212> TYPE: PRT <213>
ORGANISM: Artificial <220> FEATURE: <223> OTHER
INFORMATION: CDP435 Heavy chain <400> SEQUENCE: 16 Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Thr Thr Tyr 20
25 30 Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ala Val Ile Trp Ser Asp Gly Tyr Thr Thr Tyr Asn Ser
Ala Leu Lys 50 55 60 Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asn Asp Gly Asp Tyr Phe
Tyr Ser Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150
155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys 210 215 220 <210> SEQ ID NO 17
<211> LENGTH: 657 <212> TYPE: DNA <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
CDP435 Light chain <400> SEQUENCE: 17 gatgtgcaga tgacccagag
tccaagcagt ctctccgcca gcgtaggcga tcgtgtgact 60 attacctgta
gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg 120
tatcagcaaa aaccgggcaa agccccgaag ctgctcatct ataaagtttc caaccgattt
180 tctggtgtgc catctcgttt cagtggcagt ggcagcggta ccgactttac
cctcacaatt 240 tcgtctctcc agccggaaga tttcgccact tactattgtt
ctcaaagtac acatgttccg 300 acgttcggtc agggcactaa agtagaaatc
aaacgtacgg tagcggcccc atctgtcttc 360 atcttcccgc catctgatga
gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 420 aataacttct
atcccagaga ggccaaagta cagtggaagg tggataacgc cctccaatcg 480
ggtaactccc aggagagtgt cacagagcag gacagcaagg acagcaccta cagcctcagc
540 agcaccctga cgctgagcaa agcagactac gagaaacaca aagtctacgc
ctgcgaagtc 600 acccatcagg gcctgagctc accagtaaca aaaagtttta
atagagggga gtgttaa 657 <210> SEQ ID NO 18 <211> LENGTH:
218 <212> TYPE: PRT <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: CDP435 light
chain <400> SEQUENCE: 18 Asp Val Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr
Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala 35 40 45 Pro Lys Leu
Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Ser
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70
75 80 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ser Gln
Ser 85 90 95 Thr His Val Pro Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195
200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210>
SEQ ID NO 19 <211> LENGTH: 1516 <212> TYPE: DNA
<213> ORGANISM: Artificial <220> FEATURE: <223>
OTHER INFORMATION: CDP435 Heavy and Light chain in pTTOD
<400> SEQUENCE: 19 atgaaaaaga cagctatcgc aattgcagtg
gccttggctg gtttcgctac cgtagcgcaa 60 gctgatgtgc agatgaccca
gagtccaagc agtctctccg ccagcgtagg cgatcgtgtg 120 actattacct
gtagatctag tcagagcctt gtacacagta atggaaacac ctatttacat 180
tggtatcagc aaaaaccggg caaagccccg aagctgctca tctataaagt ttccaaccga
240 ttttctggtg tgccatctcg tttcagtggc agtggcagcg gtaccgactt
taccctcaca 300 atttcgtctc tccagccgga agatttcgcc acttactatt
gttctcaaag tacacatgtt 360 ccgacgttcg gtcagggcac taaagtagaa
atcaaacgta cggtagcggc cccatctgtc 420 ttcatcttcc cgccatctga
tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg 480 ctgaataact
tctatcccag agaggccaaa gtacagtgga aggtggataa cgccctccaa 540
tcgggtaact cccaggagag tgtcacagag caggacagca aggacagcac ctacagcctc
600 agcagcaccc tgacgctgag caaagcagac tacgagaaac acaaagtcta
cgcctgcgaa 660 gtcacccatc agggcctgag ctcaccagta acaaaaagtt
ttaatagagg ggagtgttaa 720 aatgaagaag actgctatag caattgcagt
ggcgctagct ggtttcgcca ccgtggcgca 780 agctgaggtt cagctggtcg
agtctggagg cggggttgtc cagcctggag ggagcctgcg 840 tctctcttgt
gcagttagcg gcttctcatt aactacctat ggtgtacact gggtgcggca 900
ggcacctggg aagggcctgg agtgggtggc cgtgatttgg agtgatggat acacaaccta
960 taattcagct ctcaaatccc gtttcaccat ttcccgcgac aattctaaga
acaccgttta 1020 cctccagatg aactctctcc gcgcagagga cacagcagtc
tattactgtg cacggaatga 1080 tgaggtctac ttgagagagg cgcgtctcct
gtgtcgtcag ataatgacac gtgccttact 1140 aggtgactac ttctattcta
tggactactg gggacagggg acccttgtga cagtctcgag 1200 tgcttctaca
aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg 1260
gggcacagcg gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc
1320 gtggaactca ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc
tacagtcctc 1380 aggactctac tccctcagca gcgtggtgac cgtgccctcc
