U.S. patent application number 14/107314 was filed with the patent office on 2014-07-03 for nucleic acids encoding antibody molecules which bind il-17a and il-17f.
This patent application is currently assigned to UCB Pharma S.A.. The applicant listed for this patent is UCB Pharma S.A.. Invention is credited to Ralph Adams, Andrew George Popplewell, Stephen Edward Rapecki.
Application Number | 20140186355 14/107314 |
Document ID | / |
Family ID | 37507977 |
Filed Date | 2014-07-03 |
United States Patent
Application |
20140186355 |
Kind Code |
A1 |
Adams; Ralph ; et
al. |
July 3, 2014 |
Nucleic Acids Encoding Antibody Molecules Which Bind IL-17A and
IL-17F
Abstract
The invention relates to antibody molecules having specificity
for antigenic determinants of both IL-17A and IL-17F, therapeutic
uses of the antibody molecules and methods for producing said
antibody molecules.
Inventors: |
Adams; Ralph; (Berkshire,
GB) ; Popplewell; Andrew George; (Berkshire, GB)
; Rapecki; Stephen Edward; (Berkshire, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UCB Pharma S.A. |
Brussels |
|
BE |
|
|
Assignee: |
UCB Pharma S.A.
Brussels
BE
|
Family ID: |
37507977 |
Appl. No.: |
14/107314 |
Filed: |
December 16, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13632702 |
Oct 1, 2012 |
8617847 |
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14107314 |
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12446143 |
Jun 11, 2010 |
8303953 |
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PCT/GB2007/003983 |
Oct 18, 2007 |
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13632702 |
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Current U.S.
Class: |
424/136.1 ;
435/252.33; 435/320.1; 435/69.6; 530/387.3; 536/23.53 |
Current CPC
Class: |
A61P 17/02 20180101;
A61P 33/00 20180101; A61P 1/16 20180101; A61P 31/00 20180101; A61P
19/10 20180101; A61P 25/28 20180101; A61P 31/04 20180101; C07K
2317/24 20130101; C07K 2317/31 20130101; A61P 1/04 20180101; A61P
7/02 20180101; A61P 43/00 20180101; A61P 3/10 20180101; A61P 31/12
20180101; A61P 1/02 20180101; A61P 25/00 20180101; C07K 16/244
20130101; A61P 19/08 20180101; A61P 35/02 20180101; A61P 37/06
20180101; A61K 39/3955 20130101; C07K 2317/33 20130101; A61P 9/00
20180101; A61P 11/06 20180101; A61P 37/00 20180101; C07K 2317/92
20130101; A61P 35/00 20180101; A61P 37/02 20180101; C07K 2317/90
20130101; A61P 11/04 20180101; A61P 17/06 20180101; A61P 1/18
20180101; C07K 2317/76 20130101; C07K 2317/565 20130101; A61P 19/02
20180101; A61P 31/10 20180101; C07K 16/468 20130101; A61P 25/02
20180101; A61P 29/00 20180101; A61P 9/10 20180101; A61P 1/14
20180101 |
Class at
Publication: |
424/136.1 ;
530/387.3; 536/23.53; 435/320.1; 435/252.33; 435/69.6 |
International
Class: |
C07K 16/24 20060101
C07K016/24 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 18, 2006 |
GB |
0620729.4 |
Claims
1. A neutralising antibody which binds human IL-17A and human
IL-17F, comprising a heavy chain and a light chain, wherein a
variable domain of the heavy chain comprises a sequence given in
SEQ ID NO:1 for CDR-H1, a sequence given in SEQ ID NO:2 for CDR-H2
and a sequence given in SEQ ID NO:3 for CDR-H3, wherein a variable
domain of the light chain comprises a sequence given in SEQ ID
NO:4, for CDR-L1, a sequence given in SEQ ID NO:5 for CDR-L2 and a
sequence given in SEQ ID NO:6 for CDR-L3 or an antigen binding
fragment thereof.
2. A neutralising antibody which binds human IL-17A and human
IL-17F, wherein a heavy chain comprises a sequence given in SEQ ID
NO:9.
3. The neutralising antibody according to claim 2, additionally
comprising a light chain, wherein the light chain comprises a
sequence given in SEQ ID NO:7.
4. The neutralising antibody according to claim 1, having the heavy
chain comprising the sequence given in SEQ ID NO:9 and the light
chain comprising the sequence given in SEQ ID NO:7.
5. A neutralising antibody which binds human IL-17A and human
IL-17F, having a heavy chain comprising a sequence given in SEQ ID
NO:15 and a light chain comprising a sequence given in SEQ ID
NO:11.
6. A neutralising antibody which binds human IL-17A and human
IL-17F, wherein the antibody binds to a same epitope on human
IL-17A and human IL-17F as a neutralizing antibody which binds
human IL-17A and human IL-17F, and has a heavy chain comprising the
sequence given in SEQ ID NO:9 and the light chain comprising the
sequence given in SEQ ID NO:7.
7. The neutralising antibody according to claim 1, wherein the
antibody fragment is a Fab, Fab', F(ab').sub.2, scFv or an
epitope-binding fragment thereof.
8. The neutralising antibody according to claim 7, wherein the
antibody or fragment thereof is a CDR-grafted antibody.
9. The antibody of claim 1, wherein the antibody or fragment
thereof is conjugated to one or more effector molecule(s).
10. An isolated DNA molecule encoding the heavy and/or light
chain(s) of an antibody of claim 1.
11. A cloning or expression vector comprising the DNA molecule
according to claim 13.
12. The vector according to claim 11, wherein the vector comprises
a sequences given in SEQ ID NO:14 and the sequence given in SEQ ID
NO:18.
13. An isolated host cell comprising one or more cloning or
expression vectors according to claim 11.
14. A process for the production of the antibody encoded by the DNA
molecule in the vector of the isolated host cell of claim 13,
comprising culturing the isolated host cell under conditions
suitable for expression of the antibody, and isolating the
antibody.
15. A pharmaceutical composition comprising an antibody of claim 1,
in combination with one or more of a pharmaceutically acceptable
excipient, diluent or carrier.
16. The pharmaceutical composition according to claim 15,
additionally comprising other active ingredients.
Description
[0001] The present invention relates to antibody molecules having
specificity for antigenic determinants of both IL-17A and IL-17F.
The present invention also relates to the therapeutic uses of the
antibody molecules and methods for producing them.
[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-17A 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-17A 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). It may also act through binding
to a complex of IL-17RA and IL-17RC (Toy et al., 2006, J. Immunol.
177(11); 36-39). IL-17 producing T cells called `TH17 cells` have
been implicated in the pathogenesis of certain cancers (Weaver et
al., 2006, Immunity, 24, 677-688; Langowski et al., 2006, 442,
461-465; Iwakura and Ishigame, 2006, J. Clin. Invest. 116, 5,
1218-1222).
[0003] 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. One such
homologue is IL-17F, also known as IL-24 and ML-1, which is around
55% identical to IL-17A and is thought to share the same receptors
as IL-17A (Kolls and Linden 2004, Immunity, 21, 467-476; Hymowitz,
at al., 2001, EMBO J. 20(19), 5332-5341; Kuestner et al., 2007,
Journal of Immunology, 179, 5462-5473).
[0004] Both IL-17A and IL-17F can form both homodimeric and
heterodimeric proteins, all of which are produced by activated
human CD4+ T cells (Wright at al., 2007, J Biol Chem. 282 (18),
13447-13455).
[0005] 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). Rat derived anti-human IL-17 antibodies were
described in WO04/106377. A humanised anti-IL-17 antibody with an
affinity of around 220 pM was described in WO2006/054059. A
monoclonal anti-IL-17 fully human antibody with an affinity of
around 188 pM was described in WO2006/013107. Antibodies which bind
IL-17F and IL-17A/IL-17F heterodimers were described in
WO2006/088833. Antibodies which specifically bind the IL-17A/IL-17F
heterodimer were described in WO2005/010044.
[0006] IL-17F antagonism has been associated with protection
against asthma (Kawaguchi et al., 2006, J. Allergy Clin. Immunol.
117(4); 795-801) and IL-17F is also thought to play a role in
arthritis pathology (Lubberts 2003, Current Opinion in
Investigational Drugs, 4 (5), 572-577).
[0007] Accordingly dual antagonists of IL-17A and IL-17F may be
more effective than a sole antagonist in treating IL-17 mediated
diseases. Antibodies which bind IL-17A and IL-17F were described in
WO2007/106769 published 20 Sep. 2007.
[0008] We have been able to demonstrate that it is possible to
isolate an antibody which is capable of binding to both IL-17A and
IL-17F and is capable of neutralising the activity of both isoforms
of IL-17. Hence the present invention provides an anti-IL-17
antibody which is capable of binding to both IL-17A and IL-17F. In
particular, the antibody of the present invention is capable of
specifically binding to both IL-17A and IL-17F i.e. the antibody
does not bind to other isoforms of IL-17. Preferably the antibody
of the present invention also binds the IL-17A/IL-17F heterodimer.
Preferably, the antibody of the present invention neutralises the
activity of both IL-17A and IL-17F. In one embodiment the antibody
of the present invention also neutralises the activity of the
IL-17A/IL-17F/heterodimer. The antibodies of the present invention
therefore have the advantageous property that they can inhibit the
biological activity of both IL-17A and IL-17F. Accordingly, the
present invention also provides the use of such antibodies in the
treatment of and/or prophylaxis of a disease mediated by either or
both of IL-17A or IL-17F such as autoimmune or inflammatory disease
or cancer.
[0009] As used herein, the term `neutralising antibody` describes
an antibody that is capable of neutralising the biological
signalling activity of both IL-17A and IL17F for example by
blocking binding of IL-17A and IL17F to one or more of their
receptors and by blocking binding of the IL-17A/IL-17F heterodimer
to one or more of its receptors. It will be appreciated that the
term `neutralising` as used herein refers to a reduction in
biological signalling activity which may be partial or complete.
Further, it will be appreciated that the extent of neutralisation
of IL-17A and IL-17F activity by the antibody may be the same or
different. In one embodiment the extent of neutralisation of the
activity of the IL-17A/IL-17F heterodimer may be the same or
different as the extent of neutralisation of IL-17A or IL-17F
activity.
[0010] In one embodiment the antibodies of the present invention
specifically bind to IL-17A and IL-17F. Specifically binding means
that the antibodies have a greater affinity for IL-17A and IL-17F
polypeptides (including the IL-17A/IL-17F heterodimer) than for
other polypeptides. Preferably the IL-17A and IL-17F polypeptides
are human. In one embodiment the antibody also binds cynomolgus
IL-17F.
[0011] IL-17A or IL-17F polypeptides or a mixture of the two or
cells expressing one or both of said polypeptides can be used to
produce antibodies which specifically recognise both polypeptides.
The IL-17 polypeptides (IL-17A and IL-17F) may be `mature`
polypeptides or biologically active fragments or derivatives
thereof which preferably include the receptor binding site.
Preferably the IL-17 polypeptides are the mature polypeptides.
IL-17 polypeptides may be prepared by processes well known in the
art from genetically engineered host cells comprising expression
systems or they may be recovered from natural biological sources.
In the present application, the term "polypeptides" includes
peptides, polypeptides and proteins. These are used interchangeably
unless otherwise specified. The IL-17 polypeptide may in some
instances be part of a larger protein such as a fusion protein for
example fused to an affinity tag. Antibodies generated against
these polypeptides may be obtained, where immunisation of an animal
is necessary, by administering the polypeptides to an animal,
preferably a non-human animal, using well-known and routine
protocols, see for example Handbook of Experimental Immunology, D.
M. Weir (ed.), Vol 4, Blackwell Scientific Publishers, Oxford,
England, 1986). Many warm-blooded animals, such as rabbits, mice,
rats, sheep, cows or pigs may be immunized. However, mice, rabbits,
pigs and rats are generally preferred.
[0012] Antibodies for use in the present invention include whole
antibodies and functionally active fragments or derivatives thereof
and may be, but are not limited to, monoclonal, multi-valent,
multi-specific, humanized or chimeric antibodies, domain antibodies
e.g. VH, VL, VHH, single chain antibodies, Fab fragments, Fab' and
F(ab).sub.2 fragments and epitope-binding fragments of any of the
above. Other antibody fragments include those described in
International patent applications WO2005003169, WO2005003170 and
WO2005003171. Antibody fragments and methods of producing them are
well known in the art, see for example Verma et al., 1998, Journal
of Immunological Methods, 216, 165-181; Adair and Lawson, 2005.
Therapeutic antibodies. Drug Design Reviews--Online
2(3):209-217.
[0013] Antibodies for use in the present invention include
immunoglobulin molecules and immunologically active portions of
immunoglobulin molecules, i.e. molecules that contain an antigen
binding site that specifically binds an antigen. The immunoglobulin
molecules of the invention can be of any class (e.g. IgG, IgE, IgM,
IgD and IgA) or subclass of immunoglobulin molecule.
[0014] Monoclonal antibodies may be prepared by any method known in
the art such as the hybridoma technique (Kohler & Milstein,
1975, Nature, 256:495-497), the trioma technique, the human B-cell
hybridoma technique (Kozbor et al., 1983, Immunology Today, 4:72)
and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies
and Cancer Therapy, pp 77-96, Alan R Liss, Inc., 1985).
[0015] Antibodies for use in the invention may also be generated
using single lymphocyte antibody methods by cloning and expressing
immunoglobulin variable region cDNAs generated from single
lymphocytes selected for the production of specific antibodies by
for example the methods described by Babcook, J. et al., 1996,
Proc. Natl. Acad. Sol. USA 93(15):7843-78481; WO92/02551;
WO2004/051268 and International Patent Application number
WO2004/106377.
[0016] Humanized antibodies are antibody molecules from non-human
species having one or more complementarity determining regions
(CDRs) from the non-human species and a framework region from a
human immunoglobulin molecule (see, e.g. U.S. Pat. No. 5,585,089;
WO91/09967).
[0017] Chimeric antibodies are those antibodies encoded by
immunoglobulin genes that have been genetically engineered so that
the light and heavy chain genes are composed of immunoglobulin gene
segments belonging to different species. These chimeric antibodies
are likely to be less antigenic. Bivalent antibodies may be made by
methods known in the art (Milstein et al., 1983, Nature
305:537-539; WO 93/08829, Traunecker et al., 1991, EMBO J.
10:3655-3659). Multi-valent antibodies may comprise multiple
specificities or may be monospecific (see for example WO 92/22853
and WO05/113605).
[0018] The antibodies for use in the present invention can also be
generated using various phage display methods known in the art and
include those disclosed by Brinkman et al. (in J. Immunol. Methods,
1995, 182: 41-50), Ames et al. (J. Immunol. Methods, 1995,
184:177-186), Kettleborough et al. (Eur. J. Immunol. 1994,
24:952-958), Persic et al. (Gene, 1997 187 9-18), Burton et al.
(Advances in Immunology, 1994, 57:191-280) and WO 90/02809; WO
91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO
95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484;
5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908;
5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.
Techniques for the production of single chain antibodies, such as
those described in U.S. Pat. No. 4,946,778 can also be adapted to
produce single chain antibodies which bind to IL-17A and IL-17F.
Also, transgenic mice, or other organisms, including other mammals,
may be used to express humanized antibodies.
[0019] 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.
[0020] 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.
[0021] 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.
[0022] 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.
[0023] In one embodiment the present invention provides a
neutralising antibody having specificity for human IL-17A and human
IL-17F, 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:1 for CDR-H1, a CDR having the sequence given in
SEQ ID NO:2 for CDR-H2 and a CDR having the sequence given in SEQ
ID NO:3 for CDR-H3.
[0024] In another embodiment the present invention provides a
neutralising antibody having specificity for human IL-17A and human
IL-17F, comprising 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:1 for
CDR-H1, the sequence given in SEQ ID NO:2 for CDR-H2 and the
sequence given in SEQ ID NO:3 for CDR-H3. For example, the antibody
may comprise a heavy chain wherein CDR-H1 has the sequence given in
SEQ ID NO:1 and CDR-H2 has the sequence given in SEQ ID NO:2.
Alternatively, the antibody may comprise a heavy chain wherein
CDR-H1 has the sequence given in SEQ ID NO:1 and CDR-H3 has the
sequence given in SEQ ID NO:3, or the antibody may comprise a heavy
chain wherein CDR-H2 has the sequence given in SEQ ID NO:2 and
CDR-H3 has the sequence given in SEQ ID NO:3. For the avoidance of
doubt, it is understood that all permutations are included.
[0025] In another embodiment the present invention provides a
neutralising antibody having specificity for human IL-17A and human
IL-17F, comprising a heavy chain, wherein the variable domain of
the heavy chain comprises the sequence given in SEQ ID NO:1 for
CDR-H1, the sequence given in SEQ ID NO:2 for CDR-H2 and the
sequence given in SEQ ID NO:3 for CDR-H3.
[0026] In one embodiment the present invention provides a
neutralising antibody having specificity for human IL-17A and human
IL-17F, 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:4 for CDR-L1, a CDR having the sequence given in
SEQ ID NO:5 for CDR-L2 and a CDR having the sequence given in SEQ
ID NO:6 for CDR-L3.
[0027] In another embodiment the present invention provides a
neutralising antibody having specificity for human IL-17A and human
IL-17F, comprising 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:4 for
CDR-L1, the sequence given in SEQ ID NO:5 for CDR-L2 and the
sequence given in SEQ ID NO:6 for CDR-L3. For example, the antibody
may comprise a light chain wherein CDR-L1 has the sequence given in
SEQ ID NO:4 and CDR-L2 has the sequence given in SEQ ID NO:5.
Alternatively, the antibody may comprise a light chain wherein
CDR-L1 has the sequence given in SEQ ID NO:4 and CDR-L3 has the
sequence given in SEQ ID NO:6, or the antibody may comprise a light
chain wherein CDR-L2 has the sequence given in SEQ ID NO:5 and
CDR.-L3 has the sequence given in SEQ ID NO:6. For the avoidance of
doubt, it is understood that all permutations are included.
[0028] In another embodiment the present invention provides a
neutralising antibody having specificity for human IL-17A and human
IL-17F, comprising a light chain, wherein the variable domain
comprises the sequence given in SEQ ID NO:4 for CDR-L1, the
sequence given in SEQ ID NO:5 for CDR-L2 and the sequence given in
SEQ ID NO:6 for CDR-L3.
[0029] The antibody molecules of the present invention preferably
comprise a complementary light chain or a complementary heavy
chain, respectively.
[0030] Hence in one embodiment, an antibody according to the
present invention comprises a heavy chain, wherein the variable
domain of the heavy chain comprises the sequence given in SEQ ID
NO:1 for CDR-H1, the sequence given in SEQ ID NO:2 for CDR-H2 and
the sequence given in SEQ ID NO:3 for CDR-H3 and a light chain
wherein the variable domain of the light chain comprises the
sequence given in SEQ ID NO:4 for CDR-L1, the sequence given in SEQ
ID NO:5 for CDR-L2 and the sequence given in SEQ ID NO:6 for
CDR-L3.
[0031] It will be appreciated that one or more amino acid
substitutions may be made to the CDRs provided by the present
invention without significantly altering the ability of the
antibody to bind to IL-17A and IL-17F and to neutralise IL-17A and
IL-17F activity. The effect of any amino acid substitutions on
binding and neutralisation can be readily tested by one skilled in
the art, for example by using the methods described herein.