agcagcttgg gcacccagac 1440 ctacatctgc aacgtgaatc acaagcccag
caacaccaag gtcgacaaga aagttgagcc 1500 caaatcttgt taatga 1516
<210> SEQ ID NO 20 <211> LENGTH: 98 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 20 Gln
Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg <210> SEQ ID
NO 21 <211> LENGTH: 15 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 21 Tyr Phe Asp Tyr Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 15 <210> SEQ
ID NO 22 <211> LENGTH: 95 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 22 Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu
Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr
Ser Thr Pro 85 90 95 <210> SEQ ID NO 23 <211> LENGTH:
12 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 23 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 1 5 10
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 23 <210>
SEQ ID NO 1 <211> LENGTH: 357 <212> TYPE: DNA
<213> ORGANISM: Mus musculus <400> SEQUENCE: 1
caggtgcagc tgaaggagtc aggacctggc ctggtggcgc cctcacagag cctgtccatc
60 acatgcaccg tctcagggtt ctcattaact acctatggtg tacactggat
tcgccagcct 120 ccaggaaagg gtctggagtg gctggtagtg atttggagtg
atggatacac aacctataat 180 tcagctctca aatccagact gagcatcacc
aaggacaact ccaagagcca agttttctta 240 aaaatgaaca gtctccaaac
tgatgacaca gccatgtact actgtgccag aaatgatggt 300 gactacttct
attctatgga ctactggggt caaggaacct cagtcaccgt ctcctca 357 <210>
SEQ ID NO 2 <211> LENGTH: 119 <212> TYPE: PRT
<213> ORGANISM: Mus musculus <400> SEQUENCE: 2 Gln Val
Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Thr Tyr 20
25 30 Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp
Leu 35 40 45 Val Val Ile Trp Ser Asp Gly Tyr Thr Thr Tyr Asn Ser
Ala Leu Lys 50 55 60 Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys
Ser Gln Val Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Gln Thr Asp Asp
Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Arg Asn Asp Gly Asp Tyr Phe
Tyr Ser Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val
Ser Ser 115 <210> SEQ ID NO 3 <211> LENGTH: 339
<212> TYPE: DNA <213> ORGANISM: Mus musculus
<400> SEQUENCE: 3 gatgttgtga tgacccaaac tccactctcc ctgcctgtca
gtcttggaga tcaagcctcc 60 ttctcttgca gatctagtca gagccttgta
cacagtaatg gaaacaccta tttacattgg 120 tacctgcaga agccaggcca
gtctccaaag ctcctgatct acaaagtttc caaccgattt 180 tctggggtcc
cagacaggtt cagtggcagt gggtcaggga cagatttcac actcaagatc 240
agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac acatgttccg
300 acgttcggtg gaggcaccaa gctggaaatc aaacgtacg 339 <210> SEQ
ID NO 4 <211> LENGTH: 112 <212> TYPE: PRT <213>
ORGANISM: Mus musculus <400> SEQUENCE: 4 Asp Val Val Met Thr
Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala
Ser Phe Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn
Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40
45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe
Cys Ser Gln Ser 85 90 95 Thr His Val Pro Thr Phe Gly Gly Gly Thr
Lys Leu Glu Ile Lys Arg 100 105 110 <210> SEQ ID NO 5
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Mus musculus <400> SEQUENCE: 5 Gly Phe Ser Leu Thr Thr Tyr
Gly Val His 1 5 10 <210> SEQ ID NO 6 <211> LENGTH: 16
<212> TYPE: PRT <213> ORGANISM: Mus musculus
<400> SEQUENCE: 6 Val Ile Trp Ser Asp Gly Tyr Thr Thr Tyr Asn
Ser Ala Leu Lys Ser 1 5 10 15 <210> SEQ ID NO 7 <211>
LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Mus musculus
<400> SEQUENCE: 7 Asn Asp Gly Asp Tyr Phe Tyr Ser Met Asp Tyr
1 5 10 <210> SEQ ID NO 8 <211> LENGTH: 16 <212>
TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE:
8 Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His 1
5 10 15 <210> SEQ ID NO 9 <211> LENGTH: 7 <212>
TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE:
9 Lys Val Ser Asn Arg Phe Ser 1 5 <210> SEQ ID NO 10
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Mus musculus <400> SEQUENCE: 10 Ser Gln Ser Thr His Val Pro
Thr 1 5 <210> SEQ ID NO 11 <211> LENGTH: 119