Accordingly, the present invention provides an antibody comprising
one or more CDRs selected from CDRH-1 (SEQ ID NO:1), CDRH-2 (SEQ ID
NO:2), CDRH-3 (SEQ ID NO:3), CDRL-1 (SEQ ID NO:4), CDRL-2 (SEQ ID
NO:5) and CDRL-3 (SEQ ID NO:6) in which one or more amino acids in
one or more of the CDRs has been substituted with another amino
acid. It will also be appreciated that the length of one or more of
the CDRs may be altered without significantly altering the ability
of the antibody to bind to IL-17A and IL-17F and to neutralise
IL-17A and IL-17F activity.
[0032] In one embodiment, an antibody of the present invention
comprises a heavy chain, wherein the variable domain of the heavy
chain comprises three CDRs wherein the sequence of CDRH-1 has at
least 60% identity or similarity to the sequence given in SEQ ID
NO:1, CDRH-2 has at least 60% identity or similarity to the
sequence given in SEQ ID NO:2 and/or CDRH-3 has at least 60%
identity or similarity to the sequence given in SEQ ID NO:3. In
another embodiment, an antibody of the present invention comprises
a heavy chain, wherein the variable domain of the heavy chain
comprises three CDRs wherein the sequence of CDRH-1 has at least
70%, 80%, 90%, 95% or 98% identity or similarity to the sequence
given in SEQ ID NO:1, CDRH-2 has at least 70%, 80%, 90%, 95% or 98%
identity or similarity to the sequence given in SEQ ID NO:2 and/or
CDRH-3 has at least 70%, 80%, 90%, 95% or 98% identity or
similarity to the sequence given in SEQ ID NO:3.
[0033] "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: [0034] phenylalanine,
tyrosine and tryptophan (amino acids having aromatic side chains);
[0035] lysine, arginine and histidine (amino acids having basic
side chains); [0036] aspartate and glutamate (amino acids having
acidic side chains); [0037] asparagine and glutamine (amino acids
having amide side chains); and [0038] 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).
[0039] In another embodiment, an antibody of the present invention
comprises a light chain, wherein the variable domain of the light
chain comprises three CDRs wherein the sequence of CDRL-1' has at
least 60% identity or similarity to the sequence given in SEQ ID
NO:4, CDRL-2 has at least 60% identity or similarity to the
sequence given in SEQ ID NO:5 and/or CDRL-3 has at least 60%
identity or similarity to the sequence given in SEQ ID NO:6. In
another embodiment, an antibody of the present invention comprises
a light chain, wherein the variable domain of the heavy chain
comprises three CDRs wherein the sequence of CDRL-1 has at least
70%, 80%, 90%, 95% or 98% identity or similarity to the sequence
given in SEQ ID NO:4, CDRL-2 has at least 70%, 80%, 90%, 95% or 98%
identity or similarity to the sequence given in SEQ ID NO:5 and/or
CDRL-3 has at least 70%, 80%, 90%, 95% or 98% identity or
similarity to the sequence given in SEQ ID NO:6.
[0040] In one embodiment the antibody provided by the present
invention is a monoclonal antibody.
[0041] In one embodiment the antibody provided by the present
invention is a chimeric antibody.
[0042] In one embodiment the antibody provided by the present
invention is a CDR-grafted antibody molecule comprising one or more
of the CDRs provided in SEQ ID NOS:1 to 6. 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. In one embodiment rather
than the entire CDR being transferred, only one or more of the
specificity determining residues from any one of the CDRs described
herein above are transferred to the human antibody framework (see
for example, Kashmiri et al., 2005, Methods, 36, 25-34). In one
embodiment only the specificity determining residues from one or
more of the CDRs described herein above are transferred to the
human antibody framework. In another embodiment only the
specificity determining residues from each of the CDRs described
herein above are transferred to the human antibody framework.
[0043] When the CDRs or specificity determining residues are
grafted, any appropriate acceptor variable region framework
sequence may be used having regard to the class/type of the donor
antibody from which the CDRs are derived, including mouse, primate
and human framework regions. Preferably, the CDR-grafted antibody
according to the present invention has a variable domain comprising
human acceptor framework regions as well as one or more of the CDRs
or specificity determining residues described above. Thus, provided
in one embodiment is a neutralising CDR-grafted antibody wherein
the variable domain comprises human acceptor framework regions and
non-human donor CDRs.
[0044] Examples of human frameworks which can be used in the
present invention are KOL, NEWM, REI, EU, TUR, TEL 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/
[0045] 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.
[0046] 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 3-07 together with JH4.
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
3-07 together with JH4. The sequence of human JH4 is as follows:
(YFDY)WGQGTLVTVSS. The YFDY motif is part of CDR-H3 and is not part
of framework 4 (Ravetch, N. et al., 1981, Cell, 27, 583-591).
[0047] 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) L4 together with JK1.
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) L4 together with JK1. The JK1 sequence is as follows:
(WT)FGQGTKVEIK. The WT motif is part of CDR-L3 and is not part of
framework 4 (Hieter, P A., at al., 1982, J. Biol. Chem., 257,
1516-1522).
[0048] 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 at 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.
[0049] Preferably, in a CDR-grafted antibody molecule of the
present invention, if the acceptor heavy chain has the human VH3
sequence 1-3 3-07 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 position 94 (according to
Kabat et al., (supra)). Accordingly, provided is a CDR-grafted
antibody, wherein at least the residue at position 94 of the
variable domain of the heavy chain is a donor residue.
[0050] 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) L4 together with JK1, then no donor
residues are transferred i.e. only the CDRs are transferred.
Accordingly, provided is a CDR-grafted antibody wherein only the
CDRs are transferred to the donor framework.
[0051] Donor residues are residues from the donor antibody, i.e.
the antibody from which the CDRs were originally derived.
[0052] In one embodiment, an antibody of the present invention
comprises a heavy chain, wherein the variable domain of the heavy
chain comprises the sequence given in SEQ ID NO:9 (gH9).
[0053] In another embodiment, an antibody 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:9. In one embodiment,
an antibody of the present invention comprises a heavy chain,
wherein the variable domain of the heavy chain comprises a sequence
having at least 70%, 80%, 90%, 95% or 98% identity or similarity to
the sequence given in SEQ ID NO:9.
[0054] In one embodiment, an antibody of the present invention
comprises a light chain, wherein the variable domain of the light
chain comprises the sequence given in SEQ ID NO:7 (gL7).
[0055] In another embodiment, an antibody 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:7. In one embodiment
the antibody of the present invention comprises a light chain,
wherein the variable domain of the light chain comprises a sequence
having at least 70%, 80%, 90%, 95% or 98% identity or similarity to
the sequence given in SEQ ID NO:7.
[0056] In one embodiment an antibody of the present invention
comprises a heavy chain, wherein the variable domain of the heavy
chain comprises the sequence given in SEQ ID NO:9 and a light
chain, wherein the variable domain of the light chain comprises the
sequence given in SEQ ID NO:7.
[0057] In another embodiment 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:9 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:7. Preferably, the antibody comprises a heavy chain, wherein the
variable domain of the heavy chain comprises a sequence having at
least 70%, 80%, 90%, 95% or 98% identity or similarity to the
sequence given in SEQ ID NO:9 and a light chain, wherein the
variable domain of the light chain comprises a sequence having at
least 70%, 80%, 90%, 95% or 98% identity or similarity to the
sequence given in SEQ ID NO:7.
[0058] As described herein above, 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
domain antibody e.g. VH, VL, VHH, Fab, modified Fab, Fab',
F(ab').sub.2, Fv or scFv fragment.
[0059] 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. For example IgG4 molecules in which the
amine at position 241 has been changed to proline as described in
Angal et al., Molecular Immunology, 1993, 30 (1), 105-108 may be
used. Particularly preferred is the IgG4 constant domain comprising
this change.
[0060] In one embodiment the antibody heavy chain comprises a CH1
domain and the antibody light chain comprises a CL domain, either
kappa or lambda.
[0061] In a preferred embodiment the antibody provided by the
present invention is a neutralising antibody having specificity for
human IL-17A and human IL-17F in which the heavy chain constant
region comprises the human IgG4 constant region in which the serine
at position 241 has been substituted by proline as described in
Angal et al., supra. Accordingly, the present invention provides an
antibody in which the heavy chain comprises or consists of the
sequence given in SEQ ID NO:15.
[0062] In one embodiment of the invention, the antibody comprises a
heavy chain, wherein the heavy chain comprises a sequence having at
least 60% identity or similarity to the sequence given in SEQ ID
NO:15. Preferably, the antibody comprises a heavy chain, wherein
the heavy chain comprises a sequence having at least 70%, 80%, 90%,
95% or 98% identity or similarity to the sequence given in SEQ ID
NO:15.
[0063] In one embodiment an antibody molecule according to the
present invention comprises a light chain comprising the sequence
given in SEQ ID NO:11.
[0064] In one embodiment of the invention, the antibody comprises a
light chain, wherein the light chain comprises a sequence having at
least 60% identity or similarity to the sequence given in SEQ ID
NO:11. Preferably, the antibody comprises a light chain, wherein
the light chain comprises a sequence having at least 70%, 80%, 90%,
95% or 98% identity or similarity to the sequence given in SEQ ID
NO:11.
[0065] In one embodiment the present invention provides an antibody
in which the heavy chain comprises or consists of the sequence
given in SEQ ID NO:15 and the light chain comprises or consists of
the sequence given in SEQ ID NO:11.
[0066] In one embodiment 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:15 and the light chain comprises a sequence
having at least 60% identity or similarity to the sequence given in
SEQ ID NO:11. Preferably, the antibody comprises a heavy chain,
wherein the heavy chain comprises a sequence having at least 70%,
80%, 90%, 95% or 98% identity or similarity to the sequence given
in SEQ ID NO:15 and a light chain, wherein the light chain
comprises a sequence having at least 70%, 80%, 90%, 95% or 98%
identity or similarity to the sequence given in SEQ ID NO:11.
[0067] Also provided by the present invention is a specific region
or epitope of human IL-17A and/or a specific region or epitope of
human IL-17F and/or a specific region or epitope of human IL-17A/F
heterodimer which is bound by an antibody provided by the present
invention, in particular an antibody comprising the heavy chain
sequence gH9 (SEQ ID NO:9) and/or the light chain sequence gL7 (SEQ
ID NO:7).
[0068] The specific region or epitope of the human IL-17A
polypeptide and the specific region or epitope of the human IL-17F
polypeptide and the specific region or epitope of the human
IL-17A/F heterodimer 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-17A and IL-17F 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 appropriate 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 30 obtain additional neutralising
antibodies which bind the same epitope.
[0069] Antibodies which cross-block the binding of an antibody
according to the present invention, in particular, an antibody
comprising the heavy chain sequence gH9 (SEQ ID NO:9) and the light
chain sequence gL7 (SEQ ID NO:7), may be similarly useful in
neutralising IL-17A and IL-17F activity. Accordingly, the present
invention also provides a neutralising antibody which binds human
IL-17A and human IL-17F, which cross-blocks the binding of any one
of the antibodies described above to human IL-17A and/or human
IL-17F and/or human IL-17A/F heterodimer and/or is cross-blocked
from binding IL-17A and/or IL-17F and/or human IL-17A/F heterodimer
by any one of those antibodies. In one embodiment, such an antibody
binds to the same epitope as an antibody described herein above. In
another embodiment the cross-blocking neutralising antibody binds
to an epitope which borders and/or overlaps with the epitope bound
by an antibody described herein above. In another embodiment the
cross-blocking neutralising antibody of this aspect of the
invention does not bind to the same epitope as an antibody of the
present invention or an epitope that borders and/or overlaps with
said epitope.
[0070] Cross-blocking antibodies 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 to human
IL-17A and/or human IL-17F prevents the binding of an antibody of
the present invention or vice versa.
[0071] In one embodiment there is provided a neutralising antibody
which binds to human IL-17A and human IL-17F, which cross-blocks
the binding of an antibody whose heavy chain comprises the sequence
gH9 (SEQ ID NO:9) and whose light chain comprises the sequence gL7
(SEQ ID NO:7) to human IL-17A and to human IL-17F. In one
embodiment the cross-blocking antibodies provided by the present
invention inhibit the binding of an antibody comprising the heavy
chain sequence gH9 (SEQ ID NO:9) and the light chain sequence gL7
(SEQ ID NO:7) to IL-17A by greater than 80%, preferably by greater
than 85%, more preferably by greater than 90%, even more preferably
by greater than 95% and to IL-17F by greater than 80%, preferably
by greater than 85%, more preferably by greater than 90%, even more
preferably by greater than 95%.
[0072] In one embodiment there is provided a neutralising antibody
which binds to human IL-17A and human IL-17F, which cross-blocks
the binding of an antibody whose heavy chain comprises the sequence
gH9 (SEQ ID NO:9) and whose light chain comprises the sequence gL7
(SEQ ID NO:7) to human IL-17A and to human IL-17F and to human
IL-17A/F heterodimer. In one embodiment the cross-blocking
antibodies provided by the present invention inhibit the binding of
an antibody comprising the heavy chain sequence gH9 (SEQ ID NO:9)
and the light chain sequence gL7 (SEQ ID NO:7) to IL-17A by greater
than 80%, preferably by greater than 85%, more preferably by
greater than 90%, even more preferably by greater than 95% and to
IL-17F by greater than 80%, preferably by greater than 85%, more
preferably by greater than 90%, even more preferably by greater
than 95% and to IL-17A/F heterodimer to IL-17F by greater than 80%,
preferably by greater than 85%, more preferably by greater than
90%, even more preferably by greater than 95%.
[0073] In one embodiment there is provided a neutralising antibody
which binds to human IL-17A and human IL-17F, which cross-blocks
the binding of an antibody whose heavy chain comprises the sequence
gH9 (SEQ ID NO:9) and whose light chain comprises the sequence gL7
(SEQ ID NO:7) to human IL-17A or to human IL-17F or human IL-17A/F
heterodimer. In one embodiment the cross-blocking antibodies
provided by the present invention inhibit the binding of an
antibody comprising the heavy chain sequence gH9 (SEQ ID NO:9) and
the light chain sequence gL7 (SEQ ID NO:7) to IL-17A or IL-17F or
IL-17A/F by greater than 80%, preferably by greater than 85%, more
preferably by greater than 90%, even more preferably by greater
than 95%.
[0074] Alternatively or in addition, neutralising antibodies
according to this aspect of the invention may be cross-blocked from
binding to human IL-17A and human IL-17F by an antibody comprising
the heavy chain sequence gH9 (SEQ ID NO:9) and the light chain
sequence gL7 (SEQ ID NO:7). Also provided therefore is a
neutralising antibody molecule which binds to human IL-17A and to
human IL-17F which is cross-blocked from binding human IL-17A and
human IL-17F by an antibody comprising the heavy chain sequence gH9
(SEQ ID NO:9) and the light chain sequence gL7 (SEQ ID NO:7). In
one embodiment the neutralising antibodies provided by this aspect
of the invention are inhibited from binding to human IL-17A and
human IL-17F by an antibody comprising the heavy chain sequence gH9
(SEQ ID NO:9) and the light chain sequence gL7 (SEQ ID NO:7) by
greater than 80%, preferably by greater than 85%, more preferably
by greater than 90%, even more preferably by greater than 95%.
[0075] In another embodiment there is provided a neutralising
antibody molecule which binds to human IL-17A and to human IL-17F
which is cross-blocked from binding human IL-17A and human IL-17F
and IL-17A/F heterodimer by an antibody comprising the heavy chain
sequence gH9 (SEQ ID NO:9) and the light chain sequence gL7 (SEQ ID
NO:7). In one embodiment the neutralising antibodies provided by
this aspect of the invention are inhibited from binding to human
IL-17A and human IL-17F and human IL-17A/F heterodimer by an
antibody comprising the heavy chain sequence gH9 (SEQ ID NO:9) and
the light chain sequence gL7 (SEQ ID NO:7) by greater than 80%,
preferably by greater than 85%, more preferably by greater than
90%, even more preferably by greater than 95%.
[0076] Also provided therefore is a neutralising antibody molecule
which binds to human IL-17A and to human IL-17F which is
cross-blocked from binding human IL-17A or human IL-17F or human
IL-17A/F by an antibody comprising the heavy chain sequence gH9
(SEQ ID NO:9) and the light chain sequence gL7 (SEQ ID NO:7). In
one embodiment the neutralising antibodies provided by this aspect
of the invention are inhibited from binding to human IL-17A or
human IL-17F or human IL-17A/F by an antibody comprising the heavy
chain sequence gH9 (SEQ ID NO:9) and the light chain sequence gL7
(SEQ ID NO:7) by greater than 80%, preferably by greater than 85%,
more preferably by greater than 90%, even more preferably by
greater than 95%.
[0077] The antibody molecule of any aspect of the present invention
preferably has a high binding affinity, preferably nanomolar, even
more preferably picomolar. It will be appreciated that the binding
affinity of an antibody according to the present invention for
human IL-17A may be different from the binding affinity of the same
antibody for human IL-17F and/or the IL-17A/F heterodimer. In one
example the antibody molecule of the present invention has an
affinity for IL-17A that is greater than its affinity for IL-17F.
In one example the antibody molecule of the present invention has
an affinity for IL-17A which is at least 10 fold greater than its
binding affinity for IL-17F. In one example the antibody molecule
of the present invention has an affinity for IL-17A which is at
least 50 fold greater than its binding affinity for IL-17F. In one
example the antibody molecule of the present invention has an
affinity for IL-17A which is at least 100 fold greater than its
binding affinity for IL-17F. In one example the antibody molecule
of the present invention has an affinity for IL-17F that is greater
than its affinity for IL-17A. In one example the antibody molecule
of the present invention has an affinity for IL-17A that is the
same as its affinity for IL-17F. In one example the antibody
molecule of the present invention has a picomolar affinity for
IL-17A and a nanomolar affinity for IL-17F. In one example the
antibody molecule of the present invention has a nanomolar affinity
for IL-17F and a picomolar affinity for IL-17A. In one example the
antibody molecule of the present invention has a nanomolar affinity
for both IL-17A and IL-17F. In one example the antibody molecule of
the present invention has a picomolar affinity for both IL-17A and
IL-17F.
[0078] Preferably the antibody molecule of the present invention
has a binding affinity for IL-17A of better than 10 nM. In one
embodiment the antibody molecule of the present invention has a
binding affinity for IL-17A of better than 500 pM. In one
embodiment the antibody molecule of the present invention has a
binding affinity for IL-17A of better than 100 pM. In one
embodiment the antibody molecule of the present invention has a
binding affinity for IL-17A of better than 20 pM. In one embodiment
the antibody of the present invention has an affinity for IL-17A of
16 pM.
[0079] Preferably the antibody molecule of the present invention
has a binding affinity for IL-17F of better than 10 nM. In one
embodiment the antibody of the present invention has an affinity
for IL-17F of better than 2 nM. In one embodiment the antibody of
the present invention has an affinity for IL-17F of 1.75 nM.
[0080] Preferably the antibody molecule of the present invention
has a binding affinity for IL-17A/F heterodimer of better than 10
nM. In one embodiment the antibody molecule of the present
invention has a binding affinity for IL-17A/F heterodimer of better
than 500 pM. In one embodiment the antibody molecule of the present
invention has a binding affinity for IL-17A/F heterodimer of better
than 150 pM. In one embodiment the antibody molecule of the present
invention has a binding affinity for IL-17A/F heterodimer of 116
pM.