<212> TYPE: PRT <213> ORGANISM: Artificial <220>
FEATURE: <223> OTHER INFORMATION: gH11 <400> SEQUENCE:
11 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Thr
Thr Tyr 20 25 30 Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Ser Asp Gly Tyr Thr Thr
Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asn Asp Gly
Asp Tyr Phe Tyr Ser Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu
Val Thr Val Ser Ser 115 <210> SEQ ID NO 12 <211>
LENGTH: 357 <212> TYPE: DNA <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: gH11
<400> SEQUENCE: 12 gaggttcagc tggtcgagtc tggaggcggg
gttgtccagc ctggagggag cctgcgtctc 60 tcttgtgcag ttagcggctt
ctcattaact acctatggtg tacactgggt gcggcaggca 120 cctgggaagg
gcctggagtg ggtggccgtg atttggagtg atggatacac aacctataat 180
tcagctctca aatcccgttt caccatttcc cgcgacaatt ctaagaacac cgtttacctc
240 cagatgaact ctctccgcgc agaggacaca gcagtctatt actgtgcacg
gaatgatggt 300 gactacttct attctatgga ctactgggga caggggaccc
ttgtgacagt ctcgagt 357 <210> SEQ ID NO 13 <211> LENGTH:
112 <212> TYPE: PRT <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: gL3 <400>
SEQUENCE: 13 Asp Val Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln
Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr
Gln Gln Lys Pro Gly Lys Ala 35 40 45 Pro Lys Leu Leu Ile Tyr Lys
Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65
70 75 80 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ser
Gln Ser 85 90 95 Thr His Val Pro Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg 100 105 110 <210> SEQ ID NO 14 <211>
LENGTH: 336 <212> TYPE: DNA <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: gL3 <400>
SEQUENCE: 14 gatgtgcaga tgacccagag tccaagcagt ctctccgcca gcgtaggcga
tcgtgtgact 60 attacctgta gatctagtca gagccttgta cacagtaatg
gaaacaccta tttacattgg 120 tatcagcaaa aaccgggcaa agccccgaag
ctgctcatct ataaagtttc caaccgattt 180 tctggtgtgc catctcgttt
cagtggcagt ggcagcggta ccgactttac cctcacaatt 240 tcgtctctcc
agccggaaga tttcgccact tactattgtt ctcaaagtac acatgttccg 300
acgttcggtc agggcactaa agtagaaatc aaacgt 336 <210> SEQ ID NO
15 <211> LENGTH: 672 <212> TYPE: DNA <213>
ORGANISM: Artificial <220> FEATURE: <223> OTHER
INFORMATION: CDP435 Heavy chain <400> SEQUENCE: 15 gaggttcagc
tggtcgagtc tggaggcggg gttgtccagc ctggagggag cctgcgtctc 60
tcttgtgcag ttagcggctt ctcattaact acctatggtg tacactgggt gcggcaggca
120 cctgggaagg gcctggagtg ggtggccgtg atttggagtg atggatacac
aacctataat 180 tcagctctca aatcccgttt caccatttcc cgcgacaatt
ctaagaacac cgtttacctc 240 cagatgaact ctctccgcgc agaggacaca
gcagtctatt actgtgcacg gaatgatggt 300 gactacttct attctatgga
ctactgggga caggggaccc ttgtgacagt ctcgagtgct 360 tctacaaagg
gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc 420
acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg
480 aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca
gtcctcagga 540 ctctactccc tcagcagcgt ggtgaccgtg ccctccagca
gcttgggcac ccagacctac 600 atctgcaacg tgaatcacaa gcccagcaac
accaaggtcg acaagaaagt tgagcccaaa 660 tcttgttaat ga 672 <210>
SEQ ID NO 16 <211> LENGTH: 222 <212> TYPE: PRT
<213> ORGANISM: Artificial <220> FEATURE: <223>
OTHER INFORMATION: CDP435 Heavy chain <400> SEQUENCE: 16 Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Thr Thr Tyr
20 25 30 Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ala Val Ile Trp Ser Asp Gly Tyr Thr Thr Tyr Asn
Ser Ala Leu Lys 50 55 60 Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser
Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asn Asp Gly Asp Tyr
Phe Tyr Ser Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145
150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys 210 215 220 <210> SEQ ID NO 17
<211> LENGTH: 657 <212> TYPE: DNA <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
CDP435 Light chain <400> SEQUENCE: 17 gatgtgcaga tgacccagag
tccaagcagt ctctccgcca gcgtaggcga tcgtgtgact 60 attacctgta
gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg 120