[0081] In one embodiment the antibody molecule of the present
invention has a binding affinity for cynomolgus IL-17F of better
than 2 nM. In one embodiment the antibody molecule of the present
invention has a binding affinity for cynomolgus IL-17F of 1.03
nM.
[0082] 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-17A and/or
IL-17F. 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, 0 779-783, 1992), use of imitator strains of E.
coli (Low at al., J. Mol. Biol., 250, 359-368, 1996), DNA shuffling
(Patten at al., Curr. Opin. Biotechnol., 8 724-733, 1997), phage
display (Thompson et al., J. Mol. Biol., 250, 77-88, 1996) and
sexual PCR (Crameri et al., Nature, 291, 288-291, 1998). Vaughan et
al. (supra) discusses these methods of affinity maturation.
[0083] In one embodiment the antibody molecules of the present
invention neutralise IL-17A and IL-17F activity, for example in the
in vitro assays described in the Examples. In one embodiment the
present invention provides a neutralising antibody having
specificity for human IL-17A and IL-17F which is capable of
inhibiting the activity of 0.8 nM human IL-17A by 50% at a
concentration of less than 5 nM and the activity of 4.2 nM IL-17F
by 50% at a concentration of less than 12 nM said inhibitory
activity being measured on the IL-17A or IL-17F induced release of
IL-6 from Hela cells. In one embodiment the concentration of
antibody which inhibits IL-17A by 50% is less than 3 nM. In one
embodiment the concentration of antibody which inhibits IL-17F by
50% is less than 11 nM. In one embodiment the human IL-17A and
human IL-17F used in the assay are recombinant human IL-17A and
IL-17F. In one embodiment the neutralising antibody is a humanised
or fully human antibody.
[0084] If desired an antibody for use in the present invention may
be conjugated to one or more effector molecule(s). It will be
appreciated that the effector molecule may comprise a single
effector molecule or two or more such molecules so linked as to
form a single moiety that can be attached to the antibodies of the
present invention. Where it is desired to obtain an antibody
fragment linked to an effector molecule, this may be prepared by
standard chemical or recombinant DNA procedures in which the
antibody fragment is linked either directly or via a coupling agent
to the effector molecule. Techniques for conjugating such effector
molecules to antibodies are well known in the art (see, Hellstrom
et al., Controlled Drug Delivery, 2nd Ed., Robinson at al., eds.,
1987, pp. 623-53; Thorpe at al., 1982, Immunol. Rev., 62:119-58 and
Dubowchik at 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.
[0085] 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.
[0086] 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.
[0087] 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), 20
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).
[0088] 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.
[0089] 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
(1'-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 (NOF) or other
growth factor and immunoglobulins.
[0090] 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.
[0091] 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 bather to the immune system. Examples of suitable
effector molecules of this type include polymers, albumin, albumin
binding proteins or albumin binding compounds such as those
described in WO05/117984.
[0092] 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.
[0093] Particular optional substituents which may be present on the
above-mentioned synthetic polymers include one or more hydroxy,
methyl or methoxy groups.
[0094] 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.
[0095] Particular naturally occurring polymers include lactose,
amylose, dextran, glycogen or derivatives thereof.
[0096] "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.
[0097] 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.
[0098] 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.
[0099] 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.
[0100] 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).
[0101] 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.
[0102] In one embodiment, the present invention provides a
neutralising antibody molecule having specificity for human IL-17A
and human IL-17F, which is a modified Fab fragment having a heavy
chain comprising the sequence given in SEQ ID NO:9 and a light
chain comprising the sequence given in SEQ ID NO:7 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.
[0103] 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.
[0104] 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.
[0105] 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.
[0106] 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.
[0107] 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.
[0108] Examples of suitable sequences are provided in SEQ ID NO:8;
SEQ ID NO:10; SEQ ID NO:13; SEQ ID NO:14; SEQ ID NO:17 and SEQ ID
NO:18. Nucleotides 1-57 in SEQ ID NO 18 and 1-60 in SEQ ID NO 14
encode the signal peptide sequence from mouse antibody B72.3
(Whittle et al., 1987, Protein Eng. 1(6) 499-505.) which is cleaved
to give a neutralising antibody molecule of the present
invention.
[0109] 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 sequences given in SEQ ID NO:14 and SEQ ID
NO:18. Nucleotides 1-57 in SEQ ID NO 18 and 1-60 in SEQ ID NO 14
encode the signal peptide sequence from mouse antibody B72.3
(residues 1-19 in SEQ ID NO: 16 and 1-20 in SEQ ID NO:12
respectively) which is most preferably cleaved to give a
neutralising antibody molecule of the present invention.
[0110] 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.
[0111] 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.
[0112] 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.
[0113] 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.
[0114] 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 according to the present 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.
[0115] 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.
[0116] 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.
[0117] 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.
[0118] 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 judgement 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.
[0119] 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.
[0120] 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.
[0121] 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.
[0122] 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.
[0123] 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.
[0124] 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.
[0125] 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.
[0126] 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.
[0127] 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.
[0128] 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.
[0129] 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.
[0130] A thorough discussion of pharmaceutically acceptable
carriers is available in Remington's Pharmaceutical Sciences (Mack
Publishing Company, N.J. 1991).
[0131] 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.
[0132] 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.
[0133] 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-17A and/or IL-17F or
is associated with an increased level of IL-17A and/or IL-17F.
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, 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 (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.
[0134] The present invention also provides an antibody molecule
according to the present invention for use in the treatment or
prophylaxis of pain.
[0135] 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-17A and/or IL-17F or
associated with an increased level of IL-17A and/or IL-17F.
Preferably the pathological disorder is rheumatoid arthritis or
multiple sclerosis.
[0136] 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.
[0137] An antibody molecule of the present invention may be
utilised in any therapy where it is desired to reduce the effects
of IL-17A and/or IL-17F in the human or animal body. IL-17 A and/or
IL-17F 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.
[0138] An antibody molecule according to the present invention is
preferably used for the control of inflammatory disease, autoimmune
disease or cancer.
[0139] The present invention also provides a method of treating
human or animal subjects suffering from or at risk of a disorder
mediated by IL-17A and/or IL-17F, the method comprising
administering to the subject an effective amount of an antibody
molecule of the present invention.
[0140] An antibody molecule according to the present invention may
also be used in diagnosis, for example in the in vivo diagnosis and
imaging of disease states involving IL-17A and/or IL-17F.
[0141] The present invention is further described by way of
illustration only in the following examples, which refer to the
accompanying Figures, in which:
[0142] FIG. 1 [0143] a) Light chain V region of antibody
CA028.sub.--0496 (SEQ ID NO:7) [0144] b) Heavy chain V region of
antibody CA028.sub.--0496 (SEQ ID NO:9) [0145] c) CDRH1 (SEQ ID
NO:1), CDRH12 (SEQ ID NO:2), CDRH3 (SEQ ID NO:3), CDRL1 (SEQ ID
NO:4), CDRL2 (SEQ ID NO:5), CDRL3 (SEQ ID NO:6) of antibody
CA028.sub.--496. [0146] d) Light chain of antibody CA028.sub.--496
(SEQ ID NO:11). [0147] e) Heavy chain of antibody CA028.sub.--496
(SEQ ID NO:15). [0148] f) DNA encoding light chain of antibody
CA028.sub.--496 including signal sequence (SEQ ID NO:14). [0149] g)
DNA encoding heavy chain of antibody CA028.sub.--496 including
signal sequence (SEQ ID NO:18)
[0150] FIG. 2 a) The effect of antibody CA028.sub.--0496
(designated Ab#496 in legend) on human IL-17 induced IL-6
production from Hela cells. b) The effect of antibody
CA028.sub.--0496 (designated Ab#496 in legend) on human IL-17F
induced IL-6 production from Hela cells
DNA MANIPULATIONS AND GENERAL METHODS
[0151] 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 Invitrogen. The concentration
of IgG was determined using IgG assembly ELISA.
IL-17 Isoforms
[0152] Recombinant IL-17A and IL-17F were purchased from R&D
Systems.
[0153] Recombinant IL-17A/F heterodimer was produced by linking
IL-17A and IL-17F using a GS linker. The heterodimer had the
following sequence (SEQ ID NO:19)
TABLE-US-00001 MGITIPRNPGCPNSEDKNFPRTVMVNLNIHNRNTNTNPKRSSDYYNRSTS
PWNLHRNEDPERYPSVIWEAKCRHLGCINADGNVDYHMNSVPIQQEILVL
RREPPHCPNSFRLEKILVSVGCTCVTPIVHHVAGGGGSGGGGSGGGGSGG
GGSRKIPKVGHTFFQKPESCPPVPGGSMKLDIGIINENQRVSMSRNIESR
STSPWNYTVTWDPNRYPSEVVQAQCRNLGCINAQGKEDISMNSVPIQQET
LVVRRKHQGCSVSFQLEKVLVTVGCTCVTPVIHHVQ
[0154] Recombinant Cynomolgus IL-17F (SEQ ID NO:20)
TABLE-US-00002 MRKIPKVGHTFFQKPESCPPVPEGSMKLDTGIINENQRVSMSRNIESRST
SPWNYTVTWDPNRYPSEVVQAQCKHLGCINAQGKEDISMNSVPIQQETLV
LRRKHQGCSVSFQLEKVLVTVGCTCVTPVIHHVQ
[0155] The DNA sequence encoding IL-17A/F heterodimer was
chemically synthesised by Entelechon GmbH and was subcloned into
pET43.1a at the NdeI/XhoI sites. The DNA sequence encoding cyno
L-17F was amplified by PCR using primers that introduced NdeI and
XhoI restriction sites. The PCR products were ligated into
pCR4Blunt-TOPO and sequence verified before digestion and ligation
into pET43.1a at the NdeI/XhoI sites.
[0156] pET43.1a DNA encoding IL-17 isoforms was used to transfect
BL21(DE3) cells and selected carbenicillin-resistant clones were
grown at 37.degree. C. overnight in 2TY broth containing 2% 25
glucose and 50 .mu.g/ml carbenicillin. The cultures were then
diluted and grown in the same medium to an OD.sub.600 of 0.5-0.7,
induced with 1 mM IPTG and grown at 37.degree. C. for a further 4-5
hours.
[0157] Cells were harvested by centrifugation and inclusion bodies
prepared from the cells. Inclusion bodies were solubilised in 50 mM
Tris-HCl, 5M guanidinium hydrochloride, 50 mM NaCl, 1 mM EDTA, 2 mM
reduced glutathione, 0.2 mM oxidised glutathione, pH 8.5. IL-17
protein was refolded by dropwise addition of the solubilised
protein to the above buffer without guanidinium hydrochloride, with
vigorous stirring. The final volume was chosen such that the final
protein concentration was no more than 0.1 mg/ml.
[0158] The refolded protein solution was concentrated if required,
before buffer exchange with 10 mM MES pH6. The protein was then
applied to a column of Sepharose SP HP equilibrated with 20 mM MES
pH6. Protein was eluted with a linear gradient of 0-500 mM NaCl in
MES pH6 over 10 column volumes. For IL-17F the gradient was
extended to 600 mM NaCl. In order to further purify IL-17, the
relevant fraction from the Sepharose SP HP column were pooled,
concentrated and diluted with 20 mM CAPSO (pH10) and applied to a
Mono Q column equilibrated with 20 mM CAPSO. Protein was eluted
with a linear gradient of 0-250 mM NaCl in 20 mM CAPSO over 20
column volumes. Fractions containing IL-17 were pooled and
neutralised using 1M MES pH6.
Example 1
Production of a Neutralising Anti-IL-17 Antibody
[0159] Female Sprague Dawly rats were immunised with recombinant
human IL-17 (purchased from R & D systems). Rats received four
immunisations of 20 .mu.g IL-17 in 100 .mu.l Freund's adjuvant.
Antibody 225 which binds human IL-17 was isolated using the methods
described in WO04/051268. Genes for the heavy chain variable domain
(VH) and light chain variable domain (VL) of antibody 225 were
isolated and sequenced following cloning via reverse transcription
PCR.
[0160] A series of humanised VL and VH regions were designed using
human V-region acceptor frameworks and by varying the number of
donor residues in the framework regions. Eight grafted VL regions
(gL1-8) and 9 grafted VH regions (gH1-9) were designed and genes
were built by oligonucleotide assembly and PCR mutagenesis.
[0161] The light chain grafted sequences were sub-cloned into the
human light chain expression vector pKH10.1 which contains the DNA
encoding the human C-Kappa constant region (Km3 allotype). The
heavy chain grafted sequences were sub-cloned into the human
gamma-4 expression vector pVhg4P FL, which contains the DNA
encoding the human gamma-4 constant region containing the hinge
stabilising mutation S241P (Angal et al., supra). Plasmids were
co-transfected into CHO cells and the antibodies produced screened
for activity in IL-17 binding and neutralisation assays.
Transfections of CHO cells were performed using the
Lipofectamine.TM. 2000 procedure according to manufacturer's
instructions (InVitrogen, catalogue No. 11668).
[0162] The most optimal graft based on expression, affinity and
neutralisation potency (gL7gH9) was selected and named
CA028.sub.--0496. The V region sequences of this antibody are shown
in FIGS. 1 (a) and (b) and in SEQ ID NOs: 7 and 9 for the light
chain (gL7) and heavy chains (gH9) respectively.
[0163] The heavy chain acceptor framework is the human germline
sequence VH3 1-3 3-07 with framework 4 corning from this portion of
the human JH-region germline JH4. The light chain acceptor
framework is the human germline sequence VK1 2-1-(1) L4, with
framework 4 coming from this portion of the human JK-region
germline JK1.
Example 2
Antibody CA028.sub.--0496 neutralises IL-17 and IL-17F and IL-17A/F
Heterodimer
Hela Cells
[0164] The potency of antibody CA028.sub.--0496 against human
recombinant IL-17 and human recombinant IL-17F in Hela cells was
tested and compared to antibody CDP435 (WO06/054059). 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-17A (25 ng ml.sup.-1) or
human IL-17F (125 ng ml.sup.-1) was incubated in the presence of a
fixed concentration of human TNF-.alpha. this mixture was
preincubated with antibody CA028.sub.--0496 or antibody 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-17A/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.
[0165] The data (FIGS. 2a and 2b) indicates that antibody
CA028.sub.--0496 potently neutralised human recombinant IL-17A and
also had some activity against human IL-17F. The data from these
experiments indicated that antibody CA028.sub.--0496 gave an
IC.sub.50 of 43/ng/ml against human recombinant IL-17 (25 ng
ml.sup.-1) and 1477 ng/ml against recombinant IL-17F (125 ng
ml.sup.-1).
[0166] Accordingly, antibody CA028.sub.--0496 gave an IC50 of 0.29M
against human recombinant IL-17 (0.78 nM) and 10.18 nM against
human recombinant IL-17F (4.16 nM) in this assay (calculation based
on per IgG assuming a molecular weight of 145,000 as an average
IgG4 and assuming that IL-17A and IL-17F are dimers).
Human Microglia Cells
[0167] Human microglia cells (TCS Cellworks) were plated out in a
flat bottom 96-well plate at 5,000 cells per well in a total volume
of 100 .mu.l and left for 24 hours to attach to the plastic, At
this time titrations (5, 1, 0.2 and 0.04 .mu.g/ml) of human
recombinant IL-17A, human recombinant IL-17F, cynomolgus
recombinant IL-17F and human recombinant IL-17A/F heterodimer in
the presence and absence of 10 ng/ml human recombinant TNF.alpha.
were added to wells in triplicate. Control wells contained no
stimulation, IL-17A alone (100 ng/ml), TNF.alpha. alone and IL-17A
and TNF.alpha. together. All cytokines were added in a total volume
of 110 .mu.l/well, making the total well volume 210 .mu.l. In
experiments involving antibodies, cells were plated out in the same
way. After 24 hours antibodies and cytokines were added at the same
time to give the stated final concentrations in a total final
volume of 200 .mu.l.
[0168] After a further 24 hours incubation at 37.degree. C.,
supernatants were harvested and frozen at -20.degree. C. until
analysis. For analysis, supernatants were diluted 1/10 and measured
for IL-6 using a human IL-6 MSD kit, according to manufacturer's
instructions.
[0169] All isoforms of IL-17 tested were found to be active in the
assay, particularly in the presence of TNF.alpha..
[0170] The potency of antibody CA028.sub.--0496 against human
recombinant IL-17A and human recombinant IL-17F, cynomolgus
recombinant IL-17F and human recombinant IL-17A/F heterodimer in
human microglia cells was tested in the presence of TNF.alpha. and
compared to a control antibody and an IL-17A specific antibody
using the method described above.
[0171] The control antibody had no effect on the activity of any of
the cytokines tested.
[0172] Antibody CA028.sub.--0496 had inhibitory activity against
all three cytokines IL-17, IL-17F and IL-17A/F, including
cynomolgus IL-17F while the IL-17A specific antibody only had
inhibitory activity against IL-17A and IL-17A/F heterodimer.
Example 3
Affinity of Antibody CA028 0496 (Human IgG4 Constant Regions) for
IL-17A and IL-17F
[0173] BIA (Biamolecular Interaction Analysis) was performed using
a Biacore 3000 (Biacore AB). All experiments were performed at
25.degree. C. Affinipure Fc Fragment goat anti-human IgG, Fc
fragment specific (Jackson ImmunoResearch) was immobilised on a CM5
Sensor Chip via amine coupling chemistry to a capture level of
.apprxeq.6000 response units (RUs). HBS-EP buffer (10 mM HEPES pH
7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, Biacore AB) was
used as the running buffer with a flow rate of 10 .mu.l/min. A 10
.mu.l injection of antibody CA028.sub.--0496 (1.81 mg/ml) was used
for capture by the immobilised anti-human IgG-Fc. Human IL-17A and
IL-17 isoforms were titrated over the captured CA028.sub.--0496 at
doubling dilutions from 50 nM to sub nM at a flow rate of 30
.mu.L/min. The surface was regenerated by a 30 .mu.L injections of
40 mM HCl, followed by one 5 .mu.L injection of 5 mM NaOH.
[0174] Background subtraction binding curves were double referenced
and analysed using the BIAevaluation software (version 3.2)
following standard procedures. Kinetic parameters were determined
from the fitting algorithm.
[0175] The affinity value determined for antibody CA028.sub.--0496
binding IL-17A was 16 pM and 1750 pM for IL-17F. Antibody
CA028.sub.--0496 did not bind to the other IL-17 isoforms (IL-17 B,
C, D and E). Antibody CA028.sub.--0496 therefore specifically binds
IL-17A and IL-17F.
Example 4
Affinity of Antibody CA028 0496 (Murine IgG1 Constant Regions) for
IL-17A, Cynomolgus IL-17F and IL-17A/F Heterodimer
[0176] BIA (Biamolecular Interaction Analysis) was performed using
a Biacore 3000 (Biacore AB).