tatcagcaaa aaccgggcaa agccccgaag ctgctcatct ataaagtttc caaccgattt
180 tctggtgtgc catctcgttt cagtggcagt ggcagcggta ccgactttac
cctcacaatt 240 tcgtctctcc agccggaaga tttcgccact tactattgtt
ctcaaagtac acatgttccg 300 acgttcggtc agggcactaa agtagaaatc
aaacgtacgg tagcggcccc atctgtcttc 360 atcttcccgc catctgatga
gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 420 aataacttct
atcccagaga ggccaaagta cagtggaagg tggataacgc cctccaatcg 480
ggtaactccc aggagagtgt cacagagcag gacagcaagg acagcaccta cagcctcagc
540 agcaccctga cgctgagcaa agcagactac gagaaacaca aagtctacgc
ctgcgaagtc 600 acccatcagg gcctgagctc accagtaaca aaaagtttta
atagagggga gtgttaa 657 <210> SEQ ID NO 18 <211> LENGTH:
218 <212> TYPE: PRT <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: CDP435 light
chain <400> SEQUENCE: 18 Asp Val Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr
Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala 35 40 45 Pro Lys Leu
Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Ser
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70
75 80 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ser Gln
Ser 85 90 95 Thr His Val Pro Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195
200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210>
SEQ ID NO 19 <211> LENGTH: 1516 <212> TYPE: DNA
<213> ORGANISM: Artificial <220> FEATURE: <223>
OTHER INFORMATION: CDP435 Heavy and Light chain in pTTOD
<400> SEQUENCE: 19 atgaaaaaga cagctatcgc aattgcagtg
gccttggctg gtttcgctac cgtagcgcaa 60 gctgatgtgc agatgaccca
gagtccaagc agtctctccg ccagcgtagg cgatcgtgtg 120 actattacct
gtagatctag tcagagcctt gtacacagta atggaaacac ctatttacat 180
tggtatcagc aaaaaccggg caaagccccg aagctgctca tctataaagt ttccaaccga
240 ttttctggtg tgccatctcg tttcagtggc agtggcagcg gtaccgactt
taccctcaca 300 atttcgtctc tccagccgga agatttcgcc acttactatt
gttctcaaag tacacatgtt 360 ccgacgttcg gtcagggcac taaagtagaa
atcaaacgta cggtagcggc cccatctgtc 420 ttcatcttcc cgccatctga
tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg 480 ctgaataact
tctatcccag agaggccaaa gtacagtgga aggtggataa cgccctccaa 540
tcgggtaact cccaggagag tgtcacagag caggacagca aggacagcac ctacagcctc
600 agcagcaccc tgacgctgag caaagcagac tacgagaaac acaaagtcta
cgcctgcgaa 660 gtcacccatc agggcctgag ctcaccagta acaaaaagtt
ttaatagagg ggagtgttaa 720 aatgaagaag actgctatag caattgcagt
ggcgctagct ggtttcgcca ccgtggcgca 780 agctgaggtt cagctggtcg
agtctggagg cggggttgtc cagcctggag ggagcctgcg 840 tctctcttgt
gcagttagcg gcttctcatt aactacctat ggtgtacact gggtgcggca 900
ggcacctggg aagggcctgg agtgggtggc cgtgatttgg agtgatggat acacaaccta
960 taattcagct ctcaaatccc gtttcaccat ttcccgcgac aattctaaga
acaccgttta 1020 cctccagatg aactctctcc gcgcagagga cacagcagtc
tattactgtg cacggaatga 1080 tgaggtctac ttgagagagg cgcgtctcct
gtgtcgtcag ataatgacac gtgccttact 1140 aggtgactac ttctattcta
tggactactg gggacagggg acccttgtga cagtctcgag 1200 tgcttctaca
aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg 1260
gggcacagcg gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc
1320 gtggaactca ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc
tacagtcctc 1380 aggactctac tccctcagca gcgtggtgac cgtgccctcc
agcagcttgg gcacccagac 1440 ctacatctgc aacgtgaatc acaagcccag
caacaccaag gtcgacaaga aagttgagcc 1500 caaatcttgt taatga 1516
<210> SEQ ID NO 20 <211> LENGTH: 98 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 20 Gln
Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg <210> SEQ ID
NO 21 <211> LENGTH: 15 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 21 Tyr Phe Asp Tyr Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 15 <210> SEQ
ID NO 22 <211> LENGTH: 95 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 22 Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu
Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr
Ser Thr Pro 85 90 95 <210> SEQ ID NO 23 <211> LENGTH:
12 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 23 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 1 5 10
* * * * *
References