[0177] All experiments were performed at 25.degree. C. Affinipure
F(ab').sub.2 fragment goat anti-mouse IgG, Fc fragment specific
(Jackson ImmunoResearch) was immobilised on a CM5 Sensor Chip
(Biacore AB) via amine coupling chemistry to a capture level of
.apprxeq.6000 response units (RUs). HBS-EP buffer (10 mM HEPES pH
7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, Biacore AB) was
used as the running buffer with a flow rate of 10 .mu.L/min. A 10
.mu.L injection of antibody CA028.sub.--0496 at 4 ug/mlL was used
for capture by the immobilised anti-mouse IgG, Fc. Human IL-17A,
cyno IL-17F and heterodimerA/F were titrated over the captured
CA028.sub.--0496 at doubling dilutions from 25 nM to sub nM at a
flow rate of 30 .mu.L/min. The surface was regenerated at a
flowrate of 10 uL/min by a 10 .mu.L injection of 40 mM HCl,
followed by a 5 .mu.L injection of 5 mM NaOH.
[0178] Double referenced background subtracted binding curves were
analysed using the BIAevaluation software (version 3.2) following
standard procedures. Kinetic parameters were determined from the
fitting algorithm.
[0179] Antibody CA028.sub.--0496 had an affinity of 21 .mu.M for
IL-17A, 116 .mu.M for IL-17A/F heterodimer and 1030 pM for
cynomolgus IL-17F.
[0180] 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
1
20110PRTRattus rattus 1Gly Phe Thr Phe Ser Asp Tyr Asn Met Ala 1 5
10 217PRTArtificialCDRH2 2Thr Ile Thr Tyr Glu Gly Arg Asn Thr Tyr
Tyr Arg Asp Ser Val Lys 1 5 10 15 Gly 316PRTArtificialCDRH3 3Pro
Pro Gln Tyr Tyr Glu Gly Ser Ile Tyr Arg Leu Trp Phe Ala His 1 5 10
15 411PRTArtificialCDRL1 4Arg Ala Asp Glu Ser Val Thr Thr Leu Met
His 1 5 10 57PRTRattus rattus 5Leu Val Ser Asn Arg Glu Ser 1 5
69PRTRattus rattus 6Gln Gln Thr Trp Ser Asp Pro Trp Thr 1 5
7108PRTArtificialgL7 7Ala Ile Gln Leu 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
Asp Glu Ser Val Thr Thr Leu 20 25 30 Met His Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Leu Val Ser Asn
Arg Glu 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 Thr Trp Ser Asp Pro Trp 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
8324DNAArtificialgL7 8gccatccagc tgacccagag cccttcctct ctcagcgcca
gtgtcggaga cagagtgact 60attacctgca gggctgacga aagcgtgacc acattgatgc
actggtacca acagaagcct 120ggcaaagccc ccaagctcct gatctatctg
gtttccaatc gggagtctgg agtccccagc 180aggttcagcg gcagtgggtc
tggaactgac tttaccctga caatctcctc actccagccc 240gaagatttcg
ccacctacta ttgccagcag acttggagcg acccttggac atttggacag
300ggcacaaaag tggagatcaa gcgt 3249125PRTArtificialgH9 9Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25
30 Asn Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ala Thr Ile Thr Tyr Glu Gly Arg Asn Thr Tyr Tyr Arg Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser 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 Ser Pro Pro Gln Tyr Tyr Glu
Gly Ser Ile Tyr Arg Leu Trp Phe 100 105 110 Ala His Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 115 120 125 10375DNAArtificialgH9
10gaggttcagc tcgttgaatc cggaggcgga ctcgtgcagc ctgggggctc cttgcggctg
60agctgcgctg ccagtggctt cactttcagc gattacaata tggcctgggt gcgccaggcc
120ccaggcaagg gtctggagtg ggtggccaca attacctatg agggcagaaa
cacttattac 180cgggattcag tgaaagggcg atttaccatc agcagggata
atgcaaagaa cagtctgtac 240ctgcagatga actctctgag agctgaggac
accgctgtct actattgtgc aagcccaccc 300cagtactatg agggctcaat
ctacagattg tggtttgccc attggggcca gggaacactg 360gtgaccgtct cgagc
37511214PRTArtificialGL7+constant domain 11Ala Ile Gln Leu 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 Asp Glu Ser Val Thr Thr Leu 20 25 30 Met His
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45
Tyr Leu Val Ser Asn Arg Glu 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 Thr Trp Ser
Asp Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180
185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
12234PRTArtificialSignal+gL7+constant domain 12Met Ser Val Pro Thr
Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr 1 5 10 15 Asp Ala Arg
Cys Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30 Ala
Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Asp Glu Ser 35 40
45 Val Thr Thr Leu Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
50 55 60 Lys Leu Leu Ile Tyr Leu Val Ser Asn Arg Glu Ser Gly Val
Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser 85 90 95 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Thr Trp 100 105 110 Ser Asp Pro Trp Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg 115 120 125 Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 130 135 140 Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 165 170
175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys 195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro 210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230 13645DNAArtificialgL7+constant domain 13gccatccagc
tgacccagag cccttcctct ctcagcgcca gtgtcggaga cagagtgact 60attacctgca
gggctgacga aagcgtgacc acattgatgc actggtacca acagaagcct
120ggcaaagccc ccaagctcct gatctatctg gtttccaatc gggagtctgg
agtccccagc 180aggttcagcg gcagtgggtc tggaactgac tttaccctga
caatctcctc actccagccc 240gaagatttcg ccacctacta ttgccagcag
acttggagcg acccttggac atttggacag 300ggcacaaaag tggagatcaa
gcgtacggta gcggccccat ctgtcttcat cttcccgcca 360tctgatgagc
agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat
420cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg
taactcccag 480gagagtgtca cagagcagga cagcaaggac agcacctaca
gcctcagcag caccctgacg 540ctgagcaaag cagactacga gaaacacaaa
gtctacgcct gcgaagtcac ccatcagggc 600ctgagctcgc ccgtcacaaa
gagcttcaac aggggagagt gttag
64514705DNAArtificialsignal+gL7+constant domain 14atgtcagttc
ccacacaggt gctgggcctg cttctgttgt ggctcaccga tgctaggtgt 60gccatccagc
tgacccagag cccttcctct ctcagcgcca gtgtcggaga cagagtgact
120attacctgca gggctgacga aagcgtgacc acattgatgc actggtacca
acagaagcct 180ggcaaagccc ccaagctcct gatctatctg gtttccaatc
gggagtctgg agtccccagc 240aggttcagcg gcagtgggtc tggaactgac
tttaccctga caatctcctc actccagccc 300gaagatttcg ccacctacta
ttgccagcag acttggagcg acccttggac atttggacag 360ggcacaaaag
tggagatcaa gcgtacggta gcggccccat ctgtcttcat cttcccgcca
420tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa
taacttctat 480cccagagagg ccaaagtaca gtggaaggtg gataacgccc
tccaatcggg taactcccag 540gagagtgtca cagagcagga cagcaaggac
agcacctaca gcctcagcag caccctgacg 600ctgagcaaag cagactacga
gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 660ctgagctcgc
ccgtcacaaa gagcttcaac aggggagagt gttag
70515452PRTArtificialgH9+constant domain 15Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Asn Met
Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Thr Ile Thr Tyr Glu Gly Arg Asn Thr Tyr Tyr Arg Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Ser Pro Pro Gln Tyr Tyr Glu Gly Ser Ile
Tyr Arg Leu Trp Phe 100 105 110 Ala His Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Cys Ser Arg Ser Thr Ser 130 135 140 Glu Ser Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180
185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr
Cys 195 200 205 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
Arg Val Glu 210 215 220 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro
Ala Pro Glu Phe Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 Gln Glu Asp Pro
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 290 295 300
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305
310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
Ser Ser 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu
Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 405 410 415 Val
Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 420 425
430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
435 440 445 Ser Leu Gly Lys 450
16471PRTArtificialsignal+gH9+constant domain 16Met Glu Trp Ser Trp
Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly 1 5 10 15 Val His Ser
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40
45 Ser Asp Tyr Asn Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60 Glu Trp Val Ala Thr Ile Thr Tyr Glu Gly Arg Asn Thr Tyr
Tyr Arg 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn 85 90 95 Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Ser Pro Pro Gln
Tyr Tyr Glu Gly Ser Ile Tyr Arg 115 120 125 Leu Trp Phe Ala His Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 130 135 140 Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 145 150 155 160 Ser
Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 165 170
175 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
180 185 190 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 195 200 205 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Lys Thr 210 215 220 Tyr Thr Cys Asn Val Asp His Lys Pro Ser
Asn Thr Lys Val Asp Lys 225 230 235 240 Arg Val Glu Ser Lys Tyr Gly
Pro Pro Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Phe Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 290 295
300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Gly Leu 340 345 350 Pro Ser Ser Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Gln Glu Glu Met Thr Lys 370 375 380 Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420
425 430 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe
Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser 450 455 460 Leu Ser Leu Ser Leu Gly Lys 465 470
171963DNAArtificialgH9+constant domain 17gaggttcagc tcgttgaatc
cggaggcgga ctcgtgcagc ctgggggctc cttgcggctg 60agctgcgctg ccagtggctt
cactttcagc gattacaata tggcctgggt gcgccaggcc 120ccaggcaagg
gtctggagtg ggtggccaca attacctatg agggcagaaa cacttattac
180cgggattcag tgaaagggcg atttaccatc agcagggata atgcaaagaa
cagtctgtac 240ctgcagatga actctctgag agctgaggac accgctgtct
actattgtgc aagcccaccc 300cagtactatg agggctcaat ctacagattg
tggtttgccc attggggcca gggaacactg 360gtgaccgtct cgagcgcttc
tacaaagggc ccatccgtct tccccctggc gccctgctcc 420aggagcacct
ccgagagcac agccgccctg ggctgcctgg tcaaggacta cttccccgaa
480ccggtgacgg tgtcgtggaa ctcaggcgcc ctgaccagcg gcgtgcacac
cttcccggct 540gtcctacagt cctcaggact ctactccctc agcagcgtgg
tgaccgtgcc ctccagcagc 600ttgggcacga agacctacac ctgcaacgta
gatcacaagc ccagcaacac caaggtggac 660aagagagttg gtgagaggcc
agcacaggga gggagggtgt ctgctggaag ccaggctcag 720ccctcctgcc
tggacgcacc ccggctgtgc agccccagcc cagggcagca aggcatgccc
780catctgtctc ctcacccgga ggcctctgac caccccactc atgcccaggg
agagggtctt 840ctggattttt ccaccaggct ccgggcagcc acaggctgga
tgcccctacc ccaggccctg 900cgcatacagg ggcaggtgct gcgctcagac
ctgccaagag ccatatccgg gaggaccctg 960cccctgacct aagcccaccc
caaaggccaa actctccact ccctcagctc agacaccttc 1020tctcctccca
gatctgagta actcccaatc ttctctctgc agagtccaaa tatggtcccc
1080catgcccacc atgcccaggt aagccaaccc aggcctcgcc ctccagctca
aggcgggaca 1140ggtgccctag agtagcctgc atccagggac aggccccagc
cgggtgctga cgcatccacc 1200tccatctctt cctcagcacc tgagttcctg
gggggaccat cagtcttcct gttcccccca 1260aaacccaagg acactctcat
gatctcccgg acccctgagg tcacgtgcgt ggtggtggac 1320gtgagccagg
aagaccccga ggtccagttc aactggtacg tggatggcgt ggaggtgcat
1380aatgccaaga caaagccgcg ggaggagcag ttcaacagca cgtaccgtgt
ggtcagcgtc 1440ctcaccgtcc tgcaccagga ctggctgaac
ggcaaggagt acaagtgcaa ggtctccaac 1500aaaggcctcc cgtcctccat
cgagaaaacc atctccaaag ccaaaggtgg gacccacggg 1560gtgcgagggc
cacatggaca gaggtcagct cggcccaccc tctgccctgg gagtgaccgc
1620tgtgccaacc tctgtcccta cagggcagcc ccgagagcca caggtgtaca
ccctgccccc 1680atcccaggag gagatgacca agaaccaggt cagcctgacc
tgcctggtca aaggcttcta 1740ccccagcgac atcgccgtgg agtgggagag
caatgggcag ccggagaaca actacaagac 1800cacgcctccc gtgctggact
ccgacggctc cttcttcctc tacagcaggc taaccgtgga 1860caagagcagg
tggcaggagg ggaatgtctt ctcatgctcc gtgatgcatg aggctctgca
1920caaccactac acacagaaga gcctctccct gtctctgggt aaa
1963182020DNAArtificialsignal+gH9+constant domain 18atggaatggt
cctgggtctt cctgtttttc ctttctgtca caaccggggt gcacagcgag 60gttcagctcg
ttgaatccgg aggcggactc gtgcagcctg ggggctcctt gcggctgagc
120tgcgctgcca gtggcttcac tttcagcgat tacaatatgg cctgggtgcg
ccaggcccca 180ggcaagggtc tggagtgggt ggccacaatt acctatgagg
gcagaaacac ttattaccgg 240gattcagtga aagggcgatt taccatcagc
agggataatg caaagaacag tctgtacctg 300cagatgaact ctctgagagc
tgaggacacc gctgtctact attgtgcaag cccaccccag 360tactatgagg
gctcaatcta cagattgtgg tttgcccatt ggggccaggg aacactggtg
420accgtctcga gcgcttctac aaagggccca tccgtcttcc ccctggcgcc
ctgctccagg 480agcacctccg agagcacagc cgccctgggc tgcctggtca
aggactactt ccccgaaccg 540gtgacggtgt cgtggaactc aggcgccctg
accagcggcg tgcacacctt cccggctgtc 600ctacagtcct caggactcta
ctccctcagc agcgtggtga ccgtgccctc cagcagcttg 660ggcacgaaga
cctacacctg caacgtagat cacaagccca gcaacaccaa ggtggacaag
720agagttggtg agaggccagc acagggaggg agggtgtctg ctggaagcca
ggctcagccc 780tcctgcctgg acgcaccccg gctgtgcagc cccagcccag
ggcagcaagg catgccccat 840ctgtctcctc acccggaggc ctctgaccac
cccactcatg cccagggaga gggtcttctg 900gatttttcca ccaggctccg
ggcagccaca ggctggatgc ccctacccca ggccctgcgc 960atacaggggc
aggtgctgcg ctcagacctg ccaagagcca tatccgggag gaccctgccc
1020ctgacctaag cccaccccaa aggccaaact ctccactccc tcagctcaga
caccttctct 1080cctcccagat ctgagtaact cccaatcttc tctctgcaga
gtccaaatat ggtcccccat 1140gcccaccatg cccaggtaag ccaacccagg
cctcgccctc cagctcaagg cgggacaggt 1200gccctagagt agcctgcatc
cagggacagg ccccagccgg gtgctgacgc atccacctcc 1260atctcttcct
cagcacctga gttcctgggg ggaccatcag tcttcctgtt ccccccaaaa
1320cccaaggaca ctctcatgat ctcccggacc cctgaggtca cgtgcgtggt
ggtggacgtg 1380agccaggaag accccgaggt ccagttcaac tggtacgtgg
atggcgtgga ggtgcataat 1440gccaagacaa agccgcggga ggagcagttc
aacagcacgt accgtgtggt cagcgtcctc 1500accgtcctgc accaggactg
gctgaacggc aaggagtaca agtgcaaggt ctccaacaaa 1560ggcctcccgt
cctccatcga gaaaaccatc tccaaagcca aaggtgggac ccacggggtg
1620cgagggccac atggacagag gtcagctcgg cccaccctct gccctgggag
tgaccgctgt 1680gccaacctct gtccctacag ggcagccccg agagccacag
gtgtacaccc tgcccccatc 1740ccaggaggag atgaccaaga accaggtcag
cctgacctgc ctggtcaaag gcttctaccc 1800cagcgacatc gccgtggagt
gggagagcaa tgggcagccg gagaacaact acaagaccac 1860gcctcccgtg
ctggactccg acggctcctt cttcctctac agcaggctaa ccgtggacaa
1920gagcaggtgg caggagggga atgtcttctc atgctccgtg atgcatgagg
ctctgcacaa 1980ccactacaca cagaagagcc tctccctgtc tctgggtaaa
202019286PRTArtificialIL-17A/F heterodimer 19Met Gly Ile Thr Ile
Pro Arg Asn Pro Gly Cys Pro Asn Ser Glu Asp 1 5 10 15 Lys Asn Phe
Pro Arg Thr Val Met Val Asn Leu Asn Ile His Asn Arg 20 25 30 Asn
Thr Asn Thr Asn Pro Lys Arg Ser Ser Asp Tyr Tyr Asn Arg Ser 35 40
45 Thr Ser Pro Trp Asn Leu His Arg Asn Glu Asp Pro Glu Arg Tyr Pro
50 55 60 Ser Val Ile Trp Glu Ala Lys Cys Arg His Leu Gly Cys Ile
Asn Ala 65 70 75 80 Asp Gly Asn Val Asp Tyr His Met Asn Ser Val Pro
Ile Gln Gln Glu 85 90 95 Ile Leu Val Leu Arg Arg Glu Pro Pro His
Cys Pro Asn Ser Phe Arg 100 105 110 Leu Glu Lys Ile Leu Val Ser Val
Gly Cys Thr Cys Val Thr Pro Ile 115 120 125 Val His His Val Ala Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser
Gly Gly Gly Gly Ser Arg Lys Ile Pro Lys Val Gly 145 150 155 160 His
Thr Phe Phe Gln Lys Pro Glu Ser Cys Pro Pro Val Pro Gly Gly 165 170
175 Ser Met Lys Leu Asp Ile Gly Ile Ile Asn Glu Asn Gln Arg Val Ser
180 185 190 Met Ser Arg Asn Ile Glu Ser Arg Ser Thr Ser Pro Trp Asn
Tyr Thr 195 200 205 Val Thr Trp Asp Pro Asn Arg Tyr Pro Ser Glu Val
Val Gln Ala Gln 210 215 220 Cys Arg Asn Leu Gly Cys Ile Asn Ala Gln
Gly Lys Glu Asp Ile Ser 225 230 235 240 Met Asn Ser Val Pro Ile Gln
Gln Glu Thr Leu Val Val Arg Arg Lys 245 250 255 His Gln Gly Cys Ser
Val Ser Phe Gln Leu Glu Lys Val Leu Val Thr 260 265 270 Val Gly Cys
Thr Cys Val Thr Pro Val Ile His His Val Gln 275 280 285
20134PRTArtificialCynomolgus IL-17F 20Met Arg Lys Ile Pro Lys Val
Gly His Thr Phe Phe Gln Lys Pro Glu 1 5 10 15 Ser Cys Pro Pro Val
Pro Glu Gly Ser Met Lys Leu Asp Thr Gly Ile 20 25 30 Ile Asn Glu
Asn Gln Arg Val Ser Met Ser Arg Asn Ile Glu Ser Arg 35 40 45 Ser
Thr Ser Pro Trp Asn Tyr Thr Val Thr Trp Asp Pro Asn Arg Tyr 50 55
60 Pro Ser Glu Val Val Gln Ala Gln Cys Lys His Leu Gly Cys Ile Asn
65 70 75 80 Ala Gln Gly Lys Glu Asp Ile Ser Met Asn Ser Val Pro Ile
Gln Gln 85 90 95 Glu Thr Leu Val Leu Arg Arg Lys His Gln Gly Cys
Ser Val Ser Phe 100 105 110 Gln Leu Glu Lys Val Leu Val Thr Val Gly
Cys Thr Cys Val Thr Pro 115 120 125 Val Ile His His Val Gln 130
* * * * *
References